# Patent

## Title
Methods of reprogramming cells

## Abstract

The present invention provides methods of reprogramming cells, for example, directly reprogramming a somatic cell of a first cell type into a somatic cell of a second cell type, are described herein. In particular, the present invention generally relates to methods for reprogramming a cell of an endoderm origin to a cell having pancreatic β-cell characteristics. The present invention also relates to an isolated population comprising reprogrammed cells, compositions and their use in the treatment of diabetes mellitus. In particular, the present invention relates to reprogramming a cell of an endoderm origin to a cell having pancreatic β-cell characteristics by increasing the protein expression of at least one transcription factor selected from Pdx1, Ngn3 or MafA in the cell of endoderm origin to reprogram the cell of an endoderm cell to a cell which exhibits at least one or at least two characteristics of an endogenous pancreatic β-cell.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. §371 National Phase Entry Application of International Application No. PCT/US2009/054789 filed Aug. 24, 2009, which designates the United States, and which claims benefit of priority under 35 U.S.C. §119(e) of the U.S. Provisional Application No. 61/090,936 filed Aug. 22, 2008, the contents of which are incorporated herein by reference in their entirety.


GOVERNMENT SUPPORT
This invention was made with the Government Support under DK077445 awarded by the National Institutes of Health. The Government has certain rights to this invention.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 18, 2009, is named 00280606.txt, and is 334,313 bytes in size.
FIELD OF INVENTION
The invention relates to methods for reprogramming a cell of an endoderm origin to a cell having pancreatic β-cell characteristics. The present invention also relates to an isolated population comprising reprogrammed cells, compositions and their use in the treatment of diabetes mellitus.
BACKGROUND OF THE INVENTION
One goal of regenerative medicine is to be able to convert adult cells into other cell types for tissue repair and regeneration. Cells of adult organisms arise from sequential differentiation steps that are generally thought to be irreversible1. Biologists often describe this process of development as proceeding from an undifferentiated (embryonic) cell to a terminally differentiated cell that forms part of an adult tissue or organ. There are rare examples, however, in which cells of one type can be converted to another type in a process called cellular reprogramming or lineage reprogramming (Hochedlinger et al., Nature, 2006, 441, 1061-1067; Orkin S et al., Cell, 2008; 132; 631-644). Various forms of cellular reprogramming are referred to in the literature as transdifferentiation, dedifferentiation, or transdetermination2. For example, cellular reprogramming has been reported in amphibian limb regeneration and fly imaginal disc identity switches3,4, and it may be central to certain types of pathological metaplasia2. There is long standing interest and fascination in reprogramming studies, in part because of the promise of harnessing this phenomenon for regenerative medicine whereby abundant adult cells that can be easily harvested would be converted to other medically important cell types to repair diseased or damaged tissues.
Somatic cell nuclear transfer (SCNT), developed in the 1960s, demonstrated that nuclei from differentiated adult cells could be reprogrammed to a totipotent state following injection into enucleated eggs5,6. More recently, it was shown that a small number of transcription factors can reprogram cultured adult skin cells to pluripotent stem cells7-12. These studies point to the possibility of regenerating mammalian tissues by first reverting skin or other adult cells to pluripotent stem cells and then redifferentiating these into various cell types.
SUMMARY OF THE INVENTION
The present invention relates to methods for direct reprogramming (i.e. cellular reprogramming) of cells, such as a cell of endoderm origin to a cell having characteristics of a pancreatic β-cell (i.e. a pancreatic β-like cell). In particular, the present invention relates to a method for reprogramming a cell of endoderm origin, such as, a pancreatic cell (e.g. an exocrine pancreatic cell, a pancreatic duct cell, or an acinar pancreatic cell), or a liver cell by increasing the protein expression of at least two transcription factors selected from any of Ngn3, Pdx1 and MafA in the cell of endoderm origin. In some embodiments, the method comprises increasing the expression of at least three transcription factors selected from Ngn3, Pdx1 and MafA. In some embodiments, the method comprises increasing the protein expression of a functional fragment of at least two transcription factors selected from Ngn3, Pdx1 and MafA in a cell of endoderm origin. In some embodiments, the method further comprises increasing the protein expression of additional transcription factors in addition to at least two selected from Ngn3, Pdx1 and MafA.
Accordingly, the present invention relates to methods, compositions and kits for producing a pancreatic β-like cell from a cell of endoderm origin. Other embodiments of the present invention relate to an isolated population of pancreatic β-like cells produced by increasing the protein expression of at least two transcription factors selected from any combination of Ngn3, Pdx1 and MafA in the cell of endoderm origin.
In some embodiments, the pancreatic β-like cell produced by the methods as disclosed herein secretes at least 15%, or at least 25% or at least 30% of the insulin that endogenous β-cells secrete, or alternatively, in some embodiments, a pancreatic β-like cell exhibits at least two characteristics of an endogenous pancreatic β-cell, for example, but not limited to, secretion of insulin in response to glucose, and expression of β-cell markers, such as for example, c-peptide, Pdx-1, Glut-2. In some embodiments, the β-like cell express insulin. In some embodiments, the β-like cells express VEGF. In some embodiments, the β-like cell expresses other cell markers, such as GCK, PCI/3, and transcription factors NeuroD, Nkx2.2 and Nkx6.2. In some embodiments, the β-like cell express GCK, PCI/3, NeuroD, Nkx2.2 and Nkx6 at a statistically significant increased level as compared to the cell of endoderm origin from which the β-like cell arises. In some embodiments, the β-like cell expresses insulin at a statistically significant increased level as compared to the cell of endoderm origin from which the β-like cell arises. In some embodiments, the β-like cell expresses VEGF at a statistically significant increased level as compared to the cell of endoderm origin from which the β-like cell arises.
In some embodiments, the β-like cell does not express markers Amylase, Ptf1a, Ck19 (Krt19), somatastatin/pancreatic polypeptide (SomPP), glucagon, mesenchymal markers, Nestin, Vimentin or Tuji. In some embodiments, the β-like cell expresses Amylase, Ptf1a, Ck19 (Krt19), somatastatin/pancreatic polypeptide (SomPP), glucagon, mesenchymal markers, Nestin, Vimentin or Tuji at at a statistically significant decreased level as compared to the cell of endoderm origin from which the β-like cell arise.
In some embodiments, an isolated population of pancreatic β-like cells produced by the methods and compositions as disclosed herein, is a population mammalian pancreatic β-like cell, for example, a population of human pancreatic β-like cell.
In some embodiments, an isolated population of pancreatic β-like cells and compositions is produced by the methods comprising contacting a cell or a population of cells of endoderm origin with an agent, such as a nucleic acid agent, peptide, polypeptide aptamer, antibody, antibody fragment, ribosomes, small molecule and the like, which increases the protein expression of at least two transcription factors selected from any combination of Ngn3, Pdx1 and MafA in the cell of endoderm origin. In some embodiments, the method to produce an isolated population of pancreatic β-like cells comprises introducing a nucleic acid sequence encoding at least two of the transcription factors Ngn3, Pdx1 and MafA, or functional fragments thereof into the cell of endoderm origin. In some embodiments, the nucleic acid sequence encoding at least two of the transcription factors Ngn3, Pdx1 and MafA, or functional fragments thereof is expressed transiently for a transient increase the protein expression of the polypeptides Ngn3, Pdx1 and MafA in the cell of endoderm origin.
Herein, the inventors have demonstrated using a strategy of re-expressing key developmental regulators in vivo, specific combinations are identified of three transcription factors (Ngn3, Pdx1 and MafA) that reprogram differentiated pancreatic exocrine cells in adult animals into cells that closely resemble β-cells. The pancreatic β-like cells are substantially indistinguishable from endogenous islet β-cells in size, shape, and ultrastructure. The pancreatic β-like cells express genes essential for β-cell function and can ameliorate hyperglycemia by remodeling local vasculature and secreting insulin. Accordingly, the present invention relates to a method of cellular reprogramming with defined factors, such as at least two of the transcription factors Ngn3, Pdx1 and MafA in an adult organ. The present invention also relates to a general paradigm for directing adult cell reprogramming without reversion to a pluripotent stem cell state.
One aspect of the present invention provides a method for reprogramming a cell of endoderm origin, the method comprising increasing the protein expression of at least two transcription factors selected from Pdx1, Ngn3, or MafA in the cell of enodermal origin, wherein the cell of enodermal origin is reprogrammed to exhibit at least two characteristics of a pancreatic β-cell. In some embodiments, the protein expression of Pdx1, Ngn3, and MafA are increased in the cell of enodermal origin. In some embodiments, the cell of endoderm origin is a pancreatic cell, such as, for example, a exocrine cell, a pancreatic duct cell, and an acinar pancreatic cell. In some embodiments, the cell of endoderm origin is a liver cell. In some embodiments, the cell of endoderm origin is a gall bladder cell.
In some embodiments, a characteristic of a pancreatic β-cell phenotype is secreting insulin in response to glucose. In all aspects of the present invention, a characteristic of a pancreatic β-cell phenotype is expression of at least one marker selected from the group consisting of: Ngn3−, Pdx1+ and MafA+.
In some embodiments, in increase in the protein expression of a transcription factor selected from Pdx1, Ngn3, or MafA is achieved by contacting the cell of endoderm origin with an agent which increases the expression of the transcription factor, where an agent can be selected from the group consisting of: a nucleotide sequence, a protein, an aptamer and small molecule, ribosome, RNAi agent and peptide-nucleic acid (PNA) and anologues or variants thereof. In some embodiments, protein expression is increased by introducing at least one nucleic acid sequence encoding a transcription factor protein selected from Pdx1, Ngn3, or MafA, or encoding a functional fragment thereof, in the cell of endoderm origin.
In some embodiments, protein expression of Pdx1 is increased by introducing a nucleic acid sequence encoding a Pdx1 polypeptide comprising SEQ ID NO: 2 or 32 or a functional fragment of SEQ ID NO: 2 or 32 into the cell of endoderm origin.
In some embodiments, the protein expression of Ngn3 is increased by introducing a nucleic acid sequence encoding a Ngn3 polypeptide comprising SEQ ID NO: 2 or 32 or a functional fragment of SEQ ID NO: 2 or 32 into the cell of endoderm origin.
In some embodiments, the protein expression of MafA is increased by introducing a nucleic acid sequence encoding a MafA polypeptide comprising SEQ ID NO: 3 or 33 or a functional fragment of SEQ ID NO: 3 or 33 into the cell of endoderm origin.
In some embodiments, a nucleic acid sequence is in a vector, such as a viral vector or a non-viral vector. In some embodiments, the vector is a viral vector comprising a genome that does not integrate into the host cell genome.
In some embodiments, cell of endoderm origin is in vitro. In some embodiments, cell of endoderm origin is ex vivo.
The method of any of paragraphs 1 to 19, wherein the cell of endoderm origin is in vivo or present in a subject. In some embodiments, the subject is a human subject. In some embodiments, the subject has, or is at risk of developing, diabetes, such as Type I diabetes, Type II diabetes or pre-diabetes. In some embodiments, the subject has or is at risk of developing a metabolic disorder. In some embodiments, a cell of endoderm origin is a mammalian cell, such as a human cell.
In some embodiments, the methods as disclosed herein for reprogramming a cell of endoderm origin further comprises contacting the cell of endoderm origin with at least one agent which increases the protein expression of at least one of the transcription factors selected from the group consisting of: NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 or Isl1. In some embodiments, the pancreatic β-cell expresses a marker selected from the group consisting of: NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 or Isl1.
In some embodiments, the cell of endoderm origin is reprogrammed to a pancreatic β-cell which is a pancreatic β-like cell. In some embodiments, the pancreatic β-like cell has an increased expression of a marker selected from the group consisting of: c-peptide, glucose transporter 2 (Glut2), glucokinase (GCK), prohormone convertase 1⁄3 (PC1/3), β-cell transcription factors NeuroD, Nkx2.2 and Nkx6.1 by a statistically significant amount relative to the cell of endoderm origin from which the pancreatic β-like cell was derived. In some embodiments, the pancreatic β-like cell has a decreased expression of a marker selected from the group consisting of: Amylase (Amy), glucagon, somatostatin/pancreatic polypeptide (SomPP), Ck19, Nestin, Vimentin and Tuji by a statistically significant amount relative to the cell of endoderm origin from which the pancreatic β-like cell was derived.
Another aspect of the present invention relates to an isolated population of pancreatic β-like cells obtained from a population of non-insulin producing cells of endoderm origin by a process comprising increasing the protein expression of at least two transcription factors selected from Ngn3, Pdx1 or MafA, or a functional fragment thereof, in the non-insulin producing cells of endoderm origin. In some embodiments, expression is transient expression.
In some embodiments, the isolated population of pancreatic β-like cells comprise pancreatic β-like cells which secrete insulin in response to an increase in glucose. In some embodiments, the isolated population of pancreatic β-like cells comprise pancreatic β-like cells which have a distinct morphology and localization as compared to endogenous pancreatic β-cells, for example, where the pancreatic β-like cells have at least one characteristic selected from the group consisting of: cobblestone cell morphology, a diameter of between 17-25 μm and an intercalated location within exocrine acincar rosettes. In some embodiments, the isolated population of pancreatic β-like cells is produced by increasing the protein expression in the a population of cells of endoderm origin of at least two transcription factors, which can be accomplished using a vector, such as a viral vector or a non-viral vector.
In some embodiments, the vector comprises a nucleic acid sequence encoding a Ngn3 polypeptide or a functional fragment thereof, and/or comprises a nucleic acid sequence encoding a Pdx1 polypeptide or a functional fragment thereof and/or comprises a nucleic acid sequence encoding a MafA polypeptide or a functional fragment thereof.
In some embodiments, the isolated population of pancreatic β-like cells comprise pancreatic β-like cells from a population of non-insulin producing cells of endoderm origin, such as a population of pancreatic cells. In some embodiments, such pancreatic cells are selected from the group consisting of: exocrine cells, pancreatic duct cells and an acinar pancreatic cells or a heterogeneous population thereof. In some embodiments, a population of non-insulin producing cells of endoderm origin are a population of liver cells and/or a population of gall bladder cells. In some embodiments, a population of non-insulin producing cells of endoderm origin is a heterogenous population of non-insulin producing cells of endoderm origin. In some embodiments, a population of non-insulin producing cells of endoderm origin is a heterogenous population of pancreas cells, which may include or exclude endogenous pancreatic β-cells. In some embodiments, a population of non-insulin producing cells of endoderm origin is a heterogenous cell population consisting of cells selected from the group consisting of: pancreas cells, liver cells and gall bladder cells.
In some embodiments, the population of non-insulin producing cells of endoderm origin is a population of mammalian cells, for example, human cells. In some embodiments, a population of non-insulin producing cells of endoderm origin is obtained from a subject that has diabetes, or has an increased risk of developing diabetes, such as a subject at increased risk of Type I diabetes, Type II diabetes and pre-diabetes. In some embodiments, a population of non-insulin producing cells of endoderm origin is obtained from a subject that has a metabolic disorder, or has an increased risk of developing a metabolic disorder.
Another aspect of the present invention relates to a method for the treatment of a subject with diabetes, the method comprising administering a composition comprising an isolated population of pancreatic β-like cells according to the methods as disclosed herein.
Another aspect of the present invention relates to the use of the isolated population of pancreatic β-like cells produced by the methods as disclosed herein for administering to a subject in need thereof.
In some embodiments, pancreatic β-like cells are produced from non-insulin producing endoderm cells obtained from the same subject as the composition is administered to. In some embodiments, the subject has has, or has an increased risk of developing, diabetes, for example, where the subject has, or has increased risk of getting diabetes from the group consisting of: Type I diabetes, Type II diabetes and pre-diabetes. In some embodiments, the subject has, or has an increased risk of developing, a metabolic disorder.
Another aspect of the present invention relates to kits for producing pancreatic β-like cells as disclose herein. In some embodiments, a kit comprises (i) a nucleic acid sequence encoding a Ngn3 polypeptide or a functional fragment thereof; and/or (ii) a nucleic acid sequence encoding a Pdx1 polypeptide or a functional fragment thereof; and/or (iii) a nucleic acid sequence encoding a MafA polypeptide or a functional fragment thereof. In some embodiments, the kit further comprises instructions for reprogramming a cell of endoderm origin to a cell with at least two characteristics of a pancreatic β-cell according to the methods as disclosed herein.
Another aspect of the present invention relates to a composition comprising at least one non-insulin producing endodermal cell and at least one agent which increases the protein expression of at least two transcription factors selected from Ngn3, Pdx1 or MafA.
Another aspect of the present invention relates to methods of identifying agents that alone or in combination with other agents reprogram a cell of endoderm origin to a pancreatic β-like cell. In some embodiments, the method includes contacting one or more cells of endoderm origin with one or more test agents (simultaneously or at separate times) and determining the level of expression of one or more reprogramming genes as defined herein. In some embodiments, the β-cell reprogramming genes include, Pdx1, Ngn3, mafA, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 of Isl1. Where one or more test agents increase the level of expression of one or more of the foregoing genes above the level of expression found in the cell of endoderm origin, in the absence of one or more test agents, are considered agents for reprogramming cells of endoderm origin to a pancreatic β-like cell. The test agents may include, but are not limited to, small molecules, nucleic acids, peptides, polypeptides, immunoglobulins, and oligosaccarides. In some embodiments, the just-mentioned method includes determining the level of expression of one or more of Pdx1, Ngn3 or MafA. In some embodiments, the method includes determining the level of expression of one or more of Pdx1, Ngn3 or MafA. Expression levels can be determined by any means known by one of ordinary skill in the art, for example, by RT-PCR or immunological methods.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C shows a combination of three transcription factors induces insulin+ cells in adult mouse pancreas in vivo. FIG. 1A shows a schematic diagram of experimental strategy. Adenoviruses encoding bicistronic transcription factor (T.F.) and nuclear GFP (nGFP) via an IRES element (I) were injected into the pancreas of an adult mouse (Rag−/−). FIG. 1B is a microscope pictorial image showing wild type (WT) pancreas is predominantly exocrine tissue with insulin+ β-cells in islet (outlined). Nuclei stained blue with DAPI. FIG. 1C is a microscope pictorial image showing one month after infection with a combination of Ngn3, Pdx1, and MafA viruses (pAd-M3), numerous insulin+ cells appear outside islets. FIG. 1D and FIG. 1E are graphs showing quantification of induction one month after infection. M9, M6: mixture of 9 and 6 different viruses, respectively. Data presented as mean±s.d. n=3 animals.  ̃1,000 nGFP+ cells/animal. Asterisk, P<0.05; two asterisks, p<0.01; three asterisks, p<0.001.
FIGS. 2A-2B show induced new β-cells originate from differentiated exocrine cells. FIG. 2A is a bar graph showing quantification of nGFP-infected cell types 10 days after nGFP viral infection. Data presented as mean±s.d. n=3 animals.  ̃1,000 nGFP+ cells/animal. FIG. 2B is a schematic demonstrating that double heterozygous Cpa1CreERT2; R26R adults mice are injected with TamoxifenTM, which labels mature exocrine cells with β-galactosidase (β-Gal). Reprogramming is subsequently induced by infection with pAd-M3.
FIGS. 3A-3C show endogenous and pancreatic β-like cells are indistinguishable in morphology and ultrastructure. FIG. 3A is a bar graph showing a size comparison of exocrine cells (white bar), islet β-cells (gray bar), and pancreatic β-like cells (black bar). Data presented as mean±s.d. n=3 animals. >100 cells/animal. Three asterisks: p<0.001. FIG. 3B is electron micrograph of a β-cell (outlined) in an islet. FIG. 3C shows an example of a pancreatic β-like cell situated between two exocrine cells. Endogenous and pancreatic β-like cells contain small insulin granules (In) and lack zymogen granules (Zy) of exocrine cells and extensive endoplasmic reticulum (ER). Nuc: nucleus. Scale bar: 2 um.
FIGS. 4A-4C show induced new pancreatic β-like cells remodel vasculature and ameliorate hyperglycemia. FIG. 4A is a graph showing improvement of fasting blood glucose level in diabetic mice after injection with pAd-M3 (diamond), compared with controls with nGFP virus (square). Triangle: non-diabetic controls. STZ: streptozotocin. Arrows indicate timing of injection. n=6-8 animals. FIG. 4B shows a histogram comparing non-fasting serum insulin levels 6 weeks after injection. n=6-8 animals. FIG. 4C is a histogram showing the average insulin+ β-cell number per section 8 weeks after injection. n=3 animals. Both islet and pancreatic β-like cells were counted for the pAd-M3 samples. One asterisk, p<0.05; two asterisks, p<0.01; three asterisks, p<0.001. Data presented as mean±s.d.
FIGS. 5A-5F show immuno-electron microscopy of pancreatic β-like cells. FIG. 5A shows a pancreatic β-like cell (outlined) with a nucleus (N) and small granules on a section double immunostained for GFP (15 nm gold particles) and insulin (5 nm gold particles). FIG. 5B shows a region of the nucleus (green square in 5A) showing GFP staining. FIG. 5C shows a high magnified picture of the secretory granules (red square in FIG. 5A) showing insulin staining. Arrows in FIGS. 5B and 5C indicate gold particles. FIG. 5D shows an exocrine cell on the same section with large zymogen granules and dense endoplasmic reticulum. FIG. 5E and 5F show high magnification images of examples of nucleus and granules, no gold particles found. Samples harvested one month after M3 induction.
FIGS. 6A-6B show the expression profile comparison of pancreatic β-like cells and islet cells. FIG. 6A shows a yen diagram of the number of differentially expressed genes in pancreatic β-like cells (550 genes) and islet cells (560 genes), and number of genes similarly expressed by both the pancreatic β-like cells and islet cells (1501 genes). The inventors prepared cRNA from three samples; (i) FACS sorted GFP+ cells from M3 infected samples (1 month) that contain  ̃22% reprogrammed insulin+ cells (e.g. pancreatic β-like cells); (ii) isolated whole islets (islet cells); and (iii) pancreatic non-islet cells (non-islet cells). As there are no FACS markers for mouse endocrine or β-cells, the GFP+ cells could not be further purified. Expression profile analysis was carried out with Illumina arrays and showed that in comparison to non-islet samples, 2051 genes are enriched in reprogrammed cells whereas 2061 genes are enriched in whole islets. Of these endocrine enriched genes, 1501 transcripts overlap. Due to contamination by exocrine cells, many exocrine transcripts were detected in the GFP+ sample. FIG. 6B shows a histogram of an example of the array analysis. Genes specific to endocrine cells are highly enriched in both the islet samples and the reprogrammed cell samples. N=3 independent repeats. Data presented as mean±s.d.
FIG. 7 is a graph showing results of a glucose tolerance test. Improved glucose tolerance in diabetic mice after injection with pAd-M3 (diamond), compared with controls with nGFP virus (square). Triangle: non-diabetic controls. Diabetic mice were generated by streptozotocin administration. n=6-8 animals. One asterisk, p<0.05; two asterisks, p<0.01. Data presented as mean±s.d.
FIG. 8 shows lack of spread of virus from pancreas to other organs and lack of spontaneous conversion of other cell types to β-cells in streptozotocin treated animals. FIG. 8 shows an example of RT-PCR analysis with primer set specific for Ngn3 viral transgene showed that virus injected into the pancreas does not spread to other internal organs such as liver, intestine, spleen and kidney. Samples harvested 10 days after injection of pAd M3 into the pancreas. On immuofluresence analysis, GPF was detected in the pancreas only, and no GFP was detected in the liver, spleen or kidney (data not shown).
FIG. 9 shows down regulation of viral genes from pancreatic β-like cells. FIG. 9 shows RT-PCR analysis performed with primer sets specific for viral transgenes. Transgene expression was strongly reduced by 30 days and no longer detectable by 60 days in samples infected with pAd-M3. GADPH serves as control.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions and methods for producing pancreatic β-like cells from a cell of endoderm origin. In some embodiments, the present invention provides compositions and methods for direct reprogramming of cells of endoderm origin to a pancreatic β-like cell, without the cell of endoderm origin becoming an induced pluripotent stem cell (iPS) intermediate prior to being reprogrammed to a pancreatic β-like cell.
The present invention relates to a population of pancreatic β-like cells from cells of endoderm origin, and methods and compositions for the direct reprogramming cells, such as cells of endoderm origin to a pancreatic β-like cell. In particular, the present invention relates to a method for reprogramming a cell of endoderm origin, such as a pancreatic cell (e.g. an exocrine pancreatic cell, a pancreatic duct cell, acinar pancreatic cell), or a liver cell by increasing the protein expression of at least two transcription factors selected from any of Ngn3, Pdx1 and MafA in the cell of endoderm origin. In some embodiments, the method comprises increasing the expression of at least three transcription factors selected from Ngn3, Pdx1 and MafA. In some embodiments, the method comprises increasing the protein expression of a functional fragment of at least two transcription factors selected from Ngn3, Pdx1 and MafA in a cell of endoderm origin. In some embodiments, the method further comprises increasing the protein expression of additional transcription factors in addition to at least two selected from Ngn3, Pdx1 and MafA.
Accordingly, the present invention relates to methods, compositions and kits for producing a reprogrammed pancreatic β-cell from a cell of endoderm origin. Other embodiments of the present invention relate to an isolated population of a reprogrammed pancreatic β-cell produced by increasing the protein expression of at least two transcription factors selected from any combination of Ngn3, Pdx1 and MafA in the cell of endoderm origin.
In some embodiments, the pancreatic β-like cell produced by the methods as disclosed herein expresses at least 15% of the amount of insulin expressed by an endogenous pancreatic β-cell, or at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or greater than 100%, such as at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 3-fold, or at least about 4-fold or at least about 5-fold or more than about 5-fold the amount of the insulin secreted by an endogenous pancreatic β-cell, or alternatively exhibits at least two characteristics of an endogenous pancreatic β-cell, for example, but not limited to, secretion of insulin in response to glucose, and expression of β-cell markers, such as for example, c-peptide, Pdx-1 and glut-2.
In some embodiments, an isolated population of pancreatic β-like cells produced by the methods and compositions as disclosed herein is a mammalian pancreatic β-like cell, for example, a human pancreatic β-like cell.
In some embodiments, an isolated population of pancreatic β-like cells and compositions are produced by a method comprising contacting a cell or a population of cells of endoderm origin with an agent, such as a nucleic acid agent, peptide, polypeptide aptamer, antibody, antibody fragment, ribosomes, small molecules, RNAi agents, ribosomes and the like, which increase the protein expression of at least two transcription factors selected from any combination of Ngn3, Pdx1 and MafA in the cell of endoderm origin. In some embodiments, the method to produce an isolated population of pancreatic β-like cells comprises introducing a nucleic acid sequence encoding at least two of the transcription factors Ngn3, Pdx1 and MafA, or functional fragments thereof into the cell of endoderm origin. In some embodiments, the nucleic acid sequence encoding at least two of the transcription factors Ngn3, Pdx1 and MafA, or functional fragments thereof is expressed transiently for a transient increase the protein expression of the polypeptides Ngn3, Pdx1 and MafA in the cell of endoderm origin.
In some embodiments, the method comprises increasing the protein expression of any of the transcription factors Pdx1, Ngn3, and MafA of SEQ ID NOs 1-3, or functional fragments of proteins of SEQ ID NO: 1-3 respectively for mouse polypeptide, or SEQ ID NO: 31-33 or functional fragments of proteins 31-33 respectively for human polypeptide sequences.
In some embodiments, the method to produce an isolated population of pancreatic β-like cells comprises introducing a nucleic acid sequence encoding at least two of the transcription factors Pdx1, Ngn3, and MafA, or functional fragments thereof into the cell of endoderm origin, where a nucleic acid sequences can be selected from any of SEQ ID NO: 34-39 or fragments thereof. In some embodiments, the nucleic acid sequence encoding at least two of the transcription factors Pdx1, Ngn3, and MafA, or functional fragments thereof (i.e. any sequence selected from SEQ ID NO: 34-39 or fragments thereof) is expressed transiently for a transient increase the protein expression of at least two of the polypeptides selected from Pdx1 (SEQ ID NOs: 1 or 31), Ngn3 (SEQ ID NOs: 2 or 32), and MafA (SEQ ID NOs: 3 or 33), or functional fragments thereof in the cell of endoderm origin.
Definitions:
For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term “reprogramming” as used herein refers to the process that alters or reverses the differentiation state of a somatic cell. The cell can either be partially or terminally differentiated prior to the reprogramming. Reprogramming encompasses complete reversion of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation produces an induced pluripotent (iPS) cell. A partial reversal of differentiation produces a partially induced pluriotent (PiPS) cell. Reprogramming also encompasses partial reversion of the differentiation state, for example to a multipotent state or to a somatic cell that is neither pluripotent or multipotent, but is a cell that has lost one or more specific characteristics of the differentiated cell from which it arises, e.g. direct reprogramming of a differentiated cell to a different somatic cell type. Reprogramming generally involves alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation as a zygote develops into an adult.
The term “pluripotent” as used herein refers to a cell with the capacity, under different conditions, to differentiate to more than one differentiated cell type, and preferably to differentiate to cell types characteristic of all three germ cell layers. Pluripotent cells are characterized primarily by their ability to differentiate to more than one cell type, preferably to all three germ layers, using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers.
The term “differentiated cell” is meant any primary cell that is not, in its native form, pluripotent as that term is defined herein. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. However, simply culturing such cells does not, on its own, render them pluripotent. The transition to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character in culture. Reprogrammed pluripotent cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture. Stated another way, the term “differentiated cell” refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cellular differentiation process.
As used herein, the term “somatic cell” refers to are any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body—apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells—is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell”, by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell”, by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. Unless otherwise indicated the methods for reprogramming somatic cells of endoderm origin to a β-cell can be performed both in vivo and in vitro (where in vivo is practiced when somatic cells of endoderm origin are present within a subject, and where in vitro is practiced using isolated somatic cells of endoderm origin maintained in culture). In some embodiments, where somatic cells of endodermal origin are cultured in vitro, the somatic cells of endodermal cells are cultured in an organotypic pancreatic slice culture, as described in, e.g., meneghel-Rozzo et al., (2004), Cell Tissue Res, 316(3); 295-303, which is incorporated herein in its entirety by reference.
As used herein, the term “adult cell” refers to a cell found throughout the body after embryonic development.
As used herein, the terms “iPS cell” and “induced pluripotent stem cell” are used interchangeably and refers to a pluripotent stem cell artificially derived (e.g., induced or by complete reversal) from a non-pluripotent cell, typically an adult somatic cell, for example, by inducing a forced expression of one or more genes.
The term “endoderm cell” as used herein refers to a cell which is from one of the three primary germ cell layers in the very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. An endoderm cell differentiates to give rise first to the embryonic gut and then to the linings of respiratory and digestive tracts and the liver and pancreas.
The term “a cell of endoderm origin” as used herein refers to a cell of endoderm origin includes any cell which has developed from an endoderm cell. Without wishing to be bound by theory, liver and pancreas progenitors develop from endoderm cells in the embryonic foregut. Shortly after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular functions and regenerative capacities. These changes are elicited by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in the development and regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic β cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model organisms and humans have revealed evolutionarily conserved inductive signals and transcription factor networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to promote hepatocyte and β cell differentiation from diverse stem and progenitor cell types.
The term “pancreatic β-like cell” as used herein refers to a cell produced by the methods as disclosed herein which expresses at least 15% of the amount of insulin expressed by an endogenous pancreatic β-cell, or at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100% or greater than 100%, such as at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 3-fold, or at least about 4-fold or at least about 5-fold or more than about 5-fold the amount of the insulin secreted by an endogenous pancreatic β-cell, or alternatively exhibits at least one, or at least two characteristics of an endogenous pancreatic β-cell, for example, but not limited to, secretion of insulin in response to glucose, and expression of β-cell markers, such as for example, c-peptide, Pdx-1 and glut-2. The pancreatic β-like cell is sometimes referred herein to as the “reprogrammed cell” or “reprogrammed β-cell”, which are used interchangeably herein with the term “pancreatic β-like cell”.
As used herein, the term “endogenous β-cell” refers to an insulin producing cell of the pancreas or a cell of a pancreatic β-cell (beta cell) phenotype. The phenotype of a pancreatic β-cell is well known by persons of ordinary skill in the art, and include, for example, secretion of insulin in response to an increase in glucose level, expression of markers such as c-peptide, PDX-1 polypeptide and Glut 2, as well as distinct morphological characteristics such as organized in islets in pancreas in vivo, and typically have small spindle like cells of about 9-15 μm diameter.
As used herein, the term “insulin producing cell” includes endogenous β-cells as that term is described herein, as well as pancreatic β-like cells as that term is described herein, that synthesize (i.e., transcribe the insulin gene, translate the proinsulin mRNA, and modify the proinsulin mRNA into the insulin protein), express (i.e., manifest the phenotypic trait carried by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. A population of pancreatic β-like cells or insulin producing cells made by the present invention may contain β-cells or β-like cells (e.g., cells that have at least two characteristics of an endogenous β-cell). The novelty of the present composition and methods is not negated by the presence of cells in the population that produce insulin naturally (e.g., beta cells). It is also contemplated that the population of pancreatic β-like cells may also contain non-insulin producing cells (i.e. cells of β-cell like phenotype with the exception they do not produce or secrete insulin).
The term “non-insulin producing endodermal cell” as used herein is meant any cell of endoderm origin that does not naturally synthesize, express, or secrete insulin constitutively or inducibly. Thus, the term “non-insulin producing endodermal cells” as used herein excludes pancreatic beta cells. Examples of non-insulin producing endodermal cells that can be used in the methods of the present invention include pancreatic non-beta cells, such as amylase producing cells, acinar cells, cells of ductal adenocarcinoma cell lines (e.g., CD18, CD11, and Capan-I cells (see Busik et al., 1997; Schaffert et al. 1997). Non-pancreatic cells of endoderm origin could also be used, for example, non-pancreatic stem cells and cells of other endocrine or exocrine organs, including, for example, liver cells, tymus cells, thyroid cells, intestine cells, lung cells and pituitary cells. In some embodiments, the non-insulin producing endodermal cells can be mammalian cells or, even more specifically, human cells. Examples of the present method using mammalian pancreatic non-islet, pancreatic amylase producing cells, pancreatic acinar cells are provided herein.
The term “progenitor cell” is used herein to refer to cells that have a cellular phenotype that is more primitive (i.e., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.
The term “stem cell” as used herein, refers to an undifferentiated cell which is capable of proliferation and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. The term “stem cell” refers to a subset of progenitors that have the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating. In one embodiment, the term stem cell refers generally to a naturally occurring mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells are also “multipotent” because they can produce progeny of more than one distinct cell type, but this is not required for “stem-ness.” Self-renewal is the other classical part of the stem cell definition, and it is essential as used in this document. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype. Alternatively, some of the stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only. Formally, it is possible that cells that begin as stem cells might proceed toward a differentiated phenotype, but then “reverse” and re-express the stem cell phenotype, a term often referred to as “dedifferentiation” or “reprogramming” or “retrodifferentiation” by persons of ordinary skill in the art.
In the context of cell ontogeny, the adjective “differentiated”, or “differentiating” is a relative term meaning a “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, stem cells can differentiate to lineage-restricted precursor cells (such as a mesodermal stem cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as an cardiomyocyte precursor), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
The term “embryonic stem cell” is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Pat. Nos. 5,843,780, 6,200,806). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970). The distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.
The term “adult stem cell” or “ASC” is used to refer to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As indicated above, stem cells have been found resident in virtually every tissue. Accordingly, the present invention appreciates that stem cell populations can be isolated from virtually any animal tissue.
The term “exocrine cell” as used herein refers to a cell of an exocrine gland, i.e. a gland that discharges its secretion via a duct. In particular embodiments, an exocrine cells refers to a pancreatic exocrine cell, which is a pancreatic cell that produces enzymes that are secreted into the small intestine. These enzymes help digest food as it passes through the gastrointestinal tract. Pancratic exocrine cells are also known as islets of Langerhans, that secrete two hormones, insulin and glucagon. A pancreatic exocrine cell can be one of several cell types: alpha-2 cells (which produce the hormone glucagon); or β-cells (which manufacture the hormone insulin); and alpha-1 cells (which produce the regulatory agent somatostatin). Non-insulin producing exocrine cells as used herein refers to alpha-2 cells or alpha-1 cells. Note, the term pancreatic exocrine cells encompasses “pancreatic endocrine cells” which refer to a pancreatic cell that produces hormones (e.g., insulin (produced from β-cells) and glucagon (produced by alpha-2 cells) that are secreted into the bloodstream.
The term “pancreas” refers to a glandular organ that secretes digestive enzymes and hormones. In humans, the pancreas is a yellowish organ about 7 in. (17.8 cm) long and 1.5 in. (3.8 cm) wide. It lies beneath the stomach and is connected to the small intestine intestine, muscular hoselike portion of the gastrointestinal tract extending from the lower end of the stomach (pylorus) to the anal opening. Most of the pancreatic tissue consists of grapelike clusters of cells that produce a clear fluid (pancreatic juice) that flows into the duodenum through a common duct along with bile from the liver. Pancreatic juice contains three digestive enzymes: tryptase, amylase, and lipase, that, along with intestinal enzymes, complete the digestion of proteins, carbohydrates, and fats, respectively. Scattered among the enzyme-producing cells of the pancreas are small groups of endocrine cells, called the islets of Langerhans, that secrete two hormones, insulin and glucagon. The pancreatic islets contain several types of cells: alpha-2 cells, which produce the hormone glucagon; beta cells, which manufacture the hormone insulin; and alpha-1 cells, which produce the regulatory agent somatostatin. These hormones are secreted directly into the bloodstream, and together, they regulate the level of glucose in the blood. Insulin lowers the blood sugar level and increases the amount of glycogen (stored carbohydrate) in the liver; glucagon has the opposite action. Failure of the insulin-secreting cells to function properly results in diabetes diabetes or diabetes mellitus.
The term a “β-cell reprogramming gene”, as used herein, refers to a gene whose expression, contributes to the direct reprogramming of cells of endoderm origin, i.e. somatic cells of endoderm origin to a cell with a pancreatic β-cell like phenotype, i.e. a cell which exhibits at least two characteristics of an endogenous pancreatic β-cell. A β-cell reprogramming gene can be, for example, genes encoding transcription factors Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32), or MafA (SEQ ID NO: 3 or 33). Other β-cell reprogramming genes encode transcription factors including, but are not limited to, NeuroD (SEQ ID NO:4), Nkx2.2 (SEQ ID NO:5), Nkx6.1 (SEQ ID NO:6), Pax4 (SEQ ID NO:7), Pax6 (SEQ ID NO:8) or Isl (SEQ ID NO:9).
The term “β-cell reprogramming factor” or “β-cell reprogramming polypeptide” refers to an expression product of a β-cell reprogramming gene. Expression of an exogenously introduced β-cell reprogramming factor may be transient, i.e., it may be needed during at least a portion of the direct reprogramming process in order to induce the direct reprogramming of a cell of endoderm origin to a pancreatic β-like cell like phenotype and/or establish a stable pancreatic β-like cell but afterwards not required to maintain the survival or propagation of the pancreatic β-like cell. For example, the β-cell reprogramming factor may induce expression of endogenous genes whose function is associated with reprogramming cells of endoderm origin to a pancreatic β-cell phenotype. These genes may then maintain the reprogrammed cells as differentiated pancreatic β-like cells, as disclosed herein. A β-cell reprogramming factor can be, for example, transcription factors Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32), or MafA (SEQ ID NO: 3 or 33). Other β-cell reprogramming factors are transcription factors including, but are not limited to, NeuroD (SEQ ID NO:4), Nkx2.2 (SEQ ID NO:5), Nkx6.1 (SEQ ID NO:6), Pax4 (SEQ ID NO:7), Pax6 (SEQ ID NO:8) or Isl (SEQ ID NO:9).
The term “β-cell reprogramming agent” refers to any agent which increases the protein expression of a β-cell reprogramming gene, as that term is described herein. Preferably, a β-cell reprogramming agent increases the expression of a β-cell reprogramming factor selected from transcription factors Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32), or MafA (SEQ ID NO: 3 or 33). A β-cell reprogramming agent includes a β-cell reprogramming factor as that term is defined herein, or any other agent which increases the expression of a β-cell reprogramming gene, such as increases the protein expression of Pdx1, Ngn3 or MafA.
The term “agent” as used herein means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof etc. In certain embodiments, agents are small molecule having a chemical moiety. For example, chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
The term “cell culture medium” (also referred to herein as a “culture medium” or “medium”) as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art.
The term “cell line” refers to a population of largely or substantially identical cells that has typically been derived from a single ancestor cell or from a defined and/or substantially identical population of ancestor cells. The cell line may have been or may be capable of being maintained in culture for an extended period (e.g., months, years, for an unlimited period of time). It may have undergone a spontaneous or induced process of transformation conferring an unlimited culture lifespan on the cells. Cell lines include all those cell lines recognized in the art as such. It will be appreciated that cells acquire mutations and possibly epigenetic changes over time such that at least some properties of individual cells of a cell line may differ with respect to each other.
The term “exogenous” refers to a substance present in a cell or organism other than its native source. For example, the terms “exogenous nucleic acid” or “exogenous protein” refer to a nucleic acid or protein that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found or in which it is found in lower amounts. A substance will be considered exogenous if it is introduced into a cell or an ancestor of the cell that inherits the substance. In contrast, the term “endogenous” refers to a substance that is native to the biological system.
The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, translation, folding, modification and processing. “Expression products” include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.
The term “genetically modified” or “engineered” cell as used herein refers to a cell into which an exogenous nucleic acid has been introduced by a process involving the hand of man (or a descendant of such a cell that has inherited at least a portion of the nucleic acid). The nucleic acid may for example contain a sequence that is exogenous to the cell, it may contain native sequences (i.e., sequences naturally found in the cells) but in a non-naturally occurring arrangement (e.g., a coding region linked to a promoter from a different gene), or altered versions of native sequences, etc. The process of transferring the nucleic into the cell can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments the polynucleotide or a portion thereof is integrated into the genome of the cell. The nucleic acid may have subsequently been removed or excised from the genome, provided that such removal or excision results in a detectable alteration in the cell relative to an unmodified but otherwise equivalent cell.
The term “identity” as used herein refers to the extent to which the sequence of two or more nucleic acids or polypeptides is the same. The percent identity between a sequence of interest and a second sequence over a window of evaluation, e.g., over the length of the sequence of interest, may be computed by aligning the sequences, determining the number of residues (nucleotides or amino acids) within the window of evaluation that are opposite an identical residue allowing the introduction of gaps to maximize identity, dividing by the total number of residues of the sequence of interest or the second sequence (whichever is greater) that fall within the window, and multiplying by 100. When computing the number of identical residues needed to achieve a particular percent identity, fractions are to be rounded to the nearest whole number. Percent identity can be calculated with the use of a variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. ScL USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc. Natl. Acad. ScL USA 90:5873-5877, 1993 is incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J. MoI. Biol. 215:403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. A PAM250 or BLOSUM62 matrix may be used. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). See the Web site having URL www.ncbi.nlm.nih.gov for these programs. In a specific embodiment, percent identity is calculated using BLAST2 with default parameters as provided by the NCBI.
The term “isolated” or “partially purified” as used herein refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and/or that would be present with the nucleic acid or polypeptide when expressed by a cell, or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription/translation is considered “isolated”.
The term “isolated cell” as used herein refers to a cell that has been removed from an organism in which it was originally found or a descendant of such a cell. Optionally the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally the cell is later introduced into a second organism or re-introduced into the organism from which it (or the cell from which it is descended) was isolated.
The term “isolated population” with respect to an isolated population of cells as used herein refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched from.
The term “substantially pure”, with respect to a particular cell population, refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, with respect to the cells making up a total cell population. Recast, the terms “substantially pure” or “essentially purified”, with regard to a population of pancreatic β-like cells, refers to a population of cells that contain fewer than about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are not pancreatic β-like cells or their progeny as defined by the terms herein. In some embodiments, the present invention encompasses methods to expand a population of pancreatic β-like cells, wherein the expanded population of pancreatic β-like cells is a substantially pure population of pancreatic β-like cells.
The term “modulate” is used consistently with its use in the art, i.e., meaning to cause or facilitate a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, or change in relative strength or activity of different components or branches of the process, pathway, or phenomenon. A “modulator” is an agent that causes or facilitates a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.
As used herein, the term “DNA” is defined as deoxyribonucleic acid.
The term “polynucleotide” is used herein interchangeably with “nucleic acid” to indicate a polymer of nucleosides. Typically a polynucleotide of this invention is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and complements of each single-stranded molecule) are provided. “Polynucleotide sequence” as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e. the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5′ to 3′ direction unless otherwise indicated.
The terms “polypeptide” as used herein refers to a polymer of amino acids. The terms “protein” and “polypeptide” are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a “polypeptide”. Exemplary modifications include glycosylation and palmitoylation. Polypeptides may be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc. The term “polypeptide sequence” or “amino acid sequence” as used herein can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated.
The terms “polypeptide variant” refers to any polypeptide differing from a naturally occurring polypeptide by amino acid insertion(s), deletion(s), and/or substitution(s). Variants may be naturally occurring or created using, e g., recombinant DNA techniques or chemical synthesis. In some embodiments amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in any of a variety or properties such as side chain size, polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathicity of the residues involved. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine. The polar (hydrophilic), neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Insertions or deletions may range in size from about 1 to 20 amino acids, e.g., 1 to 10 amino acids. In some instances larger domains may be removed without substantially affecting function. In certain embodiments of the invention the sequence of a variant can be obtained by making no more than a total of 5, 10, 15, or 20 amino acid additions, deletions, or substitutions to the sequence of a naturally occurring enzyme. In some embodiments not more than 1%, 5%, 10%, 15% or 20% of the amino acids in a polypeptide are insertions, deletions, or substitutions relative to the original polypeptide. Guidance in determining which amino acid residues may be replaced, added, or deleted without eliminating or substantially reducing activities of interest, may be obtained by comparing the sequence of the particular polypeptide with that of homologous polypeptides (e.g., from other organisms) and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with those found in homologous sequences since amino acid residues that are conserved among various species are more likely to be important for activity than amino acids that are not conserved.
By “amino acid sequences substantially homologous” to a particular amino acid sequence (e.g. Pdx1, Ngn3 or MafA) is meant polypeptides that include one or more additional amino acids, deletions of amino acids, or substitutions in the amino acid sequence of Pdx1, Ngn3 or MafA without appreciable loss of functional activity as compared to wild-type Pdx1, Ngn3 or MafA polypeptides in terms of the ability to produce pancreatic β-like cells from cells of endoderm origin (i.e. directly reprogram cells of endoderm origin to pancreatic β-like cell like cells). For example, the deletion can consist of amino acids that are not essential to the presently defined differentiating activity and the substitution(s) can be conservative (i.e., basic, hydrophilic, or hydrophobic amino acids substituted for the same). Thus, it is understood that, where desired, modifications and changes may be made in the amino acid sequence of Pdx1, Ngn3 or MafA, and a protein having like characteristics still obtained. It is thus contemplated that various changes may be made in the amino acid sequence of the Pdx1, Ngn3 or MafA amino acid sequence (or underlying nucleic acid sequence) without appreciable loss of biological utility or activity and possibly with an increase in such utility or activity. In some embodiments, the amino acid sequences substantially homologous to a particular amino acid sequence are at least 70%, e.g., 75%, 80%85%, 90%, 95% or another percent from 70% to 100%, in intergers thereof, identical to the particular amino acid sequence.
As used herein, “Pdx1” is refers to the Pdx1 protein of Genebank accession No: NM_008814 (mouse) (SEQ ID NO:1) or NP_000200.1 (Human)(SEQ ID NO: 31), or Gene ID: 3651. The term Pdx1 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Pdx1 is referred in the art as aliases; pancreatic and duodenal homeobox 1, IDX-1, STF-1, PDX-1, MODY4, Ipf1. Human Pdx1 is encoded by nucleic acid corresponding to GenBank Accession No: NM_000209 (human) (SEQ ID NO:34) or NM_008814 (mouse)(SEQ ID NO: 37). Pdx1 protein is a transcriptional activator of several genes, including insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2 (GLUT2). Pdx1 is a nuclear protein is involved in the early development of the pancreas and plays a major role in glucose-dependent regulation of insulin gene expression. Defects in the gene encoding the Pdx1 protein are a cause of pancreatic agenesis, which can lead to early-onset insulin-dependent diabetes mellitus (NIDDM), as well as maturity onset diabetes of the young type 4 (MODY4). The term “Pdx1”, or “Pdx1 protein” as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Pdx1 protein or a fragment, variant, or derivative thereof that at least in part retains the ability of the naturally occurring protein to bind to DNA and activate gene transcription of insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2 (GLUT2). In addition to naturally-occurring allelic variants of the pdx1 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 1 or SEQ ID NO: 31 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms “Pdx1”, “Pdx1 protein”, etc.
As used herein, “Ngn3” is refers to the Ngn3 protein of Genebank accession No: NM_009719 (mouse) (SEQ ID NO:2) or NP_066279.2 (Human)(SEQ ID NO: 32); GeneID No: 50674. The term Ngn3 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Ngn3 is referred in the art as aliases; neurogenin 3; Atoh5; Math4B; bHLHa7; NEUROG3. Human Ngn3 is encoded by nucleic acid corresponding to GenBank Accession No: NM_020999 (human) (SEQ ID NO:35) or NM_009719 (mouse)(SEQ ID NO: 38). Neurogenin-3 (NEUROG3) is expressed in endocrine progenitor cells and is required for endocrine cell development in the pancreas and intestine (Wang et al., 2006). It belongs to a family of basic helix-loop-helix transcription factors involved in the determination of neural precursor cells in the neuroectoderm (Gradwohl et al., 2000). The term “Ngn3”, or “Ngn3 protein” as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Ngn3 protein or a fragment, variant, or derivative thereof that at least in part retains the ability of the naturally occurring protein to bind to DNA and activate gene transcription of NeuroD, Delta-like 1 (Dll1), HeyL, insulinoma-assiciated-1 (IA1), Nk2.2, Notch, Hes5, Isl1, Somatastain receptor 2 (Sstr2) and other genes as disclosed in Serafimidis et al., Stem cells; 2008; 26; 3-16, which is incorporated herein in its entirety by reference. In addition to naturally-occurring allelic variants of the Ngn3 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 2 or SEQ ID NO: 32 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms “Ngn3”, “Ngn3 protein”, etc.
As used herein, “MafA” is refers to the MafA protein of Genebank accession No: NM_194350 (mouse) (SEQ ID NO:3) or NP_963883.2 (Human)(SEQ ID NO: 33); GeneID No: 389692. The term MafA also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. MafA is referred in the art as aliases; v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (avian), hMafA; RIPE3b1; MAFA. Human MafA is encoded by nucleic acid corresponding to GenBank Accession No: NM_201589 (human) (SEQ ID NO:36) or NM_194350 (mouse) (SEQ ID NO: 39). MAFA is a transcription factor that binds RIPE3b, a conserved enhancer element that regulates pancreatic beta cell-specific expression of the insulin gene (INS; MIM 176730) (Olbrot et al., 2002). The term “MafA”, or “MafA′′ protein” as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a MafA′′ protein or a fragment, variant, or derivative thereof that at least in part retains the ability of the naturally occurring protein to bind to DNA and activate gene transcription of Glut2 and pyruvate carboxylase, and other genes such as Glut2, Pdx-1, Nkx6.1, GLP-1 receptor, prohormone convertase-1/3 as disclosed in Wang et al., Diabetologia. 2007 February; 50(2): 348-358, which is incorporated herein by reference. In addition to naturally-occurring allelic variants of the MafA sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 3 or SEQ ID NO: 33 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms “MafA”, “MafA protein”, etc.
As used herein, “glucose transporter 2” refers to the Glut2 protein of Genebank accession NP_000331.1 (human) (SEQ ID NO: 46); NP_112474 (mouse) (SEQ ID NO: 47). The term Glut2 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Glut2 is referred in the art as aliases; Glut2, Slc2a2, solute carrier family 2 (facilitated glucose transporter). Human Glut2 is encoded by nucleic acid corresponding to GenBank Accession No: NM_000340.1 (human) (SEQ ID NO: 48); NM_031197 (mouse) (SEQ ID NO: 49). In addition to naturally-occurring allelic variants of the Glut2 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 46 or SEQ ID NO: 47 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “Glut2”, etc. Glucose transporter 2 isoform is an integral plasma membrane glycoprotein of the liver, islet beta cells, intestine, and kidney epithelium. It mediates facilitated bidirectional glucose transport. Because of its low affinity for glucose, it has been suggested as a glucose sensor. (GeneID: 6514). The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “glucokinase” or “GCK” are used interchangeably herein and refer to the GCK protein of Genebank accession NP_000153.1 (human) (SEQ ID NO: 50); NP_034422.2 (mouse) (SEQ ID NO: 51). The term GCK also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. GCK is referred in the art as aliases; glucokinase (hexokinase 4); GK; GLK; HK4; HHF3; HKIV; HXKP; MODY2; GCK. Human GCK is encoded by nucleic acid corresponding to GenBank Accession No: NM_000162.3 (human) (SEQ ID NO: 52); NM_010292.4 (mouse) (SEQ ID NO: 53). In addition to naturally-occurring allelic variants of the GCK sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 50 or SEQ ID NO: 51 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “GCK”, etc. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “prohormone convertase” or “PC1/3” are used interchangeably herein and refer to the PC1/3 protein of Genebank accession NP_000430.3 (human) (SEQ ID NO: 54); NP_038656.1 (mouse) (SEQ ID NO: 55). The term PC1/3 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. PC1/2 is referred in the art as aliases; proprotein convertase subtilisin/kexin type 1; PC1, PC3, SPC3; PCSK1; Nec-1, Nec1, Phpp-1. Human PC1/3 is encoded by nucleic acid corresponding to GenBank Accession NM_000439 (human) (SEQ ID NO: 56); NM_013628.2 (mouse) (SEQ ID NO: 57). In addition to naturally-occurring allelic variants of the PC1/3 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 54 or SEQ ID NO: 55 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “PC1/3”, etc. The protein encoded by this gene belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. This encoded protein is a type I proinsulin-processing enzyme that plays a key role in regulating insulin biosynthesis. It is also known to cleave proopiomelanocortin, prorenin, proenkephalin, prodynorphin, prosomatostatin and progastrin. Mutations in this gene are thought to cause obesity. This encoded protein is associated with carcinoid tumors. The use of alternate polyadenylation sites has been found for this gene, as well as multiple alternatively spliced transcript variants. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “NeuroD” refers to the NeuroD protein of Genebank accession NP_002491.2 (human) (SEQ ID NO: 58); NP_035024.1 (mouse) (SEQ ID NO: 59). The term NeuroD also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. NeuroD is referred in the art as aliases; neurogenic differentiation 1; BETA2; BHF-1; NEUROD; bHLHa3; NEUROD1. NeuroD is encoded by nucleic acid corresponding to GenBank Accession No: NM_002500.2 (human) (SEQ ID NO: 60); NM_010894.2 (mouse) (SEQ ID NO: 61). In addition to naturally-occurring allelic variants of the NeuroD sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 58 or SEQ ID NO: 59 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “NeuroD”, etc. The NeuroD gene encodes a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors, and the NeuroD protein forms heterodimers with other bHLH proteins and activates transcription of genes that contain a specific DNA sequence known as the E-box. It regulates expression of the insulin gene, and mutations in this gene result in type II diabetes mellitus. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “NK2 homeobox 2” or “Nkx2.2” are used interchangeably herein and refer to the Nkx2.2 protein of Genebank accession Nos: NP_002500.1 (human) (SEQ ID NO: 62); NP_001071100.1 (mouse) (SEQ ID NO: 63). The term Nkx2.2 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Nkx2.2 is referred in the art as aliases; NK2 homeobox 2; NKX2B; NKX2.2; NKX2-2; tinman. Nkx2.2 is encoded by nucleic acid corresponding to GenBank Accession No: NM_002509.2 (human) (SEQ ID NO: 64); NM_001077632.1 (mouse) (SEQ ID NO: 65). In addition to naturally-occurring allelic variants of the Nkx2.2 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 62 or SEQ ID NO: 63 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “Nkx2.2”, etc. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “NK6 homeobox 1” or “Nkx6.1” are used interchangeably herein and refer to the Nkx6.1 protein of Genebank accession Nos: NP_006159.2 (human) (SEQ ID NO: 66); NP_659204.1 (mouse) (SEQ ID NO: 67). The term Nxk6.1 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Nkx6.1 is referred in the art as aliases; NK6 homeobox 1; NKX6A; NKX6.1 and NKX6-1. Nkx6.1 is encoded by nucleic acid corresponding to GenBank Accession No: NM_006168.2 (human) (SEQ ID NO: 68); NM_144955.2 (mouse) (SEQ ID NO: 69). In addition to naturally-occurring allelic variants of the Nkx6.1 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 66 or SEQ ID NO: 67 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “Nkx6.1”, etc. In the pancreas, NKX6.1 is required for the development of beta cells and is a potent bifunctional transcription regulator that binds to AT-rich sequences within the promoter region of target genes Iype et al. (2004). The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein the term “c-peptide” refers to the connecting peptide which connects the A and B chains of the insulin protein hormone involved in the regulation of blood sugar levels. By way of background, insulin is produced in the liver as: its precursor proinsulin, consisting of the B and A chains of insulin linked together via a connecting C-peptide (hereinafter this C-peptide derived from the proinsulin molecule is referred to as “insulin C-peptide”). The term c-peptide also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. The protein sequence for human C-peptide is disclosed as SEQ ID NO: 1 in U.S. Pat. No. 6,558,924 (which is incorporated herein in its entirity by reference and is herein referred to as SEQ ID NO: 102). The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, the term “insulin” or “INS” refers to the protein of Genebank Nos: NM_000207.2 (human) (SEQ ID NO: 70); NM_008386.3 (mouse) (SEQ ID NO: 71). The term insulin also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Insulin is referred in the art as aliases; Ins-1; Ins2-rs1; Ins1. Insulin is encoded by nucleic acid corresponding to GenBank Accession No: NM_000207.2 (human) (SEQ ID NO: 72); NM_008386.3 (mouse) (SEQ ID NO: 73). In addition to naturally-occurring allelic variants of the insulin sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 72 or SEQ ID NO: 73 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “insulin”, etc. Insulin is a protein hormone involved in the regulation of blood sugar levels. Insulin is produced in the liver or pancreas as: its precursor proinsulin, consisting of the B and A chains of insulin linked together via a connecting C-peptide. Insulin itself is comprised of only the B and A chains. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “Amylase” or “AMY” are used interchangeably herein and refer to the amylase protein of Genebank accession NP_000690.1 (human) (SEQ ID NO: 74); NP_001036177.1 (mouse) (SEQ ID NO: 75). The term amylase also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. GCK is referred in the art as aliases; AMY2; AMY2B; amylase, alpha 2B (pancreatic). Amylase is encoded by nucleic acid corresponding to GenBank Accession No: NM_000699.2 (human) (SEQ ID NO: 76); NM_001042712.1 (mouse) (SEQ ID NO: 77). In addition to naturally-occurring allelic variants of the amylase sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 74 or SEQ ID NO: 75 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “amylase”, etc. Amylases are secreted proteins that hydrolyze 1,4-alpha-glucoside bonds in oligosaccharides and polysaccharides, and thus catalyze the first step in digestion of dietary starch and glycogen. The human genome has a cluster of several amylase genes that are expressed at high levels in either salivary gland or pancreas. This gene encodes an amylase isoenzyme produced by the pancreas. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “Glucagon” refers to the glucagon protein of Genebank accession Nos: NP_002045.1 (pro-protein) (human) (SEQ ID NO: 78); NP_032126.1 (mouse) (SEQ ID NO: 79). The term Glucagon also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. glucagon is referred in the art as aliases; GLP1; GLP2; GRPP; GCG; glicentin-related polypeptide, glucagon-like peptide 1, glucagon-like peptide 2. Glucagon is encoded by nucleic acid corresponding to GenBank Accession No: NM_002054.2 (human) (SEQ ID NO: 80); NM_008100.3 (mouse) (SEQ ID NO: 81). In addition to naturally-occurring allelic variants of the glucagon sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 78 or SEQ ID NO: 78 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “glucagon”, etc. The glucagon protein encoded by this gene is actually a preproprotein that is cleaved into four distinct mature peptides. One of these, glucagon, is a pancreatic hormone that counteracts the glucose-lowering action of insulin by stimulating glycogenolysis and gluconeogenesis. Glucagon is a ligand for a specific G-protein linked receptor whose signalling pathway controls cell proliferation. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “somatostatin/pancreatic polypeptide” or “SomPP” are used interchangeably herein and refer to the pancreatic polypeptide protein of Genebank accession Nos: NP_001039.1 (human) (SEQ ID NO: 82); NP_033241.1 (mouse) (SEQ ID NO: 83). The term pancreatic polypeptide also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Pancreatic polypeptide is referred in the art as aliases; somatostatin-14; somatostatin-28; SST. Pancreatic polypeptide is encoded by nucleic acid corresponding to GenBank Accession No: NM_001048.3 (human) (SEQ ID NO: 84); NM_009215.1 (mouse) (SEQ ID NO: 85). In addition to naturally-occurring allelic variants of the Pancreatic polypeptide sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 82 or SEQ ID NO: 83 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “pancreatic polypeptide”, etc. The hormone somatostatin/pancreatic polypeptide has active 14 aa and 28 aa forms that are produced by alternate cleavage of the single preproprotein encoded by this gene. Somatostatin is expressed throughout the body and inhibits the release of numerous secondary hormones by binding to high-affinity G-protein-coupled somatostatin receptors. This hormone is an important regulator of the endocrine system through its interactions with pituitary growth hormone, thyroid stimulating hormone, and most hormones of the gastrointestinal tract. Somatostatin also affects rates of neurotransmission in the central nervous system and proliferation of both normal and tumorigenic cells. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “Ptf1a” or “pancreas specific transcription factor, 1a” are used interchangeably herein and refer to the Pf1a protein of Genebank accession NP_835455.1 (human) (SEQ ID NO: 86); NP_061279.1 (mouse) (SEQ ID NO: 87). The term Ptf1a also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Ptf1a is referred in the art as aliases; pancreas specific transcription factor, 1a; PTF1-p48, bHLHa29 PTF1p48. Ptf1a is encoded by nucleic acid corresponding to GenBank Accession No: NM_178161.2 (human) (SEQ ID NO: 88); NM_018809.1 (mouse) (SEQ ID NO: 89). In addition to naturally-occurring allelic variants of the Ptf1a sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 50 or SEQ ID NO: 51 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “Ptf1a”, etc. This gene encodes a protein that is a component of the pancreas transcription factor 1 complex (PTF1) and is known to have a role in mammalian pancreatic development. The protein plays a role in determining whether cells allocated to the pancreatic buds continue towards pancreatic organogenesis or revert back to duodenal fates. The protein is thought to be involved in the maintenance of exocrine pancreas-specific gene expression including elastase 1 and amylase. Mutations in this gene cause cerebellar agenesis and loss of expression is seen in ductal type pancreas cancers. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “Ck19” refers to the Ck19 protein of Genebank accession NP_002267.2 (human) (SEQ ID NO: 90); NP_032497.1 (mouse) (SEQ ID NO: 91). The term Ck19 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Ck19 is referred in the art as aliases; keratin 19, K19, CK19, K1CS, MGC15366; Krt19. Ck19 is encoded by nucleic acid corresponding to GenBank Accession NM_002276.4 (human) (SEQ ID NO: 92); NM_008471.2 (mouse) (SEQ ID NO: 93). In addition to naturally-occurring allelic variants of the Ck19 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 90 or SEQ ID NO: 91 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “Ck19”, etc. The Ck19 protein encoded by this gene is a member of the keratin family. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. The type I cytokeratins consist of acidic proteins which are arranged in pairs of heterotypic keratin chains Unlike its related family members, this smallest known acidic cytokeratin is not paired with a basic cytokeratin in epithelial cells. It is specifically expressed in the periderm, the transiently superficial layer that envelopes the developing epidermis. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “nestin” refers to the nestin protein of Genebank accession NP_006608.1 (human) (SEQ ID NO: 94); NP_057910.3 (mouse) (SEQ ID NO: 95). The term nestin also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Nestin is referred in the art as aliases; FLJ21841; Nbla00170; NES. Nestin is encoded by nucleic acid corresponding to GenBank Accession No: NM_006617.1 (human) (SEQ ID NO: 96); NM_016701.3 (mouse) (SEQ ID NO: 97). In addition to naturally-occurring allelic variants of the Nestin sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 94 or SEQ ID NO: 95 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “Nestin”, etc. Nestin is an intermediate filament protein that was first identified with a monoclonal antibody by Hockfield and McKay (1985). It is expressed predominantly in stem cells of the central nervous system in the neural tube. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
As used herein, “vimentin” refers to the vimentin of Genebank accession NP_003371.2 (human) (SEQ ID NO: 98); NP_035831.2 (mouse) (SEQ ID NO: 99). The term vimentin also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Vemintin is referred in the art as aliases; FLJ36605; VIM. Vimentin is encoded by nucleic acid corresponding to GenBank Accession No: NM_003380.2 (human) (SEQ ID NO: 100); NM_011701.3 (mouse) (SEQ ID NO: 101). In addition to naturally-occurring allelic variants of the vementin sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 98 or SEQ ID NO: 99 (referred to as “wild type” sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms “vimentin”, etc. The vimentin gene encodes a member of the intermediate filament family. Intermediate filamentents, along with microtubules and actin microfilaments, make up the cytoskeleton. The protein encoded by this gene is responsible for maintaining cell shape, integrity of the cytoplasm, and stabilizing cytoskeletal interactions. It is also involved in the immune response, and controls the transport of low-density lipoprotein (LDL)-derived cholesterol from a lysosome to the site of esterification. It functions as an organizer of a number of critical proteins involved in attachment, migration, and cell signaling. Mutations in this gene causes a dominant, pulverulent cataract. The sequences of the accession Nos. that have been assigned Sequence Identifier numbers are incorporated herein in their entirety.
The term a “variant” in referring to a polypeptide could be, e.g., a polypeptide at least 80%, 85%, 90%, 95%, 98%, or 99% identical to full length polypeptide. The variant could be a fragment of full length polypeptide The variant could be a naturally occurring splice variant. The variant could be a polypeptide at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of the polypeptide, wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% as long as the full length wild type polypeptide or a domain thereof having an activity of interest such as the ability to directly reprogram endodermal cells (e.g. pancreatic exocrine cells) to pancreatic β-like cells. In some embodiments the domain is at least 100, 200, 300, or 400 amino acids in length, beginning at any amino acid position in the sequence and extending toward the C-terminus. Variations known in the art to eliminate or substantially reduce the activity of the protein are preferably avoided. In some embodiments, the variant lacks an N- and/or C-terminal portion of the full length polypeptide, e.g., up to 10, 20, or 50 amino acids from either terminus is lacking. In some embodiments the polypeptide has the sequence of a mature (full length) polypeptide, by which is meant a polypeptide that has had one or more portions such as a signal peptide removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments wherein the protein is produced other than by purifying it from cells that naturally express it, the protein is a chimeric polypeptide, by which is meant that it contains portions from two or more different species. In some embodiments wherein a protein is produced other than by purifying it from cells that naturally express it, the protein is a derivative, by which is meant that the protein comprises additional sequences not related to the protein so long as those sequences do not substantially reduce the biological activity of the protein.
One of skill in the art will be aware of, or will readily be able to ascertain, whether a particular polypeptide variant, fragment, or derivative is functional using assays known in the art. For example, the ability of a variant of a Pdx1 (SEQ ID NO: 1 or SEQ ID NO:31) or Ngn3 (SEQ ID NO:2 or 32) or MafA (SEQ ID NO: 3 or 33) polypeptide to reprogram cells of endoderm origin to pancreatic β-like cells can be assessed using the assays as disclose herein in the Examples. Other convenient assays include measuring the ability to activate transcription of a reporter construct containing a Pdx1 or Ngn3 or MafA binding site operably linked to a nucleic acid sequence encoding a detectable marker such as luciferase. One assay involves determining whether the Pdx1 or Ngn3 or MafA variant induces a cell of endoderm origin (such as a pancreatic exocrine cell) to secrete insulin in the presence of a polypeptide at least two wild type polypeptides of Pdx1, Ngn3 or MafA (i.e. not selecting the polypeptide of which the variant is being assessed). Insulin secretion can be determined using any suitable method, e.g., immunoblotting. Such assays may readily be adapted to identify or confirm activity of agents that directly reprogram cells of endoderm origin to pancreatic β-like cells. In certain embodiments of the invention a functional variant or fragment has at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the full length wild type polypeptide.
The term “functional fragments” as used herein regarding Pdx1, Ngn3 or MafA polypeptides or other β-cell reprogramming factors having amino acid sequences substantially homologous thereto means a polypeptide sequence of at least 5 contiguous amino acids of Pdx1 (SEQ ID NO: 1 or 31), Ngn3 (SEQ ID NO: 2 or 32) or MafA (SEQ ID NO: 3 or 33), or other β-cell reprogramming factors having amino acid sequences substantially homologous thereto, wherein the functional fragment polypeptide sequence is about at least 50%, or 60% or 70% or at 80% or 90% or 100% or greater, for example 1.5-fold, 2-fold, 3-fold, 4-fold or greater than 4-fold as effective at direct reprogramming cells of endoderm origin as the corresponding wild type Pdx1 (SEQ ID NO: 1 or 31), Ngn3 (SEQ ID NO: 2 or 32) or MafA (SEQ ID NO: 3 or 33) polypeptides as described herein. The functional fragment polypeptide may have additional functions that can include decreased antigenicity, increased DNA binding (as in transcription factors), or altered RNA binding (as in regulating RNA stability or degradation).
The term “vector” refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. Thus, an “expression vector” is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell. In some embodiments the gene of interest is operably linked to another sequence in the vector. Vectors can be viral vectors or non-viral vectors. Should viral vectors be used, it is preferred the viral vectors are replication defective, which can be achieved for example by removing all viral nucleic acids that encode for replication. A replication defective viral vector will still retain its infective properties and enters the cells in a similar manner as a replicating adenoviral vector, however once admitted to the cell a replication defective viral vector does not reproduce or multiply. Vectors also encompass liposomes and nanoparticles and other means to deliver DNA molecule to a cell.
The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. The term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to permit expression of the polynucleotide sequence under the control of the expression control sequence, and production of the desired polypeptide encoded by the polynucleotide sequence.
The term “viral vectors” refers to the use of viruses, or virus-associated vectors as carriers of a nucleic acid construct into a cell. Constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including reteroviral and lentiviral vectors, for infection or transduction into cells. The vector may or may not be incorporated into the cell's genome. The constructs may include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors.
As used herein, the term “adenovirus” refers to a virus of the family Adenovirida. Adenoviruses are medium-sized (90-100 nm), nonenveloped (naked) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome.
As used herein, the term “non-integrating viral vector” refers to a viral vector that does not integrate into the host genome; the expression of the gene delivered by the viral vector is temporary. Since there is little to no integration into the host genome, non-integrating viral vectors have the advantage of not producing DNA mutations by inserting at a random point in the genome. For example, a non-integrating viral vector remains extra-chromosomal and does not insert its genes into the host genome, potentially disrupting the expression of endogenous genes. Non-integrating viral vectors can include, but are not limited to, the following: adenovirus, alphavirus, picornavirus, and vaccinia virus. These viral vectors are “non-integrating” viral vectors as the term is used herein, despite the possibility that any of them may, in some rare circumstances, integrate viral nucleic acid into a host cell's genome. What is critical is that the viral vectors used in the methods described herein do not, as a rule or as a primary part of their life cycle under the conditions employed, integrate their nucleic acid into a host cell's genome. It goes without saying that an iPS cell generated by a non-integrating viral vector will not be administered to a subject unless it and its progeny are free from viral remnants.
As used herein, the term “viral remnants” refers to any viral protein or nucleic acid sequence introduced using a viral vector. Generally, integrating viral vectors will incorporate their sequence into the genome; such sequences are referred to herein as a “viral integration remnant”. However, the temporary nature of a non-integrating virus means that the expression, and presence of, the virus is temporary and is not passed to daughter cells. Thus, upon passaging of a re-programmed cell the viral remnants of the non-integrating virus are essentially removed.
As used herein, the term “free of viral integration remnants” and “substantially free of viral integration remnants” refers to iPS cells that do not have detectable levels of an integrated adenoviral genome or an adenoviral specific protein product (i.e., a product other than the gene of interest), as assayed by PCR or immunoassay. Thus, the iPS cells that are free (or substantially free) of viral remnants have been cultured for a sufficient period of time that transient expression of the adenoviral vector leaves the cells substantially free of viral remnants.
The terms “regulatory sequence” and “promoter” are used interchangeably herein, and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operatively linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of a protein. In some instances the promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required for initiating transcription of a specific gene.
As used herein, the term “tissue-specific promoter” means a nucleic acid sequence that serves as a promoter, i.e., regulates expression of a selected nucleic acid sequence operably linked to the promoter, and which selectively affects expression of the selected nucleic acid sequence in specific cells of a tissue, such as cells of neural origin, e.g. neuronal cells. The term also covers so-called “leaky” promoters, which regulate expression of a selected nucleic acid primarily in one tissue, but cause lesser expression in other tissues as well.
The term “phenotype” refers to one or a number of total biological characteristics that define the cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
As used herein, “the presence of lower amounts of a marker in the β-like cell as compared to the cell of endoderm origin from which is was derived” refers to an amount of a marker protein or gene product (e.g. mRNA) that is significantly decreased in the β-like cell as compared to the amount of the same marker present in the cell of endoderm origin from which is was derived. “significantly decreased” means that the differences between the compared levels is statistically significant. The levels of the marker level can be represented by arbitrary units, for example as units obtained from a densitometer, luminometer, or an Elisa plate reader.
As used herein, “the presence of higher amounts of a marker in the β-like cell as compared to the cell of endoderm origin from which is was derived” refers to an amount of a marker protein or gene product (e.g. mRNA) that is significantly increased in the β-like cell as compared to the amount of the same marker present in the cell of endoderm origin from which is was derived. “significantly increased” means that the differences between the compared levels is statistically significant. The levels of the marker level can be represented by arbitrary units, for example as units obtained from a densitometer, luminometer, or an Elisa plate reader.
As used herein, the term “transcription factor” refers to a protein that binds to specific parts of DNA using DNA binding domains and is part of the system that controls the transfer (or transcription) of genetic information from DNA to RNA.
As used herein, “proliferating” and “proliferation” refer to an increase in the number of cells in a population (growth) by means of cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens. Cell proliferation may also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.
The terms “enriching” or “enriched” are used interchangeably herein and mean that the yield (fraction) of cells of one type is increased by at least 10% over the fraction of cells of that type in the starting culture or preparation.
The terms “renewal” or “self-renewal” or “proliferation” are used interchangeably herein, are used to refer to the ability of stem cells to renew themselves by dividing into the same non-specialized cell type over long periods, and/or many months to years. In some instances, proliferation refers to the expansion of cells by the repeated division of single cells into two identical daughter cells.
The term “lineages” as used herein describes a cell with a common ancestry or cells with a common developmental fate. In the context of a cell that is of endoderm origin or is “endodermal linage” this means the cell was derived from an endodermal cell and can differentiate along the endodermal lineage restricted pathways, such as one or more developmental lineage pathways which give rise to definitive endoderm cells, which in turn can differentiate into liver cells, thymus, pancreas, lung and intestine.
The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced”, “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
The terms “increased”, “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
As used herein, the term “xenogeneic” refers to cells that are derived from different species.
The term “β-cell reprogramming factor” refers to a gene, RNA, or protein that promotes or contributes to cell reprogramming, e.g., in vitro. In aspects of the invention relating to reprogramming factor(s), the invention provides embodiments in which the reprogramming factor(s) are of interest for reprogramming somatic cells to pluripotency in vitro. Examples of reprogramming factors of interest for reprogramming somatic cells to pluripotency in vitro are Oct4, Nanog, Sox2, Lin28, Klf4, c-Myc, and any gene/protein that can substitute for one or more of these in a method of reprogramming somatic cells in vitro. “Reprogramming to a pluripotent state in vitro”, or “reprogramming to pluripotency in vitro”, is used herein to refer to in vitro reprogramming methods that do not require and typically do not include nuclear or cytoplasmic transfer or cell fusion, e.g., with oocytes, embryos, germ cells, or pluripotent cells. Any embodiment or claim of the invention may specifically exclude compositions or methods relating to or involving nuclear or cytoplasmic transfer or cell fusion, e.g., with oocytes, embryos, germ cells, or pluripotent cells.
A “marker” as used herein is used to describe the characteristics and/or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interests. Markers will vary with specific cells. Markers are characteristics, whether morphological, functional or biochemical (enzymatic) characteristics of the cell of a particular cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art. However, a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art. Markers can also be the absence of a morphological characteristic or absence of proteins, lipids etc. Markers can be a combination of a panel of unique characteristics of the presence and absence of polypeptides and other morphological characteristics.
The term “selectable marker” refers to a gene, RNA, or protein that when expressed, confers upon cells a selectable phenotype, such as resistance to a cytotoxic or cytostatic agent (e.g., antibiotic resistance), nutritional prototrophy, or expression of a particular protein that can be used as a basis to distinguish cells that express the protein from cells that do not. Proteins whose expression can be readily detected such as a fluorescent or luminescent protein or an enzyme that acts on a substrate to produce a colored, fluorescent, or luminescent substance (“detectable markers”) constitute a subset of selectable markers. The presence of a selectable marker linked to expression control elements native to a gene that is normally expressed selectively or exclusively in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed to a pluripotent state. A variety of selectable marker genes can be used, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP) blue, sapphire, yellow, red, orange, and cyan fluorescent proteins and variants of any of these. Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also of use. As will be evident to one of skill in the art, the term “selectable marker” as used herein can refer to a gene or to an expression product of the gene, e.g., an encoded protein.
In some embodiments the selectable marker confers a proliferation and/or survival advantage on cells that express it relative to cells that do not express it or that express it at significantly lower levels. Such proliferation and/or survival advantage typically occurs when the cells are maintained under certain conditions, i.e., “selective conditions”. To ensure an effective selection, a population of cells can be maintained for a under conditions and for a sufficient period of time such that cells that do not express the marker do not proliferate and/or do not survive and are eliminated from the population or their number is reduced to only a very small fraction of the population. The process of selecting cells that express a marker that confers a proliferation and/or survival advantage by maintaining a population of cells under selective conditions so as to largely or completely eliminate cells that do not express the marker is referred to herein as “positive selection”, and the marker is said to be “useful for positive selection”. Negative selection and markers useful for negative selection are also of interest in certain of the methods described herein. Expression of such markers confers a proliferation and/or survival disadvantage on cells that express the marker relative to cells that do not express the marker or express it at significantly lower levels (or, considered another way, cells that do not express the marker have a proliferation and/or survival advantage relative to cells that express the marker). Cells that express the marker can therefore be largely or completely eliminated from a population of cells when maintained in selective conditions for a sufficient period of time.
A “reporter gene” as used herein encompasses any gene that is genetically introduced into a cell that adds to the phenotype of the stem cell. Reporter genes as disclosed in this invention are intended to encompass fluorescent, luminescent, enzymatic and resistance genes, but also other genes which can easily be detected by persons of ordinary skill in the art. In some embodiments of the invention, reporter genes are used as markers for the identification of particular stem cells, cardiovascular stem cells and their differentiated progeny. A reporter gene is generally operatively linked to sequences that regulate its expression in a manner dependent upon one or more conditions which are monitored by measuring expression of the reporter gene. In some cases, expression of the reporter gene may be determined in live cells. Where live cell reporter gene assays are used, reporter gene expression may be monitored at multiple timepoints, e.g., 2, 3, 4, 5, 6, 8, or 10 or more timepoints. In some cases, where a live cell reporter assay is used, reporter gene expression is monitored with a frequency of at least about 10 minutes to about 24 hours, e.g., 20 minutes, 1 hour, 2 hrs, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, or another frequency from any interger between about 10 minutes to about 24 hours.
The terms “subject” and “individual” are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained and/or to whom treatment, including prophylactic treatment, with the cells as described herein, is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human subject, the term subject refers to that specific animal. The “non-human animals” and “non-human mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g. dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like.
The terms “treat”, “treating”, “treatment”, etc., as applied to an isolated cell, include subjecting the cell to any kind of process or condition or performing any kind of manipulation or procedure on the cell. As applied to a subject, the terms refer to providing medical or surgical attention, care, or management to an individual. The individual is usually ill or injured, or at increased risk of becoming ill relative to an average member of the population and in need of such attention, care, or management.
As used herein, the term “treating” and “treatment” refers to administering to a subject an effective amount of a composition so that the subject as a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term “treatment” includes prophylaxis. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already diagnosed with a cardiac condition, as well as those likely to develop a cardiac condition due to genetic susceptibility or other factors such as weight, diet and health.
As used herein, the terms “administering,” “introducing” and “transplanting” are used interchangeably in the context of the placement of pancreatic β-like cells of the invention into a subject, by a method or route which results in at least partial localization of the pancreatic β-like cells at a desired site. The pancreatic β-like cells can be directly to the pancreas, or alternatively be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e. g. twenty-four hours, to a few days, to as long as several years.
The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. The phrases “systemic administration,” “administered systemically”, “peripheral administration” and “administered peripherally” as used herein mean the administration of cardiovascular stem cells and/or their progeny and/or compound and/or other material other than directly into the central nervous system, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
The term “tissue” refers to a group or layer of specialized cells which together perform certain special functions. The term “tissue-specific” refers to a source of cells from a specific tissue.
As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.
Direct Reprogramming:
The process of altering the cell phenotype of a differentiated cell (i.e. a first cell), such as a somatic cell to a differentiated cell of a different phenotype (i.e. a second cell) without the first differentiated cell being completely reprogrammed to a less differentiated phenotype intermediate is referred to as “direct reprogramming”. Stated another way, cells of one type can be converted to another type in a process by what is commonly referred to in the art as cellular reprogramming or lineage reprogramming.
Direct reprogramming (i.e. cellular reprogramming), which encompasses a process of switching the phenotype of a differentiated cell to the phenotype of a different differentiated cell, without the complete reversal of the differentiation state of the somatic cell, is distinct from “reprogramming to a pluripotent state” which typically refers to a process that completely reverses the differentiation state of a somatic cell to a cell with a stem cell-like phenotype.
As disclosed herein, the present invention relates to compositions and methods for the direct reprogramming of a cell of endoderm origin, such as a somatic cell which is of endoderm origin (e.g. pancreatic exocrine cells, pancreatic acinar cell, liver cells etc.) to a cell of an insulin producing phenotype, such as a cell with pancreatic β-cell characteristics. Such a direct reprogrammed cell as disclosed herein is referred to as a “pancreatic β-like cell”. In certain embodiments of the invention, reprogramming a cell of endoderm origin causes the cell of endoderm origin to assume a pancreatic β-cell like state, without being completely reprogrammed to a pluripotent state prior to assuming the β-cell like phenotype.
In some embodiments, the methods and compositions of the present invention can be practiced on cells of endoderm origin that are fully differentiated and/or restricted to giving rise only to cells of that particular type. The cell of endoderm origin can be either partially or terminally differentiated prior to direct reprogramming.
The present invention relates to compositions and methods for reprogramming a cell of endoderm origin, such as a somatic cell, (e.g., a pancreatic cell such as a pancreatic exocrine cell, or a pancreatic duct cell or a pancreatic acrine cell, or a liver cell) to a β-cell. The invention provides methods for reprogramming endodermal cells to a different phenotype, such as a pancreatic β-cell phenotype. The resulting cells are referred to herein as “pancreatic β-like cells” herein, or in some embodiments as induced β-cells if reprogrammed to a pluripotent state.
Cells of Endoderm Origin
The present invention relates to a method of direct reprogramming cells of an endoderm origin to pancreatic β-like cells. In some embodiments, cells of endoderm origin are the preferred starting material. In some embodiments, a population of pancreatic β-like cells is produced by increasing the protein expression of at least two transcription factors selected from Pdx1, Ngn3 and MafA in a cell of endoderm origin. Accordingly in all aspects of the present invention, the population of cells of endoderm origin can comprise any cell type of endoderm origin, for example, but not limited to, a liver cell and pancreatic cell, such as a pancreatic endodermal cell, pancreatic anciar cell, pancreatic duct cell and the like. In alternative embodiments, the population of cells of endoderm origin can comprise a mixture or combination of different cells of endoderm origin, for example a mixture of cells such as a liver cell, a pancreatic cell, such as a pancreatic endodermal cell, pancreatic anciar cell, pancreatic duct cell.
In some embodiments, the population of cells of endoderm origin is a substantially pure population of pancreatic exocrine cells, or substantially pure population of pancreatic duct cells or in some embodiments a substantially pure population of liver cells (e.g hepatocytes). In some embodiments, a population of cells of endoderm origin is substantially free of insulin-producing cells, such as β-cells.
In some embodiments, a population of cells of endoderm origin is a population of somatic cells or differentiated cells. In some embodiments, the population of cells of endoderm origin are substantially free or devoid of embryonic stem cells or pluripotent cells or iPS cells.
In some embodiments, a cell of endoderm origin is genetically modified. In some embodiments, the cell of endoderm origin comprises one or more nucleic acid sequences encoding at the proteins of least two transcription factors selected from Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32) or MafA (SEQ ID NO: 3 or 33) or functional variants or functional fragments thereof.
In some embodiments, cells of endoderm origin can be isolated from a subject, for example as a tissue biopsy, such as, for example, a liver biopsy or biopsy of the pancreas. In some embodiments, the cells of endoderm origin are maintained in culture by methods known by one of ordinary skill in the art, and in some embodiments, propagated prior to being directly reprogrammed into pancreatic β-like cells by the methods as disclosed herein.
Further, the cell of endoderm origin can be from any mammalian species, with non-limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. For clarity and simplicity, the description of the methods herein refers to pancreatic exocrine cells as cells of endoderm origin, but it should be understood that all of the methods described herein can be readily applied to other cell types of endoderm origin. In one embodiment, the cell of endoderm origin is derived from a human individual.
In some embodiments, a subject from which the cells of endoderm origin are obtained is a mammalian subject, such a human subject, and in some embodiments, the subject is suffering from diabetes and/or a metabolic disorder. In such embodiments, the cells of endoderm origin can be reprogrammed to pancreatic β-like cells ex vivo by the methods as described herein and then administered to the subject from which the cells were harvested in a method to treat the subject for diabetes or a metabolic disorder.
In some embodiments, cells of endoderm origin are located within a subject (in vivo) and are directly reprogrammed to become pancreatic β-like cells by the methods as disclosed herein in vivo, for example as disclosed in the Examples demonstrating direct reprogramming pancreatic exocrine cells to pancreatic β-like cells in vivo by transducing the pancreatic cells with a viral vector, such as adenovirus which has the ability to express three transcription factors Pdx1, Ngn3 and MafA in the pancreatic exocrine cell.
In some embodiments, such contacting may be performed by maintaining the cell of endoderm origin in culture medium comprising the agent(s). In some embodiments the cells of endoderm origin can be genetically engineered. In some embodiments, a cells of endoderm origin can be genetically engineered to express one or more β-cell reprogramming factors as disclosed herein, for example express at least one a polypeptide selected from Pdx1 (SEQ ID NO:1 or 31) or Ngn3 (SEQ ID NO: 2 or 32) or MafA (SEQ ID NO: 3 or 33), or an amino acid sequences substantially homologous thereof, or functional fragments or functional variants thereof.
Where the cell of endoderm origin is maintained under in vitro conditions, conventional tissue culture conditions and methods can be used, and are known to those of skill in the art. Isolation and culture methods for various cells are well within the abilities of one skilled in the art.
In the methods of the present invention cells of endoderm origin (e.g. exocrine cells, pancreatic exocrine cells, acinar pancreatic cells, liver cells and gall bladder cells) can, in general, be cultured under standard conditions of temperature, pH, and other environmental conditions, e.g., as adherent cells in tissue culture plates at 37° C. in an atmosphere containing 5-10% CO2. The cells and/or the culture medium are appropriately modified to achieve direct reprogramming to pancreatic β-like cells as described herein. In certain embodiments, cells of endoderm origin (e.g. exocrine cells, pancreatic exocrine cells, acinar pancreatic cells, liver cells and gall bladder cells) can be cultured on or in the presence of a material that mimics one or more features of the extracellular matrix or comprises one or more extracellular matrix or basement membrane components. In some embodiments MatrigelTM is used. Other materials include proteins or mixtures thereof such as gelatin, collagen, fibronectin, etc. In certain embodiments of the invention, cells of endoderm origin (e.g. exocrine cells, pancreatic exocrine cells, acinar pancreatic cells, liver cells and gall bladder cells) can be cultured in the presence of a feeder layer of cells. Such cells may, for example, be of murine or human origin. They can also be irradiated, chemically inactivated by treatment with a chemical inactivator such as mitomycin c, or otherwise treated to inhibit their proliferation if desired. In other embodiments cells of endoderm origin (e.g. exocrine cells, pancreatic exocrine cells, acinar pancreatic cells, liver cells and gall bladder cells) are cultured without feeder cells.
Methods of Direct Reprogramming Cells of Endodermal Origin to a Pancreatic β-Like Cell
Generating reprogrammed pancreatic β-cells by reprogramming cells of endoderm origin (e.g, pancreatic exocrine cells, pancreatic duct cells, liver cells etc.) using the methods of the present invention has a number of advantages. First, the methods of the present invention allow one to generate autologous pancreatic β-like cells, which are cells specific to and genetically matched with an individual. The cells are derived from cells of endoderm origin (e.g, pancreatic exocrine cells, pancreatic duct cells, liver cells etc.) obtained from the individual. In general, autologous cells are less likely than non-autologous cells to be subject to immunological rejection.
Second, the methods of the present invention allow the artisan to generate pancreatic β-like cells without using embryos, oocytes, and/or nuclear transfer technology. Herein, the applicants' results demonstrate that (i) cells of endoderm origin (e.g, pancreatic exocrine cells, pancreatic duct cells, liver cells etc.) can be directly reprogrammed to become pancreatic β-like cells, without the need to be fully reprogrammed to a pluripotent state, therefore minimizing the risk of differentiation into unwanted cell types or risk of teratomas formation.
Also encompassed in the methods of the present invention is a method of direct reprogramming cells of endoderm origin (e.g, pancreatic exocrine cells, pancreatic duct cells, liver cells etc.) by means other than engineering the cells to express β-cell reprogramming factors, i.e., by contacting the cells of endoderm origin with a β-cell reprogramming agent other than a nucleic acid or viral vector capable of being taken up and causing a stable genetic modification to the cells. In particular, the invention encompasses the recognition that extracellular signaling molecules, e.g., molecules that when present extracellularly bind to cell surface receptors and activate intracellular signal transduction cascades, are of use to reprogram somatic cells. The invention further encompasses the recognition that activation of such signaling pathways by means other than the application of extracellular signaling molecules is also of use to directly reprogram cells of endoderm origin. In addition, the methods of the present invention relate to methods of identification of the pancreatic β-like cells that are detectable based on morphological criteria, without the need to employ a selectable marker. The present disclosure thus reflects several fundamentally important advances in the area of somatic cell reprogramming technology, in particular direct reprogramming technology.
While certain aspects of the invention are exemplified herein using three main β-cell reprogramming factors, e.g., Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32) and MafA (SEQ ID NO: 3 or 33) transcription factors, the methods of the invention encompass use of any other β-cell reprogramming factors in replace of any one of Pdx1, Ngn3 or MafA, where the other β-reprogramming factors includes, for example, but is not limited to, NeuroD (SEQ ID NO: 4); Nkx2.2 (SEQ ID NO: 5); Nkx6.1 (SEQ ID NO: 6); Pax4 (SEQ ID NO: 7); Pax6 (SEQ ID NO: 8); Isl1 (SEQ ID NO: 9) or functional variants, homologues or functional fragments thereof for the purposes of directly reprogramming cells of endoderm origin to pancreatic β-like cells.
One aspect of the present invention relates to direct reprogramming cells of endoderm origin (e.g. exocrine cells, pancreatic exocrine cells, acinar pancreatic cells, liver cells and gall bladder cells) to pancreatic β-like cells.
Another aspect of the present invention relates to methods to produce a population of isolated pancreatic β-like cells by increasing the protein expression of at least two β-cell reprogramming factors in a population of cells of endoderm origin (e.g. exocrine cells, pancreatic exocrine cells, acinar pancreatic cells, liver cells and gall bladder cells). In some embodiments, cells of endoderm origin (e.g. exocrine cells, pancreatic exocrine cells, acinar pancreatic cells, liver cells and gall bladder cells) can be treated in any of a variety of ways to cause direct reprogramming of the endodermal cell to a pancreatic β-like cells according to the methods of the present invention. For example, in some embodiments, the treatment can comprise contacting the cells with one or more agent(s), herein referred to as a “β-cell reprogramming agent” or a “β-cell reprogramming factor” which increases the protein expression of at least two of the transcription factors selected from Pdx1, Ngn3, or MafA, or increases the protein expression of a functional homologue or a functional fragment of at least two of Pdx1, Ngn3, or MafA polypeptides in the cell of endoderm origin.
Accordingly, one aspect of the present invention provides a method of direct reprogramming a cell, for example, a cell of endoderm origin by contacting a cell of endoderm origin with one or more agents, such as β-cell reprogramming agents or β-cell reprogramming factors to reprogram the cell of endoderm origin (e.g., a first cell) into a cell with pancreatic β-cell like phenotype (e.g. reprogrammed into a second cell type).
In some embodiments, the method comprises reprogramming a cell of endoderm origin by increasing the protein expression of at least two of the following transcription factors Pdx1, Ngn3 or MafA in the cell of endoderm origin, wherein the expression is for sufficient amount of time, typically transient increase in expression, to allow the reprogramming of the cell to become a cell which exhibits at least two characteristics of a endogenous pancreatic β-cell, for example at least two of the following characteristics; secretion of insulin in response to glucose, expression of c-peptide, or Pdx1 or MafA or Glut2 or lack of expression of Ngn3.
In some embodiments, the method comprises reprogramming a cell of endoderm origin by increasing the protein expression of all three of following transcription factors Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32) and MafA (SEQ ID NO: 3 or 33) in the cell of endoderm origin. The increase in expression of the transcription factors can be done all at the same time (e.g. concurrently), or alternatively, subsequently in any order.
In some embodiments, the method comprises substituting the increase in the protein expression of Ngn3 with an increase in the protein expression of Neuro D (SEQ ID NO: 4) or a peptide substantially homologous to SEQ ID NO: 4 or a functional fragment or functional variant of SEQ ID NO:4 in the cell of endoderm origin. For example, the present invention also encompasses a method comprises reprogramming a cell of endoderm origin by increasing the protein expression of at least 2, or at least 3 of following transcription factors Pdx1 (SEQ ID NO:1 or 31), Neuro D (SEQ ID NO: 4) and MafA (SEQ ID NO: 3 or 33) in the cell of endoderm origin.
In some embodiments, the method comprises substituting the increase in the protein expression of any of Pdx1, Ngn3 or MafA with an increase in the protein expression of any other β-cell reprogramming factor selected from Neuro D (SEQ ID NO: 4); Nkx2.2 (SEQ ID NO: 5); Nkx6.1 (SEQ ID NO: 6); Pax4 (SEQ ID NO: 7); Pax6 (SEQ ID NO: 8); Isl1 (SEQ ID NO: 9) or functional variants, polypeptides with amino acids substantially homologues or functional fragments thereof in the cell of endoderm origin. In some embodiments, the method comprises reprogramming a cell of endoderm origin by expressing at least 2, or at least 3, or at least 4 or at least 5, or at least 6, or at least 7 or at least 8, or at least 9 or any combination of transcription factors selected from, for example, but is not limited to, Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32) and MafA (SEQ ID NO: 3 or 33); NeuroD (SEQ ID NO: 4); Nkx2.2 (SEQ ID NO: 5); Nkx6.1 (SEQ ID NO: 6); Pax4 (SEQ ID NO: 7); Pax6 (SEQ ID NO: 8); Isl1 (SEQ ID NO: 9) or functional variants, polypeptides with amino acids substantially homologues or functional fragments thereof in a cell of endoderm origin to reprogram to a β-like cell.
In some embodiments, increasing the protein expression can be by any means known by one of ordinary art, for example can include introduction of nucleic acid encoding one or more of the transcription factors, or contacting the cell of endoderm origin with an agent (e.g. a β-cell reprogramming agent or factor) which reprograms the cell of endoderm origin to a cell with β-cell like phenotype.
In some embodiments, a β-cell reprogramming agent is a vector comprising a nucleotide sequence encoding the polypeptide Pdx1 (SEQ ID NO:1 or 31) or encoding a polypeptide substantially homologous to SEQ ID NO:1 or 31 or a functional variant or functional fragment of polypeptides of sequences SEQ ID NO:1 or 31. In such embodiments, the nucleotide sequence comprises SEQ ID NO: 34 or 35 or a fragment or variant thereof.
In some embodiments, a β-cell reprogramming agent is a vector comprising a nucleotide sequence encoding the polypeptide Ngn3 (SEQ ID NO:2 or 32) or encoding a polypeptide substantially homologous to SEQ ID NO:2 or 32 or a functional variant or functional fragment of polypeptides of sequences SEQ ID NO:2 or 32. In such embodiments, the nucleotide sequence comprises SEQ ID NO: 36 or 37 or a fragment or variant thereof.
In some embodiments, a β-cell reprogramming agent is a vector comprising a nucleotide sequence encoding the polypeptide MafA (SEQ ID NO:3 or 33) or encoding a polypeptide substantially homologous to SEQ ID NO:1 or 31 or a functional variant or functional fragment of polypeptides of sequences SEQ ID NO:3 or 33. In such embodiments, the nucleotide sequence comprises SEQ ID NO: 38 or 39 or a fragment or variant thereof.
In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a non-integrating viral vector. While retroviral vectors incorporate into the host cell genome and can potentially disrupt normal gene function, non-integrating vectors have the advantage of controlling expression of a gene product by extra-chromosomal transcription. It follows that since non-integrating vectors do not become part of the host genome, non-integrating vectors tend to express a nucleic acid transiently in a cell population. This is due in part to the fact that the non-integrating vectors as used herein are rendered replication deficient. Thus, non-integrating vectors have several advantages over retroviral vectors including but not limited to: (1) no disruption of the host genome, and (2) transient expression, and (3) no remaining viral integration products.
Some non-limiting examples of non-integrating vectors include adenovirus, baculovirus, alphavirus, picornavirus, and vaccinia virus. In one embodiment, the non-integrating viral vector is an adenovirus. The advantages of non-integrating viral vectors further include the ability to produce them in high titers, their stability in vivo, and their efficient infection of host cells.
While it is known that some non-integrating vectors integrate into the host genome at extremely low frequencies (i.e., 10−4 to 10−5), a non-integrating vector, as the term is used herein, refers to vectors having a frequency of integration of less than 0.1% of the total number of infected cells; preferably the frequency of integration is less than 0.01%, less than 0.001%, less than 0.0001%, or less than 0.000001% (or lower) of the total number of infected cells. In one embodiment, the vector does not integrate at all. In another embodiment, the viral integration remnants of the virus are below the detection threshold as assayed by PCR (for nucleic acid detection) or immunoassay (for protein detection). In general, pancreatic β-like cells produced by the methods described herein should be assayed for an integration event by the viral vector using, for example, PCR-mediated detection of the viral genome prior to administering a population of pancreatic β-like cells to a subject. Any pancreatic β-like cells with detectable integration products should not be administered to a subject.
The viral titer necessary to achieve a desired (i.e., effective) level of gene expression in a host cell is dependent on many factors, including, for example, the cell type, gene product, culture conditions, co-infection with other viral vectors, and co-treatment with other agents, among others. It is well within the abilities of one skilled in the art to test a range of titers for each virus or combination of viruses by detecting the expression levels of either (a) a marker expression product, or (b) a test gene product. Detection of protein expression in cells can be achieved by several techniques including Western blot analysis, immuno-cytochemistry, and fluorescence-mediated detection, among others. It is contemplated that experiments are first optimized by testing a variety of titer ranges for each cell type under the desired culture conditions. Once an optimal titer of a virus or a cocktail of viruses is determined, then that protocol will be used to induce the reprogramming of somatic cells.
In addition to viral titers, it is also important that the infection and induction times are appropriate with respect to different cells. For example, as discussed in the Examples section herein, initial attempts with an adenoviral vector were deemed unsuccessful due to an inadequate induction time. Upon recognition of this important consideration and considerable lengthening of induction time, induced pluripotent stem cells were produced using an adenoviral vector. With the knowledge provided herein that length of time is an important variable in induced pluripotent stem cell induction, one of skill in the art can test a variety of time points for infection or induction using a non-integrating vector and recover induced pluripotent stem cells from a given somatic cell type.
In some embodiments, the vector is a non-viral polycystronic vector as disclosed in Gonzalez et al., Proc. Natl. Acad. Sci. USA 2009 106:8918-8922; Carey et al., PNAS, 2009; 106; 157-162, WO/2009/065618 and WO/2000/071096 and Okita et al., Science 7, 2008: 322; 949-953, which are all incorporated herein in their entirety by reference.
In other embodiments, the methods or the present invention encompass non-viral means to increase the expression of the transcription factors (e.g. Pdx1, Ngn3 or MafA) in a cell of endoderm origin for the purposes for reprogramming to a pancreatic β-like cell as disclosed herein. For example, in one embodiment, naked DNA technology can be used, for example nucleic acid encoding the polypeptides of least two transcription factors of SEQ ID NOs 1-3 or 31-33 can be introduced into a cell of endoderm origin for the purposes of reprogramming the cell to a β-like cell. Methods of naked DNA technology are well known in the art, and are disclosed in U.S. Pat. No. 6,265,387 (which is incorporated herein in its entirety by reference) which describes a method of delivering naked DNA into a hepatocyte in vivo the via bile duct. U.S. Pat. No. 6,372,722 (which is incorporated herein in its entirety by reference) describes a method of naked DNA delivery to a secretory gland cell, for example, a pancreatic cell, a mammary gland cell, a thyroid cell, a thymus cell, a pituitary gland cell, and a liver cell.
In some embodiments, another non-viral means to increase the expression of the transcription factors (e.g. Pdx1, Ngn3 or MafA) in a cell of endoderm origin include use of piggyBac transposon vectors, as disclosed in U.S. Pat. Nos. 7,129,083, and 6,5518,25; U.S. Patent Application 2009/0042297 and International Patent Application WO/2007/100821 which are incorporated herein in their entirety by reference.
Other non-viral means to increase the expression of the transcription factors (e.g. Pdx1, Ngn3 or MafA) in a cell of endoderm origin for the purposes for reprogramming to a pancreatic β-like cell are also encompassed for use in the methods as disclosed herein.
In one embodiment, one can contact the cells of endoderm origin with polypeptides or peptides of Pdx1 (SEQ ID NO:1 or 31), Neuro D (SEQ ID NO: 4) and MafA (SEQ ID NO: 3 or 33) or functional variants, polypeptides with amino acids substantially homologues or functional fragments thereof in a cell of endoderm origin to reprogram to a β-like cell. Alternatively, one can use aptamers or antibodies or any other agent which activates and increases the expression of the transcription factors (e.g. Pdx1, Ngn3 or MafA) in a cell of endoderm origin.
In alternative embodiments, one can contact the cell of endoderm origin with a small molecule or combination of small molecules (e.g. chemical complementation) to increase the expression of at least two transcription factors in the cell of endoderm origin.
Thus, in some embodiments, the contacting step will typically be for at least twenty-four hours. By “at least twenty-four hours,” is meant twenty-four hours or greater. Specifically, the cells of endoderm origin can be contacted with β-cell reprogramming agent (e.g. small molecule, polypeptide, nucleic acid, nucleic acid analogues, etc) for about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 hours up to 3, 4, 5, 6, 7, or more days or any particular intervening time in hours or minutes within the above range. Preferably the cells of endoderm origin can be contacted with the β-cell reprogramming factor for seven days.
In another embodiment, the present invention provides a method of reprogramming cells of endoderm origin comprising contacting the cell of endoderm origin with at least 1 or at least 2 or at least 3 polypeptides selected from the group of Pdx1, Ngn3 or MafA, or having amino acid sequences substantially homologous thereto, and functional fragments or functional variants thereof. In some embodiments, the present invention provides a method of reprogramming cells of endoderm origin comprising contacting the cell of endoderm origin with at least 1 or at least 2 or at least 3 polypeptides selected from the group of polypeptides of SEQ ID NO: 4-29, or having amino acid sequences substantially homologous thereto, and functional fragments or functional variants thereof.
Where the β-cell reprogramming factor is a polypeptide, e.g. a polypeptide of Pdx1, Ngn3 or MafA, the dosages of Pdx1, Ngn3 or MafA polypeptides, their active fragments or related growth factors to be used in the in vivo or in vitro methods and processes of the invention preferably range from about 1 pmoles/kg/minute to about 100 nmoles/kg/minute for continuous administration and from about 1 nmoles/kg to about 40 mmoles/kg for bolus injection. Preferably, the dosage of Pdx1, Ngn3 or MafA polypeptides in in vitro methods will be 10 pmoles/kg/min to about 100 nmoles/kg/min, and in in vivo methods from about 0.003 nmoles/kg/min to about 48 nmoles/kg/min. More preferably, the dosage of Pdx1, Ngn3 or MafA polypeptides in in vitro methods ranges from about 100 picomoles/kg/minute to about 10 nanomoles/kg/minute, and in in vivo methods from about 0.03 nanomoles/kg/minute to about 4.8 nanomoles/kg/minute. In some embodiments, the preferred dosage of Pdx1, Ngn3 or MafA polypeptides, or other polypeptides of sequences SEQ ID NO: 4-29 in in vitro methods is 1 pmoles/kg/min to about 10 nmoles/kg/mine, and in in vivo from about 1 pmole/kg to about 400 pmoles/kg for a bolus injection. The more preferred dosage of the preferred dosage of Pdx1, Ngn3 or MafA polypeptides, or other polypeptides of sequences SEQ ID NO: 4-29 in in vitro methods ranges from about 10 pmole/kg/minute to about 1 nmole/kg/minute, and in in vivo from about 10 pmoles/kg to about 40 pmoles/kg for a bolus injection.
Confirming Presence of a Pancreatic β-Like Cell from a Cell of Endoderm Origin.
A pancreatic β-like cell as disclosed herein, produced by the methods as disclosed herein, e.g. by increasing the protein expression of at least 2 transcription factors of Pdx1, Ngn3, or MafA in the cell of endoderm origin, is a cell with the phenotypic characteristics of an endogenous β-cell such that is produces insulin. The pancreatic β-like cell can have all the phenotypic characteristics of an endogenous β-cell or may have less than all the phenotypic characteristics of an endogenous β-cell. In some embodiments, the pancreatic β-like cell can produce insulin but otherwise maintain at least one phenotypic characteristic of the endodermal cell from which it as reprogrammed from. For example, in some embodiments, a cell of endoderm origin (e.g. a non-insulin producing endoderm cell, such as a pancreatic amylase producing cell (i.e., pancreatic acinar cell), that is subjected to an increase in at least two transcription factors as disclosed herein can continue to express amylase, typical of an amylase producing cell, but, unlike the typical amylase producing cell, also produces insulin. Thus, a continuum between complete phenotypic change and a single phenotypic change is possible. An increase in proliferation of cells of endoderm origin may precede the reprogramming to β-like cells which produce insulin, and “reprogramming” is not meant to exclude any proliferation that accompanies the change of the cell to an insulin producing β-cell like phenotype.
To confirm the reprogramming of a cell of endoderm origin to a β-like cells, isolated clones can be tested for the expression of a marker of β-cells. Such expression identifies the cells as pancreatic β-like cells. Markers for pancreatic β-like cells can be selected from the non-limiting group including c-peptide, Glut2 and Pdx1. Additionally, pancreatic β-like cells secrete insulin in the response to glucose, for example an increase in glucose level. Other makers for the pancreatic β-like cells include, for example, but are not limited to an increase in expression of Insulin (INS), NeuroD, NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) by a stastistially significant amount as compared to the cell of endoderm origin from which the β-like cell was reprogrammed from.
In some embodiments, pancreatic β-like cells can be identified by the expression of markers as disclosed herein in the Examples, and in FIG. 6B, which include decrease in the expression of Ins1, Ins2, IAPP as compared to endogenous pancreatic β-cells, but an increase as compared to non-insulin producing cells of endocrine origin. In some embodiments, the the pancreatic β-like cells as disclosed herein have an increased expression of a marker selected from, for example, Ins1, Ins2, IAPP, Chrom, PYY, i.e. the expression is increased by a statistically significant amount in the pancreatic β-like cell as compared to the cell of endoderm origin from which the cell was reprogrammed from.
In some embodiments, the pancreatic β-like cell produced by the methods as disclosed herein secretes at least 15%, or at least 25% or at least 30% of the insulin that endogenous β-cells secrete, or alternatively, in some embodiments, a pancreatic β-like cell exhibits at least two characteristics of an endogenous pancreatic β-cell, for example, but not limited to, secretion of insulin in response to glucose, and expression of β-cell markers, such as for example, c-peptide, Pdx-1, Glut-2. In some embodiments, the β-like cell express insulin. In some embodiments, the β-like cells express VEGF. In some embodiments, the β-like cell expresses other cell markers, such as GCK, PC1/3, and transcription factors NeuroD, Nkx2.2 and Nkx6.2. In some embodiments, the β-like cell express GCK, PC1/3, NeuroD, Nkx2.2 and Nkx6 at a statistically significant increased level as compared to the cell of endoderm origin from which the β-like cell arises. In some embodiments, the β-like cell expresses insulin at a statistically significant increased level as compared to the cell of endoderm origin from which the β-like cell arises. In some embodiments, the β-like cell expresses VEGF at a statistically significant increased level as compared to the cell of endoderm origin from which the β-like cell arises.
In some embodiments, the β-like cell does not express markers Amylase, Ptf1a, Ck19 (Krt19), somatastatin/pancreatic polypeptide (SomPP), glucagon, mesenchymal markers, Nestin, Vimentin or Tuji. In some embodiments, the β-like cell expresses Amylase, Ptf1a, Ck19 (Krt19), somatastatin/pancreatic polypeptide (SomPP), glucagon, mesenchymal markers, Nestin, Vimentin or Tuji at at a statistically significant decreased level as compared to the cell of endoderm origin from which the β-like cell arise.
In another embodiment, a pancreatic β-like cell expresses or secretes Insulin, i.e. the expression is increased by a statistically significant amount as compared to the cell of endoderm origin from which the cell was reprogrammed from. In some embodiments, the pancreatic β-like cells expresses at least about 15%, or at least about 20% or at least about 30% or at least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 100% or greater than 100%, such as at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 3-fold, or at least about 4-fold or at least about 5-fold or more than about 5-fold the amount of the insulin secreted by an endogenous pancreatic β-cell.
In another embodiment, a pancreatic β-like cell does not express or secrete a hormone (e.g., glucagon, somatostatin, pancreatic polypeptide), i.e. the expression is decreased by a statistically significant amount as compared to the cell of endoderm origin from which the cell was reprogrammed from.
In one embodiment, the pancreatic β-like cell produced by the methods as disclosed herein has decreased or absent expression of a marker selected from, for example, amylase, ptf1a, Ck19, Nestin, Vimentin, Tuji) i.e. the expression is decreased by a statistically significant amount in the pancreatic β-like cell as compared to the cell of endoderm origin from which the cell was reprogrammed from.
Methods for detecting the expression of such markers are well known in the art, and include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as ELISA.
In some embodiments, a pancreatic β-like cells produced by the methods as disclosed herein can be identified based on unique morphological characteristics. In some embodiments, the pancreatic β-like cells are located intercalated within anicar rosettes in the pancreas, as discussed in the Examples and FIG. 1B. Thus, in some embodiments, the pancreatic β-like cells have a unique localization in the pancrease as compared to endogenous pancreatic β-cells in vivo, where endogenous pancreatic β-cells are organized into islet.
In some embodiments, the pancreatic β-like cells are between about 17-25 μm in diameter, for example, at least about 14 μm, or at least about 15 μm, or about at least 16 μm, or at least about 17 μm, or at least about 18 μm, or at least about 19 μm, or at least about 20 μm, or at least about 21 μm, or at least about 22 μm, or at least about 23 μm, or at least about 24 μm, or at least about 25 μm, or at least about 26 μm, or at least about 27 μm, or greater than about 27 μm in diameter. Thus, in some embodiments, the pancreatic β-like cells have a larger diameter as compared to endogenous pancreatic β-cells in vivo. In some embodiments, the pancreatic β-like cells have a stastisically significant larger diameter as compared to endogenous pancreatic β-cells in vivo. Typically, Endogenous pancreatic β-cells are about 9-15 μm in diameter.
In some embodiments, the pancreatic β-like cells in vivo have a cobblestone like gross morphology cell appearance, as disclosed in the Examples and in FIG. 3B, which is distinct from endogenous β-cells in vivo, which have a smaller spindle like morphology (see FIG. 3A). Thus, in some embodiments, a population of pancreatic β-like cells can be distinguished from a population of endogenous β-like cells based on morphology, such as size, cell appearance and cell localization. In some embodiments, a population of pancreatic β-like cells in vivo can be distinguished from a population of endogenous β-like cells in vivo based on morphology, such as size, cell appearance and cell localization.
In some embodiments, the present invention relates to an isolated population of pancreatic β-like cells produced by the methods as disclosed herein. In some embodiments, pancreatic β-like cells can be isolated by methods known in the art, for example FACs sorting, as disclosed in Liu et al., Journal Sichuan University, medical science edition, 209; 40(1); 153-6 or Liu et al., J Biol Chem, 1998; 273, 22201-22208, which are incorporated herein by reference).
Monitoring the Production of Pancreatic β-Like Cells from Cells of Endoderm Origin
The progression of a cell of endoderm origin to a pancreatic β-like cell can be monitored by determining the expression of markers characteristic of endogenous β-cells. In some processes, the expression of certain markers is determined by detecting the presence or absence of the marker. Alternatively, the expression of certain markers can be determined by measuring the level at which the marker is present in the cells of the cell culture or cell population. In certain processes, the expression of markers characteristic of pancreatic β-like cell as well as the lack of significant expression of markers characteristic of the cell of endoderm origin from which it was derived is determined.
As described in connection with monitoring the production of a pancreatic β-like cell, qualitative or semi-quantitative techniques, such as blot transfer methods and immunocytochemistry, can be used to measure marker expression. Alternatively, marker expression can be accurately quantitated through the use of technique such as Q-PCR. Additionally, it will be appreciated that at the polypeptide level, many of the markers of pancreatic islet hormone-expressing cells are secreted proteins. As such, techniques for measuring extracellular marker content, such as ELISA, may be utilized.
As set forth in the Examples below, markers of pancreatic β-like cell include the expression of markers, but are not limited to, insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide). As set forth in the Examples below, markers of pancreatic β-like cell include the lack of expression of, but are not limited to, Amylase (Amy), Glucagon, somatostatin/pancreatic polypeptide (SomPP), Ptf1a, the duct marker Ck19 (also known as Krt19), and mesenchymal markers Nestin and Vimentin.
The pancreatic β-like cell produced by the processes described herein express one or more of the above-listed markers, thereby producing the corresponding gene products. However, it will be appreciated that pancreatic β-like cella need not express all of the above-described markers. For example, pancreatic β-like cells reprogrammed from cells of endoderm origin do not always express INS.
Because endogenous β-cells do not substantially express the Ngn3, the transition of a cell of endoderm origin to a pancreatic β-like cell can be validated by monitoring the decrease in expression of NGN3 while monitoring the increase in expression of one or more of insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide). In addition to monitoring the increase and/or decrease in expression of one or more the above-described markers, in some processes, the expression of genes indicative endogenous β-cells can also be monitored.
It will be appreciated that insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) marker expression is induced over a range of different levels in pancreatic β-cells depending on the differentiation conditions. As such, in some embodiments described herein, the expression of insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) in pancreatic β-like cells is at least about 2-fold higher to at least about 10,000-fold higher than the expression of insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) in a cell of endoderm origin from which the pancreatic β-like cell was derived.
In other embodiments, the expression of the expression of insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) in pancreatic β-like cells is at least about 4-fold higher, at least about 6-fold higher, at least about 8-fold higher, at least about 10-fold higher, at least about 15-fold higher, at least about 20-fold higher, at least about 40-fold higher, at least about 80-fold higher, at least about 100-fold higher, at least about 150-fold higher, at least about 200-fold higher, at least about 500-fold higher, at least about 750-fold higher, at least about 1000-fold higher, at least about 2500-fold higher, at least about 5000-fold higher, at least about 7500-fold higher or at least about 10,000-fold higher than the expression of insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) in a cell of endoderm origin from which the pancreatic β-like cell was derived.
In other embodiments, the expression of the expression of insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) in pancreatic β-like cells is infinitely higher than than the expression of insulin (INS), NKX6 transcription factor related, locus 1 (NKX6.1), Nkx2.2, NeuroD, Glut2, glucokinase (GCK), prohormone convertase (PC1/3) and/or connecting peptide (C-peptide) in a cell of endoderm origin from which the pancreatic β-like cell was derived.
It will also be appreciated that the MafA marker expression increases, for example, in the β-like cells over a range of different levels in β-like cells. As such, in some embodiments described herein, the expression of the MafA marker in pancreatic β-like cell is at least about 2-fold higher to at least about 10,000-fold higher than the expression of MafA marker expression in a cell of endoderm origin from which the pancreatic β-like cell was derived. In other embodiments, the expression of the MAFA marker in pancreatic β-like cell is at least about 4-fold higher, at least about 6-fold higher, at least about 8-fold higher, at least about 10-fold higher, at least about 15-fold higher, at least about 20-fold higher, at least about 40-fold higher, at least about 80-fold higher, at least about 100-fold higher, at least about 150-fold higher, at least about 200-fold higher, at least about 500-fold higher, at least about 750-fold higher, at least about 1000-fold higher, at least about 2500-fold higher, at least about 5000-fold higher, at least about 7500-fold higher or at least about 10,000-fold higher than the expression of the MAFA markers a cell of endoderm origin from which the pancreatic β-like cell was derived.
In some embodiments of the processes described herein, the amount of hormone release from a population of pancreatic β-like cell can be determined. For example, the amount of insulin release, can be monitored. In a preferred embodiment, the amount of insulin secreted in response to glucose (GSIS) is measured. In still other embodiments, secreted breakdown or by-products produced by the pancreatic β-like cell, such as c-peptide and islet amyloid protein, can be monitored.
It will be appreciated that methods of measuring the expression of secreted proteins are well known in the art. For example, an antibody against one or more hormones produced by islet cells can be used in ELISA assays.
In some embodiments of the present invention, insulin release by a pancreatic β-like cell is measured by measuring C-peptide release. C-peptide is a cleavage product that is produced in equal molar amounts to insulin during the maturation of pro-insulin. Measuring C-peptide is advantageous because its half life is longer than that of insulin. Methods of measuring C-peptide release are well known in the art, for example, ELISA using anti-C-peptide monoclonal antibody (Linco Research, St. Louis, Mo.). In some embodiments of the present invention, pancreatic β-like cells produced from cells of endoderm origin secrete at least about 50 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 100 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 150 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 200 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 250 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 300 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 350 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 400 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 450 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 500 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 550 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 600 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 650 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 700 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 750 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 800 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 850 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 900 pmol of C-peptide (insulin)/μg of cellular DNA, at least about 950 pmol of C-peptide (insulin)/μg of cellular DNA or at least about 1000 pmol of C-peptide (insulin)/.mu.g of cellular DNA. In preferred embodiments, the pancreatic β-like cells are cells that secrete a single type of islet cell hormone (for example, the cells secrete only insulin). In certain preferred embodiments, the insulin is secreted in response to glucose. In other embodiments, the pancreatic β-like cells are cells are cells that secrete insulin in addition to other factors, for example, VEGF.
In some embodiments, the pancreatic β-like cells are cells process greater than about 80% of the insulin produced by a non-insulin producing cell of endoderm origin from which it was derived. In some embodiments, a pancreatic β-like cell is a cell that process greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% or greater than about 99% of the insulin produced a non-insulin producing cell of endoderm origin from which it was derived.
Methods of Identifying Agents for Reprogramming Cells of Endodermal Origin to a Pancreatic β-Like Cell.
Another aspect of the present invention relates to methods of identifying agents that alone or in combination with other agents reprogram a cell of endoderm origin to a pancreatic β-like cell. In some embodiments, the method includes contacting one or more cells of endoderm origin with one or more test agents (simultaneously or at separate times) and determining the level of expression of one or more reprogramming genes as defined herein. In some embodiments, the β-cell reprogramming genes include, Pdx1, Ngn3, mafA, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 of Isl1. Where one or more test agents increase the level of expression of one or more of the foregoing genes above the level of expression found in the cell of endoderm origin, in the absence of one or more test agents, are considered agents for reprogramming cells of endoderm origin to a pancreatic β-like cell. The test agents may include, but are not limited to, small molecules, nucleic acids, peptides, polypeptides, immunoglobulins, and oligosaccarides. In some embodiments, the just-mentioned method includes determining the level of expression of one or more of Pdx1, Ngn3 or MafA. In some embodiments, the method includes determining the level of expression of one or more of Pdx1, Ngn3 or MafA. Expression levels can be determined by any means known by one of ordinary skill in the art, for example, by RT-PCR or immunological methods.
Of particular interest are screening assays for agents that are active reprogramming human cells of endoderm origin to pancreatic β-like cells. A wide variety of assays may be used for this purpose, including immunoassays for protein binding; determination of cell growth, differentiation and functional activity; production of factors; and the like.
In the screening method of the invention for agents, the cells of endoderm origin are contacted with the agent of interest, and the effect of the agent assessed by monitoring output parameters, such as expression of reprogramming genes such as, but not limited to Pdx1, Ngn3 or MafA, cell viability, β-like cell characteristics, and the like. The cells may be freshly isolated, cultured, genetically engineered as described above, or the like. The s cells of endoderm origin may be environmentally induced variants of clonal cultures: e.g. split into independent cultures and grown under distinct conditions, for example with or without virus; in the presence or absence of other cytokines or combinations thereof. Alternatively, the cells of endoderm origin may be variants with a desired pathological characteristic. For example, the desired pathological characteristic includes a mutation and/or polymorphism which contribute to disease pathology.
In alternative embodiments, the methods of the invention can be used to screen for agents in which some cells of endoderm origin comprising a particular mutation and/or polymorphism respond differently compared with cells of endoderm origin without the mutation and/or polymorphism, therefore the methods can be used for example, to assess an effect of a particular drug and/or agent on stem cells from a defined subpopulation of people and/or cells, therefore acting as a high-throughput screen for personalized medicine and/or pharmogenetics. The manner in which cells respond to an agent, particularly a pharmacologic agent, including the timing of responses, is an important reflection of the physiologic state of the cell.
The agent used in the screening method can be selected from a group of a chemical, small molecule, chemical entity, nucleic acid sequences, an action; nucleic acid analogues or protein or polypeptide or analogue of fragment thereof. In some embodiments, the nucleic acid is DNA or RNA, and nucleic acid analogues, for example can be PNA, pcPNA and LNA. A nucleic acid may be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, PNA, etc. Such nucleic acid sequences include, for example, but not limited to, nucleic acid sequence encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/or peptide agent or fragment thereof, can be any protein of interest, for example, but not limited to; mutated proteins; therapeutic proteins; truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell. Proteins of interest can be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof. The agent may be applied to the media, where it contacts the cell (such as cells of endoderm origin) and induces its effects. Alternatively, the agent may be intracellular within the cell (e.g. cells of endoderm origin) as a result of introduction of the nucleic acid sequence into the cell and its transcription resulting in the production of the nucleic acid and/or protein agent within the cell. An agent also encompasses any action and/or event the cells (e.g. cells of endoderm origin) are subjected to. As a non-limiting examples, an action can comprise any action that triggers a physiological change in the cell, for example but not limited to; heat-shock, ionizing irradiation, cold-shock, electrical impulse, light and/or wavelength exposure, UV exposure, pressure, stretching action, increased and/or decreased oxygen exposure, exposure to reactive oxygen species (ROS), ischemic conditions, fluorescence exposure etc. Environmental stimuli also include intrinsic environmental stimuli defined below. The exposure to agent may be continuous or non-continuous.
In some embodiments, the agent is an agent of interest including known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, including toxicity testing; and the like. Candidate agents also include organic molecules comprising functional groups necessary for structural interactions, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Also included as agents are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include, for example, chemotherapeutic agents, hormones or hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof. Exemplary of pharmaceutical agents suitable for this invention are those described in, “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992).
The agents include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g. ground water, sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates.
Parameters are quantifiable components of cells of endoderm origin, particularly the expression of genes (e.g., protein expression or mRNA expression) such as, one or more in any combination of Pdx1, Ngn3 or MafA. In some embodiments, expression of one or more, in any combination of Pdx1, Ngn3, mafA, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 of Isl1 that can be accurately measured, desirably in a high throughput system. A parameter can be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values. In some embodiments, the assay is a computerized assay or a robotic high-throughput system operated through a computer interface.
Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
Agents are screened for effect on the cells of endoderm origin by adding the agent to at least one and usually a plurality of cells of endoderm origin, usually in conjunction with cells of endoderm origin lacking the agent. The change in parameters in response to the agent is measured, and the result evaluated by comparison to reference cultures, e.g. in the presence and absence of the agent, obtained with other agents, etc.
The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method. In some embodiments, agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.
A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1:10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.
Optionally, the cells of endoderm origin used in the screen can be manipulated to express desired gene products. Gene therapy can be used to either modify a cell to replace a gene product or add or knockdown a gene product. In some embodiments the genetic engineering is done to facilitate regeneration of tissue, to treat disease, or to improve survival of the β-like cells following implantation into a subject (i.e. prevent rejection). Techniques for transfecting cells are known in the art.
A skilled artisan could envision a multitude of genes which would convey beneficial properties to the transfected β-like cell or, more indirectly, to the cells of endoderm origin used for reprogramming. The added gene may ultimately remain in the recipient cell and all its progeny, or may only remain transiently, depending on the embodiment. For example, genes encoding angiogenic factors could be transfected into cells of endoderm origin. Such genes would be useful for inducing collateral blood vessel formation as the cell was reprogrammed to a β-like cell. It some situations, it may be desirable to transfect the cell with more than one gene.
In some instances, it is desirable to have the gene product secreted. In such cases, the gene product preferably contains a secretory signal sequence that facilitates secretion of the protein. For example, if the desired gene product is an angiogenic protein, a skilled artisan could either select an angiogenic protein with a native signal sequence, e.g. VEGF, or can modify the gene product to contain such a sequence using routine genetic manipulation (See Nabel et al., 1993).
The desired gene can be transfected into the cell using a variety of techniques. Preferably, the gene is transfected into the cell using an expression vector. Suitable expression vectors include plasmid vectors (such as those available from Stratagene, Madison Wis.), viral vectors (such as replication defective retroviral vectors, herpes virus, adenovirus, adeno-virus associated virus, and lentivirus), and non-viral vectors (such as liposomes or receptor ligands).
The desired gene is usually operably linked to its own promoter or to a foreign promoter which, in either case, mediates transcription of the gene product. Promoters are chosen based on their ability to drive expression in restricted or in general tissue types, for example in cells of endoderm origin, or on the level of expression they promote, or how they respond to added chemicals, drugs or hormones. Other genetic regulatory sequences that alter expression of a gene may be co-transfected. In some embodiments, the host cell DNA may provide the promoter and/or additional regulatory sequences. Other elements that can enhance expression can also be included such as an enhancer or a system that results in high levels of expression.
Methods of targeting genes in mammalian cells are well known to those of skill in the art (U.S. Pat. Nos. 5,830,698; 5,789,215; 5,721,367 and 5,612,205). By “targeting genes” it is meant that the entire or a portion of a gene residing in the chromosome of a cell is replaced by a heterologous nucleotide fragment. The fragment may contain primarily the targeted gene sequence with specific mutations to the gene or may contain a second gene. The second gene may be operably linked to a promoter or may be dependent for transcription on a promoter contained within the genome of the cell. In a preferred embodiment, the second gene confers resistance to a compound that is toxic to cells lacking the gene. Such genes are typically referred to as antibiotic-resistance genes. Cells containing the gene may then be selected for by culturing the cells in the presence of the toxic compound.
Enrichment, Isolation and/or Purification of a Population of Pancreatic β-Like Cells.
Another aspect of the present invention relates to the isolation of a population of pancreatic β-like cells from a heterogeneous population of cells, such a comprising a mixed population of pancreatic β-like cells and cells on endoderm origin from which the pancreatic β-cells were derived. A population of pancreatic β-like cells produced by any of the above-described processes can be enriched, isolated and/or purified by using an affinity tag that is specific for such cells. Examples of affinity tags specific for pancreatic β-like cells are antibodies, ligands or other binding agents that are specific to a marker molecule, such as a polypeptide, that is present on the cell surface of pancreatic β-like cells but which is not substantially present on other cell types (i.e. on the cells of endoderm origin) that would be found in the heterogeneous population of cells produced by the methods described herein. In some processes, an antibody which binds to a cell surface antigen on human pancreatic islet cells is used as an affinity tag for the enrichment, isolation or purification of pancreatic β-like cells produced by in vitro methods, such as the methods described herein. Such antibodies are known and commercially available. For example, a monoclonal antibody that is highly specific for a cell surface marker on human islet cells is available from USBiological, Swampscott, Mass. (Catalog Number P2999-40). Other examples include the highly specific monoclonal antibodies to glycoproteins located on the pancreatic islet cell surface, which have been described by Srikanta, et al., (1987) Endocrinology, 120:2240-2244, the disclosure of which is incorporated herein by reference in its entirety. A preferred example of an affinity tag for mature pancreatic β-like cells, such as those derived from cells of endoderm origin, is NCAM. Antibodies against NCAM are commercially available, for example from Abcam (Cambridge, Mass.).
The skilled artisan will readily appreciate that the processes for making and using antibodies for the enrichment, isolation and/or purification of pancreatic β-like cells are also readily adaptable for the enrichment, isolation and/or purification of pancreatic β-like cells. For example, in some embodiments, the reagent, such as an NCAM antibody, is incubated with a cell population containing pancreatic β-like cells, wherein the cell population has been treated to reduce intercellular and substrate adhesion. The cell population are then washed, centrifuged and resuspended. In some embodiments, if the antibody is not already labeled with a label, the cell suspension is then incubated with a secondary antibody, such as an FITC-conjugated antibody that is capable of binding to the primary antibody. The cells are then washed, centrifuged and resuspended in buffer. The cell suspension is then analyzed and sorted using a fluorescence activated cell sorter (FACS). Antibody-bound, fluorescent cells are collected separately from non-bound, non-fluorescent, thereby resulting in the isolation of such cell types.
In preferred embodiments of the processes described herein, the isolated cell composition comprising pancreatic β-like cells can be further purified by using an alternate affinity-based method or by additional rounds of sorting using the same or different markers that are specific for pancreatic β-like cells. For example, in some embodiments, FACS sorting is used to first isolate Pdx1-positive pancreatic β-like cells from Pdx1 negative cells from cell populations comprising pancreatic β-like cells. Sorting the Pdx1 positive cells again using FACS to isolate cells that are Pdx1 positive enriches the cell population for pancreatic β-like cells that express markers characteristic of this cell type, including NKX6.1, MAFA, ISL1 or PAX6 and other markers as disclosed herein. In other embodiments, FACS sorting is used to separate cells by negatively sorting for a marker that is present on most cells in the cell population other than the pancreatic β-like cells. An example of such a negative sort is the use of Tuji, which is a marker that is not substantially expressed on the surface of pancreatic β-like cells after the first round of enrichment.
In some embodiments of the processes described herein, pancreatic β-like cells are fluorescently labeled without the use of an antibody then isolated from non-labeled cells by using a fluorescence activated cell sorter (FACS). In such embodiments, a nucleic acid encoding GFP, YFP or another nucleic acid encoding an expressible fluorescent marker gene, such as the gene encoding luciferase, is used to label pancreatic β-like cells using the methods described above. For example, in some embodiments, at least one copy of a nucleic acid encoding GFP or a biologically active fragment thereof is introduced into a cell of endoderm origin, preferably a human cell of endoderm origin, downstream of the NKX6.1 promoter such that the expression of the GFP gene product or biologically active fragment thereof is under control of the NKX6.1 promoter. In some embodiments, the entire coding region of the nucleic acid, which encodes NKX6.1, is replaced by a nucleic acid encoding GFP or a biologically active fragment thereof. In other embodiments, the nucleic acid encoding GFP or a biologically active fragment thereof is fused in frame with at least a portion of the nucleic acid encoding NKX6.1, thereby generating a fusion protein. In such embodiments, the fusion protein retains a fluorescent activity similar to GFP.
It will be appreciated that promoters other than the NKX6.1 promoter can be used provided that the promoter corresponds to a marker that is expressed in pancreatic β-like cells. One exemplary marker is NKX2.2 or NeuroD, or Pdx1 or MafA.
Fluorescently marked cells, such as the above-described cells of endoderm origin, are differentiated to pancreatic β-like cells as described previously above. Because pancreatic β-like cells express the fluorescent marker gene, whereas other cell types do not, pancreatic β-like cells can be separated from the other cell types. In some embodiments, cell suspensions comprising a population of a mixture of fluorescently-labeled pancreatic β-like cells and unlabeled non-pancreatic β-like cells (i.e. cells of endoderm origin from which the pancreatic β-like cells were derived) are sorted using a FACS. Pancreatic β-like cells can be collected separately from non-fluorescing cells (i.e. the cells of endoderm origin which have not been reprogrammed to become pancreatic β-like cells), thereby resulting in the isolation of pancreatic β-like cells. If desired, the isolated cell compositions comprising pancreatic β-like cells can be further purified by additional rounds of sorting using the same or different markers that are specific for pancreatic β-like cells.
In preferred processes, pancreatic β-like cells are enriched, isolated and/or purified from other non-pancreatic β-like cells (i.e. from the cells of endoderm origin which have not been reprogrammed to become pancreatic β-like cells) after the cell population is induced to reprogram towards pancreatic β-like cell using the methods and compositions as disclosed herein.
In addition to the procedures just described, pancreatic β-like cells may also be isolated by other techniques for cell isolation. Additionally, pancreatic β-like cells may also be enriched or isolated by methods of serial subculture in growth conditions which promote the selective survival or selective expansion of the pancreatic β-like cells.
Using the methods described herein, enriched, isolated and/or purified populations of pancreatic β-like cells can be produced in vitro from cells of endoderm origin, which have undergone sufficient reprogramming to produce at least some pancreatic β-like cells. In a preferred method, the cells of endoderm origin are reprogrammed primarily into pancreatic β-like cells. Some preferred enrichment, isolation and/or purification methods relate to the in vitro production of pancreatic β-like cells from human cells of endoderm origin.
Using the methods described herein, isolated cell populations of pancreatic β-like cells are enriched in pancreatic β-like cells content by at least about 2- to about 1000-fold as compared to a population before reprogramming of the cells of endoderm origin. In some embodiments, pancreatic β-like cells can be enriched by at least about 5- to about 500-fold as compared to a population before reprogramming of the cells of endoderm origin. In other embodiments, pancreatic β-like cells can be enriched from at least about 10- to about 200-fold as compared to a population before reprogramming of the cells of endoderm origin. In still other embodiments, pancreatic β-like cells can be enriched from at least about 20- to about 100-fold as compared to a population before reprogramming of the cells of endoderm origin. In yet other embodiments, pancreatic β-like cells can be enriched from at least about 40- to about 80-fold as compared to a population before reprogramming of the cells of endoderm origin. In certain embodiments, pancreatic β-like cells can be enriched from at least about 2- to about 20-fold as compared to a population before reprogramming of the cells of endoderm origin.
Compositions Comprising Pancreatic β-Like Cells
Some embodiments of the present invention relate to cell compositions, such as cell cultures or cell populations, comprising pancreatic β-like cells, wherein the pancreatic β-like cells are cells, which have been derived from cells e.g. human cells of endoderm origin, which express or exhibit one or more characteristics of an endogenous pancreatic β-cell or alternatively, express at least 15% of the insulin secreted by an endogenous pancreatic β-cell. In accordance with certain embodiments, the pancreatic β-like cells are mammalian cells, and in a preferred embodiment, such cells are human pancreatic β-like cells.
Other embodiments of the present invention relate to compositions, such as cell cultures or cell populations, comprising pancreatic β-like cells. In such embodiments, cells that are of endoderm origin comprise less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of the total cells in the cell population.
Certain other embodiments of the present invention relate to compositions, such as cell cultures or cell populations, comprising pancreatic β-like cells. In some embodiments, cells of endoderm origin from which the pancreatic β-cells are derived comprise less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of the total cells in the culture. In certain embodiments, pre-primitive streak cells comprise less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% of the total cells in the culture.
Additional embodiments of the present invention relate to compositions, such as cell cultures or cell populations, produced by the processes described herein and which comprise pancreatic β-like cells as the majority cell type. In some embodiments, the processes described herein produce cell cultures and/or cell populations comprising at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 77%, at least about 76%, at least about 75%, at least about 74%, at least about 73%, at least about 72%, at least about 71%, at least about 70%, at least about 69%, at least about 68%, at least about 67%, at least about 66%, at least about 65%, at least about 64%, at least about 63%, at least about 62%, at least about 61%, at least about 60%, at least about 59%, at least about 58%, at least about 57%, at least about 56%, at least about 55%, at least about 54%, at least about 53%, at least about 52%, at least about 51% or at least about 50% pancreatic β-like cells. In preferred embodiments, the cells of the cell cultures or cell populations comprise human cells. In other embodiments, the processes described herein produce cell cultures or cell populations comprising at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about IT %, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2% or at least about 1% pancreatic β-like cells. In preferred embodiments, the cells of the cell cultures or cell populations comprise human cells. In some embodiments, the percentage of pancreatic β-like cells in the cell cultures or populations is calculated without regard to the feeder cells remaining in the culture.
Still other embodiments of the present invention relate to compositions, such as cell cultures or cell populations, comprising mixtures of pancreatic β-like cells and cells of endoderm origin. For example, cell cultures or cell populations comprising at least about 5 pancreatic β-like cells for about every 95 cell of endoderm origin can be produced. In other embodiments, cell cultures or cell populations comprising at least about 95 pancreatic β-like cells for about every 5 cell of endoderm origin can be produced. Additionally, cell cultures or cell populations comprising other ratios of pancreatic β-like cells to cell of endoderm origin are contemplated. For example, compositions comprising at least about 1 pancreatic β-like cells for about every 1,000,000, or at least 100,000 cells, or a least 10,000 cells, or at least 1000 cells or 500, or at least 250 or at least 100 or at least 10 cells of endocrine origin can be produced.
Further embodiments of the present invention relate to compositions, such as cell cultures or cell populations, comprising human cells, including human pancreatic β-like cells which express at least 15% of the level of insulin as compared to endogenous pancreatic β-cells.
In preferred embodiments of the present invention, cell cultures and/or cell populations of pancreatic β-like cells comprise human pancreatic β-like cells, that are non-recombinant cells. In such embodiments, the cell cultures and/or cell populations are devoid of or substantially free of recombinant human pancreatic β-like cells.
In some embodiments of the cell cultures and/or cell populations described herein, the pancreatic β-like cells secrete more than one pancreatic hormone. In other embodiments of the cell cultures and/or cell populations described herein, the pancreatic β-like cells s secrete a single pancreatic hormone. In preferred embodiments, the hormone is insulin. In even more preferred embodiments, the pancreatic β-like cells are responsive to glucose. In other embodiments, human pancreatic β-like cells from cells of endoderm origin secrete insulin in an amount similar to or greater than the amount of insulin secreted by pancreatic beta cells of the human pancreas in vivo.
Using the processes described herein, compositions comprising pancreatic β-like cells are substantially free of other cell types can be produced. In some embodiments of the present invention, the pancreatic β-like cell populations or cell cultures produced by the methods described herein are substantially free of cells that significantly express the Amylase, Ck19, glucogon, somatastatin, pancreatic polypeptide, Ptf1a or other markers.
Use of the Pancreatic β-Like Cells
Another aspect of the present invention further provides a method of treating diabetes in a subject diagnosed with Type 1 diabetes, comprising administering to the subject a population of pancreatic β-like cells.
In an alternative embodiment, the present invention provides a method of treating diabetes in a subject with, or at increased risk of developing diabetes a composition comprising at least one agent which increases the protein expression of at least two transcription factors selected from the group selected from Pdx1, Ngn3 or MafA, or administering a nucleic acid sequence encoding the polypeptides of least two transcription factors selected from Pdx1 (SEQ ID NO:1 or 31), Ngn3 (SEQ ID NO: 2 or 32) or MafA (SEQ ID NO: 3 or 33), or polypeptides with amino acid sequences substantially homologous thereto, and functional fragments or functional variants thereof by continuous infusion for at least twenty-four hours.
If the transcription factor has a fairly long half-life, the β-cell reprogramming agent which increases the protein expression of the transcription factor can be administered by bolus at least once. The treatment methods are effective to treat diabetes in a subject with Type 1 diabetes, because the β-cell reprogramming agent promotes the reprogramming of cells of endoderm origin (e.g. non-insulin producing pancreatic cells) in the subject into insulin producing cells (e.g. pancreatic β-like cells), as described in detail herein.
The present invention also provides a method of treating diabetes in a subject, comprising obtaining a population of cells of endoderm origin, for example non-insulin producing cells of endoderm origin from a subject, e.g. from the subject being treated, or from a donor subject; increasing the protein expression of at least two, or all three transcription factors selected Pdx1, Ngn3, MafA in the population of cells of endoderm origin in vitro, for example by the methods as described herein, thereby promoting reprogramming of the population of cells of endoderm origin into pancreatic β-like cells (i.e. insulin-producing cells); and administering a substantially pure population of pancreatic β-like cells to the diabetic subject.
In the method of treating diabetes, wherein the cells of endoderm origin are from a donor, the donor can be a cadaver. As a further embodiment of the present invention, the cells of endoderm origin can be allowed to proliferate in vitro prior to increasing the protein expression of at least two, or in some embodiments 3 of the transcription factors selected Pdx1, Ngn3, MafA. Preferably, promoting reprogramming of cells of endoderm origin into pancreatic β-like cells as disclosed herein will result in greater than about 20% reprogramming of cells of endoderm origin into pancreatic β-like cells. Even more preferably, greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the cells of endoderm origin will be reprogrammed into β-like cells.
In some embodiments, altering the surface antigens of the pancreatic β-like cells produced by the methods as disclosed herein can reduce the likelihood that pancreatic β-like cells will cause an immune response. The pancreatic β-like cells with altered surface antigens can then be administered to the diabetic subject. The cell surface antigens can be altered prior to, during, or after the non-insulin producing cells are differentiated into insulin-producing cells.
The subject of the invention can include individual humans, domesticated animals, livestock (e.g., cattle, horses, pigs, etc.), pets (like cats and dogs).
By “diabetes” is meant diabetes mellitus, a metabolic disease characterized by a deficiency or absence of insulin secretion by the pancreas. As used throughout, “diabetes” includes Type 1, Type 2, Type 3, and Type 4 diabetes mellitus unless otherwise specified herein.
As used herein, the term diabetes refers to is a syndrome of disordered metabolism, usually due to a combination of hereditary and environmental causes, resulting in abnormally high blood sugar levels (hyperglycemia). The two most common forms of diabetes are due to either a diminished production of insulin (in type 1), or diminished response by the body to insulin (in type 2 and gestational). Both lead to hyperglycemia, which largely causes the acute signs of diabetes: excessive urine production, resulting compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism. Diabetes can cause many complications. Acute complications (hypoglycemia, ketoacidosis, or nonketotic hyperosmolar coma) may occur if the disease is not adequately controlled. Serious long-term complications (i.e. chronic side effects) include cardiovascular disease (doubled risk), chronic renal failure, retinal damage (which can lead to blindness), nerve damage (of several kinds), and microvascular damage, which may cause impotence and poor wound healing. Poor healing of wounds, particularly of the feet, can lead to gangrene, and possibly to amputation.
Type 1 diabetes (Type 1 diabetes, Type I diabetes, T1D, T1DM, IDDM, juvenile diabetes) is an autoimmune disease that results in the permanent destruction of insulin-producing beta cells of the pancreas.
Type 2 diabetes (non-insulin-dependent diabetes mellitus (NIDDM), or adult-onset diabetes) is a metabolic disorder that is primarily characterized by insulin resistance (diminished response by the body to insulin), relative insulin deficiency, and hyperglycemia.
In some embodiments, a subject is pre-diabetic, which can be characterized, for example, as having elevated fasting blood sugar or elevated post-prandial blood sugar.
In other embodiments, a subject who has diabetes or pre-diabetes is being treated with one or more agents, including, for example, a biguanide, preferably metformin, a thiazolidinedione (e.g., a TZD such as rosiglitazone or pioglitazone), an alpha glucosidase inhibitor, such as acarbose or voglibose, a glucagon-like peptide agonist (e.g., a GLP-1 agonist), a dipeptidylpeptidase 4 (e.g., DPP4) inhibitor or insulin.
Accordingly, the methods described herein can be combined with other methods of treating pre-diabetes, lowering blood glucose in a subject, decreasing hemoglobinA1c in a subject, inhibiting gluconeogenesis in a subject, decreasing post-prandial glucose in a subject (e.g., treating post-prandial hyperglycemia), treating type I diabetes, treating type II diabetes, or treating a metabolic disorder, comprising administering a treatment, for example, administering, an agent described herein, for example an anti-diabetic agent. Examples of anti-diabetic agents include a glucosidase inhibitor, a thiazolidinedione (e.g., TZD), an insulin sensitizer, a glucagon-like peptide-1 (GLP-1), insulin, a PPAR α/γ dual agonist, an aP2 inhibitor and/or a DPP4 inhibitor. Examples of a glucosidase inhibitor include acarbose (disclosed in U.S. Pat. No. 4,904,769), voglibose, miglitol (disclosed in U.S. Pat. No. 4,639,436), which may be administered in a separate dosage form or the same dosage form. Examples of a PPARγ agonist includes a thiazolidinedione (e.g., TZD) such as rosiglitazone (AVANDIA®), pioglitazone (ACTOS®), englitazone, and darglitazone, which may be administered in a separate dosage form or the same dosage form. An example of a DPP4 inhibitor includes PHX1149, which is being developed by Phenomix®.
Methods of Identifying Reprogramming Factors
Described herein is a strategy to identify adult cell reprogramming factors by re-expressing multiple embryonic genes in living adult animals. A focus on embryonic genes is based in part on regeneration studies in newts, frogs and fish, where it has been shown that dedifferentiation of adult cells to progenitors, a form of cellular reprogramming, is accompanied by reactivation of embryonic regulators3,17,18.
To search for factors that could reprogram adult cells into β-cells, transcription factors (TF) were evaluated, a class of genes enriched for factors that regulate cell fates during embryogenesis. An in situ hybridization screen of more than 1,100 TFs identified groups of TFs with cell type specific expressions in the embryonic pancreas19. There are at least twenty TFs expressed in mature β-cells and their immediate precursors, the endocrine progenitors (Table. 1). Of these, nine genes (herein referred to “β-cell reprogramming genes”) exhibit β-cell developmental phenotypes when mutated20,21, and these were selected for initial reprogramming experiments.
Mature exocrine and duct cells of the adult pancreas were chosen as target cells for reprogramming, rather than cells from other organs. It was reasoned that these targets come from the endoderm, as do β-cells22, and that any pancreatic β-like cells would reside in their native environment which might promote their survival and/or maturation. In addition, this approach allows for a direct comparison of endogenous and pancreatic β-like cells. The transcription factors were delivered into the pancreas in adenoviral vectors. It has been shown that adenovirus preferentially infects pancreatic exocrine cells, but not islet cells23, and since most endogenous β-cells reside within islets (FIG. 1b), any newly formed (reprogrammed) β-cells could be easily detected as extra-islet insulin+ cells.
Kits
The cells and components such as one or more transcription factors can be provided in a kit. The kit includes (a) the cells and components described herein, e.g., a composition(s) that includes a cell and component(s) described herein, and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of a compound(s) described herein for the methods described herein.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of a cell, the nature of the components such as the transcription factor, concentration of components, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for administering the cells or other components.
In one embodiment, the informational material can include instructions to administer a compound(s) component such as a transcription factor described herein in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein) (e.g., to a cell in vitro or a cell in vivo). In another embodiment, the informational material can include instructions to administer a component(s) described herein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disorder described herein or to a cell in vitro.
The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a compound described herein and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.
In addition to a compound(s) described herein, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, and/or an additional agent, e.g., for reprogramming a cell of endoderm origin, such as a somatic cell (e.g., in vitro or in vivo) or for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than a component described herein. In such embodiments, the kit can include instructions for admixing a component(s) described herein and the other ingredients, or for using a component(s) described herein together with the other ingredients, e.g., instructions on combining the two agents prior to administration.
The kit can include one or more containers for the composition containing a component(s) described herein. In some embodiments, the kit contains separate containers (e.g., two separate containers for the two agents), dividers or compartments for the component(s) and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a compound described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a component described herein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
The kit optionally includes a device suitable for administration of the component, e.g., a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device.
Pharmaceutical Compositions Comprising a Population of Pancreatic β-Like Cells:
In another aspect of the invention, the methods provide use of an isolated population of the pancreatic β-like cells as disclosed herein. In one embodiment of the invention, an isolated population of the pancreatic β-like cells as disclosed herein may be used for the production of a pharmaceutical composition, for the use in transplantation into subjects in need of treatment, e.g. a subject that has, or is at risk of developing diabetes, for example but not limited to subjects with congenital and acquired diabetes. In one embodiment, an isolated population of the β-like cells may be genetically modified. In another aspect, the subject may have or be at risk of diabetes and/or metabolic disorder. In some embodiments, an isolated population of the pancreatic β-like cells as disclosed herein may be autologous and/or allogenic. In some embodiments, the subject is a mammal, and in other embodiments the mammal is a human.
The use of an isolated population of the pancreatic β-like cells as disclosed herein provides advantages over existing methods because the vβ-like cells can be reprogrammed from cells of endoderm origin obtained or harvested from the subject administered an isolated population of the pancreatic β-like cells. This is highly advantageous as it provides a renewable source of cells with pancreatic β-cell characteristics for transplantation into a subject, in particular a substantially pure population of pancreatic β-like cells that do not have the risks and limitations of β-like cells derived from other systems, such as from ES cell based systems, or iPS cells which have risks of formation of teratomas (Lafamme and Murry, 2005, Murry et al, 2005; Rubart and Field, 2006).
In another embodiment, an isolated population of pancreatic β-like cells can be used as models for studying properties of islet β-cells or pancreatic β-cells, or pathways of development of cells of endoderm origin into pancreatic β-cells. In some embodiments, the pancreatic β-like cells may be genetically engineered to comprise markers operatively linked to promoters that are expressed when a marker is expressed or secreted, for example, a marker can be operatively linked to an insulin promoter, so that the marker is expressed when the pancreatic β-like cells express and secrete insulin. In some embodiments, a population of pancreatic β-like cells can be used as a model for studying the differentiation pathway of cells which differentiate into islet β-cells or pancreatic β-like cells. In other embodiments, the pancreatic β-like cells may be used as models for studying the role of islet β-cells in the pancreas and in the development of diabetes and metabolic disorders. In some embodiments, the pancreatic β-like cells can be from a normal subject, or from a subject which carries a mutation and/or polymorphism (e.g. in the gene Pdx1 which leads to early-onset insulin-dependent diabetes mellitus (NIDDM), as well as maturity onset diabetes of the young type 4 (MODY4), which can be use to identify small molecules and other therapeutic agents that can be used to treat subjects with diabetes with a mutation or polymorphism in Pdx1. In some embodiments, the pancreatic β-like cells may be genetically engineered to correct the polymorphism in the Pdx1 gene prior to being administered to a subject in the therapeutic treatment of a subject with diabetes. In some embodiments, the pancreatic β-like cells may be genetically engineered to carry a mutation and/or polymorphism.
In one embodiment of the invention relates to a method of treating diabetes or a metabolic disorder in a subject comprising administering an effective amount of a composition comprising a population of pancreatic β-like cells as disclosed herein to a subject with diabetes and/or a metabolic disorder. In a further embodiment, the invention provides a method for treating diabetes, comprising administering a composition comprising a population of pancreatic β-like cells as disclosed herein to a subject that has, or has increased risk of developing diabetes in an effective amount sufficient to produce insulin in response to increased blood glucose levels.
In one embodiment of the above methods, the subject is a human and a population of pancreatic β-like cells as disclosed herein are human cells. In some embodiments, the invention contemplates that a population of pancreatic β-like cells as disclosed herein are administered directly to the pancreas of a subject, or is administered systemically. In some embodiments, a population of pancreatic β-like cells as disclosed herein can be administered to any suitable location in the subject, for example in a capsule in the blood vessel or the liver or any suitable site where administered population of pancreatic β-like cells can secrete insulin in response to increased glucose levels in the subject.
The present invention is also directed to a method of treating a subject with diabetes or a metabolic disorder which occurs as a consequence of genetic defect, physical injury, environmental insult or conditioning, bad health, obesity and other diabetes risk factors commonly known by a person of ordinary skill in the art. Efficacy of treatment can be monitored by clinically accepted criteria and tests, which include for example, (i) Glycated hemoglobin (A1C) test, which indicates a subjects average blood sugar level for the past two to three months, by measuring the percentage of blood sugar attached to hemoglobin, the oxygen-carrying protein in red blood cells. The higher your blood sugar levels, the more hemoglobin has sugar attached. An A1C level of 6.5 percent or higher on two separate tests indicates the subject has diabetes. A test value of 6-6.5% suggest the subject has prediabetes. (ii) Random blood sugar test. A blood sample will be taken from the subject at a random time, and a random blood sugar level of 200 milligrams per deciliter (mg/dL)-11.1 millimoles per liter (mmol/L), or higher indicated the subject has diabetes. (iii) Fasting blood sugar test. A blood sample is taken from the subject after an overnight fast. A fasting blood sugar level between 70 and 99 mg/dL (3.9 and 5.5 mmol/L) is normal. If the subjects fasting blood sugar levels is 126 mg/dL (7 mmol/L) or higher on two separate tests, the subject has diabetes. A blood sugar level from 100 to 125 mg/dL (5.6 to 6.9 mmol/L) indicates the subject has prediabetes. (iv) Oral glucose tolerance test. A blood sample will be taken after the subject has fasted for at least eight hours or overnight and then ingested a sugary solution, and the blood sugar level will be measured two hours later. A blood sugar level less than 140 mg/dL (7.8 mmol/L) is normal. A blood sugar level from 140 to 199 mg/dL (7.8 to 11 mmol/L) is considered prediabetes. This is sometimes referred to as impaired glucose tolerance (IGT). A blood sugar level of 200 mg/dL (11.1 mmol/L) or higher may indicate diabetes.
In some embodiments, the effects of administration of a population of β-like cells as disclosed herein to a subject in need thereof is associated with improved exercise tolerance or other quality of life measures, and decreased mortality. The effects of cellular therapy can be evident over the course of days to weeks after the procedure. However, beneficial effects may be observed as early as several hours after the procedure, and may persist for several years.
In some embodiments, a population of β-like cells as disclosed herein may be used for tissue reconstitution or regeneration in a human patient or other subject in need of such treatment. The cells are administered in a manner that permits them to graft or migrate to the intended tissue site and reconstitute or regenerate the functionally deficient area. Special devices are available that are adapted for administering cells capable of reconstituting a population of β-like cells as disclosed herein into the pancreas or at an alternative desired location. The cells may be administered to a recipient pancreas by injection, or administered by intramuscular injection.
The compositions comprising a population of β-like cells as disclosed herein have a variety of uses in clinical therapy, research, development, and commercial purposes. For therapeutic purposes, for example, a population of β-like cells as disclosed herein may be administered to enhance insulin production in response to increase in blood glucose level for any perceived need, such as an inborn error in metabolic function, the effect of a disease condition (e.g. diabetes), or the result of significant trauma (i.e. damage to the pancreas or loss or damage to islet β-cells). In some embodiments, a population of β-like cells as disclosed herein are administered to the subject not only help restore function to damaged or otherwise unhealthy tissues, but also facilitate remodeling of the damaged tissues.
To determine the suitability of cell compositions for therapeutic administration, the cells can first be tested in a suitable animal model. At one level, cells are assessed for their ability to survive and maintain their phenotype in vivo. Cell compositions can be administered to immunodeficient animals (such as nude mice, or animals rendered immunodeficient chemically or by irradiation). Tissues are harvested after a period of regrowth, and assessed as to whether the administered cells or progeny thereof are still present.
This can be performed by administering cells that express a detectable label (such as green fluorescent protein, or beta-galactosidase); that have been prelabeled (for example, with BrdU or [3H] thymidine), or by subsequent detection of a constitutive cell marker (for example, using human-specific antibody). The presence and phenotype of the administered population of β-like cells can be assessed by immunohistochemistry or ELISA using human-specific antibody, or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for human polynucleotides, according to published sequence data.
A number of animal models for testing diabetes are available for such testing, and are commonly known in the art, for example as disclosed in U.S. Pat. No. 6,187,991 which is incorporated herein by reference, as well as rodent models; NOD (non-obese mouse), BB_DB mice, KDP rat and TCR mice, and other animal models of diabetes as described in Rees et al, Diabet Med. 2005 April; 22(4):359-70; Srinivasan K, et al., Indian J Med Res. 2007 March; 125(3):451-7; Chatzigeorgiou A, et al., In Vivo. 2009 March-April; 23(2):245-58, which are incorporated herein by reference.
In some embodiments, a population of pancreatic β-like cells as disclosed herein may be administered in any physiologically acceptable excipient, where the cells may find an appropriate site for regeneration and differentiation. In some embodiments, a population of pancreatic β-like cells as disclosed herein can be introduced by injection, catheter, or the like. In some embodiments, a population of β-like cells as disclosed herein can be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use on thawing. If frozen, a population of pancreatic β-like cells will usually be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the cells may be expanded by use of growth factors and/or feeder cells associated with culturing β-cells and pancreatic β-like cells as disclosed herein.
In some embodiments, a population of pancreatic β-like cells as disclosed herein can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Choice of the cellular excipient and any accompanying elements of the composition comprising a population of pancreatic β-like cells as disclosed herein will be adapted in accordance with the route and device used for administration. In some embodiments, a composition comprising a population of pancreatic β-like cells can also comprise or be accompanied with one or more other ingredients that facilitate the engraftment or functional mobilization of the pancreatic β-like cells. Suitable ingredients include matrix proteins that support or promote adhesion of the pancreatic β-like cells, or complementary cell types, especially endothelial cells. In another embodiment, the composition may comprise resorbable or biodegradable matrix scaffolds.
In some embodiments, a population of pancreatic β-like cells as disclosed herein may be genetically altered in order to introduce genes useful in the β-like cells, e.g. repair of a genetic defect in an individual, selectable marker, etc., or genes useful in selection against non-insulin producing β-like cells or for the selective suicide of implanted pancreatic β-like cells. In some embodiments, a population of pancreatic β-like cells can also be genetically modified to enhance survival, control proliferation, and the like. In some embodiments, a population of pancreatic β-like cells as disclosed herein can be genetically altering by transfection or transduction with a suitable vector, homologous recombination, or other appropriate technique, so that they express a gene of interest. In one embodiment, a pancreatic β-like cell is transfected with genes encoding a telomerase catalytic component (TERT), typically under a heterologous promoter that increases telomerase expression beyond what occurs under the endogenous promoter, (see International Patent Application WO 98/14592, which is incorporated herein by reference). In other embodiments, a selectable marker is introduced, to provide for greater purity of the population of pancreatic β-like cells. In some embodiments, a population of pancreatic β-like cells may be genetically altered using vector containing supernatants over a 8-16 h period, and then exchanged into growth medium for 1-2 days. Genetically altered pancreatic β-like cells can be selected using a drug selection agent such as puromycin, G418, or blasticidin, and then recultured.
Gene therapy can be used to either modify a cell to replace a gene product, to facilitate regeneration of tissue, to treat disease, or to improve survival of the cells following implantation into a subject (i.e. prevent rejection).
In an alternative embodiment, a population of pancreatic β-like cells as disclosed herein can also be genetically altered in order to enhance their ability to be involved in tissue regeneration, or to deliver a therapeutic gene to a site of administration. A vector is designed using the known encoding sequence for the desired gene, operatively linked to a promoter that is either pan-specific or specifically active in the differentiated cell type. Of particular interest are cells that are genetically altered to express one or more growth factors of various types, such as somatostatin, glucagon, and other factors.
Many vectors useful for transferring exogenous genes into target pancreatic β-like cells as disclosed herein are available. The vectors may be episomal, e.g. plasmids, virus derived vectors such as cytomegalovirus, adenovirus, etc., or may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus derived vectors such MMLV, HIV-1, ALV, etc. In some embodiments, combinations of retroviruses and an appropriate packaging cell line may also find use, where the capsid proteins will be functional for infecting the pancreatic β-like cells as disclosed herein. Usually, pancreatic β-like cells and virus will be incubated for at least about 24 hours in the culture medium. In some embodiments, the pancreatic β-like cells are then allowed to grow in the culture medium for short intervals in some applications, e.g. 24-73 hours, or for at least two weeks, and may be allowed to grow for five weeks or more, before analysis. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Replication of the vector requires growth in the packaging cell line.
The host cell specificity of the retrovirus is determined by the envelope protein, env (p120). The envelope protein is provided by the packaging cell line. Envelope proteins are of at least three types, ecotropic, amphotropic and xenotropic. Retroviruses packaged with ecotropic envelope protein, e.g. MMLV, are capable of infecting most murine and rat cell types. Ecotropic packaging cell lines include BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearing amphotropic envelope protein, e.g. 4070A (Danos et al, supra.), are capable of infecting most mammalian cell types, including human, dog and mouse. Amphotropic packaging cell lines include PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902) GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelope protein, e.g. AKR env, are capable of infecting most mammalian cell types, except murine cells. In some embodiments, the vectors may include genes that must later be removed, e.g. using a recombinase system such as Cre/Lox, or the cells that express them destroyed, e.g. by including genes that allow selective toxicity such as herpesvirus TK, Bcl-Xs, etc.
Suitable inducible promoters are activated in a desired target cell type, either the transfected cell, or progeny thereof. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least about 100 fold, more usually by at least about 1000 fold. Various promoters are known that are induced in different cell types.
In one aspect of the present invention, a population of pancreatic β-like cells as disclosed herein are suitable for administering systemically or to a target anatomical site. A population of pancreatic β-like cells can be grafted into or nearby a subject's pancreas, for example, or may be administered systemically, such as, but not limited to, intra-arterial or intravenous administration. In alternative embodiments, a population of pancreatic β-like cells of the present invention can be administered in various ways as would be appropriate to implant in the pancreatic or secretory system, including but not limited to parenteral, including intravenous and intraarterial administration, intrathecal administration, intraventricular administration, intraparenchymal, intracranial, intracisternal, intrastriatal, and intranigral administration. Optionally, a population of pancreatic β-like cells are administered in conjunction with an immunosuppressive agent.
In some embodiments, a population of pancreatic β-like cells can be administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically “effective amount” for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement, including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. A population of pancreatic β-like cells can be administered to a subject the following locations: clinic, clinical office, emergency department, hospital ward, intensive care unit, operating room, catheterization suites, and radiologic suites.
In other embodiments, a population of pancreatic β-like cells is stored for later implantation/infusion. A population of pancreatic β-like cells may be divided into more than one aliquot or unit such that part of a population of pancreatic β-like cells is retained for later application while part is applied immediately to the subject. Moderate to long-term storage of all or part of the cells in a cell bank is also within the scope of this invention, as disclosed in U.S. Patent Application Serial No. 20030054331 and Patent Application No. WO03024215, and is incorporated by reference in their entireties. At the end of processing, the concentrated cells may be loaded into a delivery device, such as a syringe, for placement into the recipient by any means known to one of ordinary skill in the art.
In some embodiments, a population of pancreatic β-like cells can be applied alone or in combination with other cells, tissue, tissue fragments, growth factors such as VEGF and other known angiogenic or arteriogenic growth factors, biologically active or inert compounds, resorbable plastic scaffolds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the population. In some embodiments, a population of pancreatic β-like cells may also be modified by insertion of DNA or by placement in cell culture in such a way as to change, enhance, or supplement the function of the cells for derivation of a structural or therapeutic purpose. For example, gene transfer techniques for stem cells are known by persons of ordinary skill in the art, as disclosed in (Morizono et al., 2003; Mosca et al., 2000), and may include viral transfection techniques, and more specifically, adeno-associated virus gene transfer techniques, as disclosed in (Walther and Stein, 2000) and (Athanasopoulos et al., 2000). Non-viral based techniques may also be performed as disclosed in (Murarnatsu et al., 1998).
In another aspect, in some embodiments, a population of pancreatic β-like cells could be combined with a gene encoding pro-angiogenic growth factor(s). Genes encoding anti-apoptotic factors or agents could also be applied. Addition of the gene (or combination of genes) could be by any technology known in the art including but not limited to adenoviral transduction, “gene guns,” liposome-mediated transduction, and retrovirus or lentivirus-mediated transduction, plasmid’ adeno-associated virus. Cells could be implanted along with a carrier material bearing gene delivery vehicle capable of releasing and/or presenting genes to the cells over time such that transduction can continue or be initiated. Particularly when the cells and/or tissue containing the cells are administered to a patient other than the patient from whom the cells and/or tissue were obtained, one or more immunosuppressive agents may be administered to the patient receiving the cells and/or tissue to reduce, and preferably prevent, rejection of the transplant. As used herein, the term “immunosuppressive drug or agent” is intended to include pharmaceutical agents which inhibit or interfere with normal immune function. Examples of immunosuppressive agents suitable with the methods disclosed herein include agents that inhibit T-cell/B-cell costimulation pathways, such as agents that interfere with the coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No 2002/0182211, which is incorporated herein by reference. In one embodiment, a immunosuppressive agent is cyclosporine A. Other examples include myophenylate mofetil, rapamicin, and anti-thymocyte globulin. In one embodiment, the immunosuppressive drug is administered with at least one other therapeutic agent. The immunosuppressive drug is administered in a formulation which is compatible with the route of administration and is administered to a subject at a dosage sufficient to achieve the desired therapeutic effect. In another embodiment, the immunosuppressive drug is administered transiently for a sufficient time to induce tolerance to the cardiovascular stem cells of the invention.
Pharmaceutical compositions comprising effective amounts of a population of pancreatic β-like cells are also contemplated by the present invention. These compositions comprise an effective number of pancreatic β-like cells, optionally, in combination with a pharmaceutically acceptable carrier, additive or excipient. In certain aspects of the present invention, a population of pancreatic β-like cells are administered to the subject in need of a transplant in sterile saline. In other aspects of the present invention, a population of pancreatic β-like cells are administered in Hanks Balanced Salt Solution (HBSS) or Isolyte S, pH 7.4. Other approaches may also be used, including the use of serum free cellular media. In one embodiment, a population of pancreatic β-like cells are administered in plasma or fetal bovine serum, and DMSO. Systemic administration of a population of pancreatic β-like cells to the subject may be preferred in certain indications, whereas direct administration at the site of or in proximity to the diseased and/or damaged tissue may be preferred in other indications.
In some embodiments, a population of pancreatic β-like cells can optionally be packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution or thawing (if frozen) of a population of pancreatic β-like cells prior to administration to a subject.
In one embodiment, an isolated population of the β-like cells as disclosed herein are administered with a differentiation agent. In one embodiment, the β-like cells are combined with the differentiation agent to administration into the subject. In another embodiment, the cells are administered separately to the subject from the differentiation agent. Optionally, if the cells are administered separately from the differentiation agent, there is a temporal separation in the administration of the cells and the differentiation agent. The temporal separation may range from about less than a minute in time, to about hours or days in time. The determination of the optimal timing and order of administration is readily and routinely determined by one of ordinary skill in the art.
The methods described herein can be used, for example, to directly reprogram a first cell (e.g., a differentiated cell, an adult cell or a somatic cell) of a first cell type (e.g. pancreatic exocrine cell) into a cell of a second cell type (e.g., pancreatic β-like cell) without reversion to a pluripotent stem cell state. The reprogrammed cells can be used for regenerative medicine (e.g., tissue repair and regeneration) and to study differentiation and disease mechanisms/pathology.
Accordingly, in one aspect, the disclosure features a method of reprogramming a cell, the method comprising: providing a first cell of a first cell type (i.e. a cell of endoderm origin) wherein the first cell is a differentiated cell; and treating the first cell with one or more components to thereby reprogram the first cell into a cell of a second cell type (e.g. a β-like cell).
In one embodiment, the second cell type is a differentiated cell, an adult cell or a somatic cell.
In one embodiment, the second cell type is a human cell or a mouse cell.
In one embodiment, the second cell type is not an iPS cell.
In one embodiment, the second cell type is reprogrammed directly from the first cell type.
In one embodiment, the reprogramming does not produce an iPS cell.
In one embodiment, the reprogramming is performed in a subject (e.g., an animal such as a human or mouse).
In one embodiment, the subject is a human.
In one embodiment, the reprogramming is performed ex-vivo.
In one embodiment, the subject is suffering from a metabolic disorder (e.g., diabetes (e.g., Type I diabetes or Type II diabetes)).
In one embodiment, the subject is being treated for a metabolic disorder (e.g., pre-diabetes, Type I diabetes or Type II diabetes).
In one embodiment, the first cell type is different from the second cell type.
In one embodiment, both the first cell type and the second cell type are derived from the endoderm.
In one embodiment, the expression of a hormone (e.g., glucagon, somatostatin, pancreatic polypeptide) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., Glut2, GCK, PC1/3, NeuroD, Nkx2.2, Nkx6.1 or C-peptide) is up-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., amylase, ptf1a, Ck19, Nestin, Vimentin, Tuji) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, both the first cell type and the second cell type are pancreatic cells.
In one embodiment, the first cell type is a pancreatic exocrine cell.
In one embodiment, the second cell has one or more properties of a pancreatic β-cell (e.g., size, shape or secretion of insulin).
In one embodiment, the second cell type (e.g. pancreatic β-like cell) is a cell type that secretes insulin.
In one embodiment, the expression of a hormone (e.g., insulin) is up-regulated by a statistically significant amount in the second cell type relative to the first cell-type.
In one embodiment, the level of glucose tolerance in the subject is increased by a statistically significant amount after the first cell type (e.g. cell of endoderm origin) is reprogrammed to the second cell type (e.g. a pancreatic β-like cell).
In one embodiment, the insulin level in the serum of the subject is increased by a statistically significant amount after the first cell type (e.g. cell of endoderm origin) is reprogrammed to the second cell type (e.g. a pancreatic β-like cell).
In one embodiment, the cells of the second cell type (e.g. pancreatic β-like cell) in a subject remain present in a statistically significant amount up to 10, 20, 30, 40, 50, 60, 70, 80, 90 or greater days.
In one embodiment, the component is a transcription factor.
In one embodiment, the transcription factor is an embryonic transcription factor.
In one embodiment, the transcription factor is selected from the group consisting of Pdx1, Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1 and MafA.
In one embodiment, the transcription factor is selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type (e.g. cell of endoderm origin) is reprogrammed to the cell of the second cell type (e.g. pancreatic β-like cell) by treating the first cell with at least one transcription factor selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell (e.g. cell of endoderm origin) type is reprogrammed to the cell of the second cell type (e.g. pancreatic β-like cell) by treating the first cell with at least two transcription factors selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type (e.g. cell of endoderm origin) is reprogrammed to the cell of the second cell type (e.g. pancreatic β-like cell) by treating the first cell with at least three transcription factors (e.g., Ngn3, Pdx1 and MafA).
In one embodiment, the transcription factor is delivered into the first cell in a viral vector.
In one embodiment, the viral vector is an adenoviral vector.
In one embodiment, a plurality of the first cells of the first cell type are reprogrammed to a plurality of the cells of the second cell type.
In one embodiment, the method further comprises isolating a population of the cells of the second cell type (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater are of the subject cell type).
In one embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the cells of the first cell type that have been treated with one or more components are reprogrammed to the cells of the second cell type.
In one embodiment, (e.g., where the method is performed ex-vivo) the method further comprises implanting the cells of the second cell type into a subject (e.g., a subject suffering from a metabolic disorder (e.g., pre-diabetes, Type I diabetes or Type II diabetes)).
In one embodiment, the cells of the first and second time are from the subject into whom the cells are implanted. In one embodiment, the cells of the second cell type are from a donor different than the subject (e.g., a relative of the subject).
In one embodiment, the cells of the second cell type (e.g. pancreatic β-like cell) are surgically implanted.
In one embodiment, the first differentiated human cell of a first cell type (e.g. cell of endoderm origin) is reprogrammed to a differentiated human cell of a second cell type (e.g. a pancreatic β-like cell) in vivo. In one embodiment, the first differentiated human cell of a first cell type (e.g. cell of endoderm origin) is reprogrammed to a differentiated human cell of a second cell type (e.g. a pancreatic β-like cell) ex vivo.
In one embodiment, the first differentiated mouse cell of a first cell type is reprogrammed to a differentiated mouse cell of a second cell type in vivo. In one embodiment, the first differentiated human cell of a first cell type is reprogrammed to a differentiated human cell of a second cell type ex vivo.
In another aspect, this invention features a method of evaluating a transcription factor (TF) for the ability to reprogram a differentiated cell of a first type (e.g. cell of endoderm origin) into a differentiated cell of a second type (e.g. pancreatic β-like cell). The method includes: selecting a TF having the following properties, it is expressed in one or both of the second cell type or a precursor of the second cell type, when mutated it results in a non-wildtype phenotype in the second cell type (or in a tissue which includes the second cell type); contacting the TF with a cell of the first type; and evaluating whether the cell has become a cell of the second type, thereby evaluating a TF for the ability to reprogram differentiated cell of a first type into a differentiated cell of a second type.
In one embodiment, the reprogramming of a cell of endoderm origin does not produce an iPS cell.
In one embodiment, the second cell type (e.g. pancreatic β-like cell) is reprogrammed directly from the first cell type (e.g. cell of endoderm origin).
In one embodiment, the first cell type is different from the second cell type.
In one embodiment, both the first cell type and the second cell type come from the endoderm.
In one embodiment, the second cell type is a differentiated cell, an adult cell or a somatic cell.
In one embodiment, the second cell type is a human cell or a mouse cell.
In one embodiment, both the first cell type and the second cell type are pancreatic cells.
In one embodiment, the first cell type is a pancreatic exocrine cell.
In one embodiment, the second cell has one or more properties of a β-cell (e.g., size, shape or secretion of insulin).
In one embodiment, the second cell type is a cell type that secretes insulin.
In one embodiment, the expression of a hormone (e.g., insulin) is up-regulated by a statistically significant amount in the second cell type relative to the first cell-type.
In one embodiment, the expression of a hormone (e.g., glucagon, somatostatin, pancreatic polypeptide) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., Glut2, GCK, PC1/3, NeuroD, Nkx2.2, Nkx6.1 or C-peptide) is up-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., amylase, ptf1a, Ck19, Nestin, Vimentin, Tuji) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the transcription factor is an embryonic transcription factor.
In one embodiment, the transcription factor is delivered into the first cell in a viral vector.
In one embodiment, the viral vector is an adenoviral vector.
In one embodiment, a plurality of the first cells of the first cell type are reprogrammed to a plurality of the cells of the second cell type.
In one embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the cells of the first cell type (e.g. cell of endoderm origin) that have been treated with one or more components (i.e. β-cell reprogramming agents or factors) are reprogrammed to the cells of the second cell type (e.g. a pancreatic β-cell”.
In another aspect, the invention features a kit comprising: a first differentiated cell of a first cell type; reagents for treating the first cell with one or more components to thereby reprogram the first cell into a cell of a second cell type; and instructions for reprogramming a cell.
In one embodiment, the second cell type is a differentiated cell.
In one embodiment, the second cell type is a differentiated cell, an adult cell or a somatic cell.
In one embodiment, the second cell type is a human cell or a mouse cell.
In one embodiment, the second cell type is not an iPS cell.
In one embodiment, the second cell type is reprogrammed directly from the first cell type.
In one embodiment, the reprogramming does not produce iPS cells.
In one embodiment, the reprogramming is performed in a subject.
In one embodiment, the subject is a human.
In one embodiment, the subject is suffering from a metabolic disorder (e.g., diabetes (e.g., Type I diabetes or Type II diabetes)).
In one embodiment, the subject is being treated for a metabolic disorder (e.g., pre-diabetes, Type I diabetes or Type II diabetes).
In one embodiment, the first cell type is different from the second cell type.
In one embodiment, both the first cell type and the second cell type are derived from the endoderm.
In one embodiment, the expression of a hormone (e.g., glucagon, somatostatin, pancreatic polypeptide) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., Glut2, GCK, PC1/3, NeuroD, Nkx2.2, Nkx6.1 or C-peptide) is up-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., amylase, ptf1a, Ck19, Nestin, Vimentin, Tuji) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, both the first cell type and the second cell type are pancreatic cells.
In one embodiment, the first cell type is a pancreatic exocrine cell.
In one embodiment, the second cell has one or more properties of a β-cell (e.g., size, shape or secretion of insulin)
In one embodiment, the second cell type is a cell type that secretes insulin.
In one embodiment, the expression of a hormone (e.g., insulin) is up-regulated by a statistically significant amount in the second cell type relative to the first cell-type.
In one embodiment, the level of glucose tolerance in the subject is increased by a statistically significant amount after the first cell type is reprogrammed to the second cell type.
In one embodiment, the insulin level in the serum of the subject is increased by a statistically significant amount after the first cell type is reprogrammed to the second cell type.
In one embodiment, the cells of the second cell type in a subject remain present in a statistically significant amount up to 10, 20, 30, 40, 50, 60, 70, 80, 90 or greater days.
In one embodiment, the component is a transcription factor.
In one embodiment, the transcription factor is an embryonic transcription factor.
In one embodiment, the transcription factor is selected from the group consisting of Pdx1, Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1 and MafA.
In one embodiment, the transcription factor is selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type is reprogrammed to the cell of the second cell type by treating the first cell with at least one transcription factor selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type is reprogrammed to the cell of the second cell type by treating the first cell with at least two transcription factors selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type is reprogrammed to the cell of the second cell type by treating the first cell with at least three transcription factors (e.g., Ngn3, Pdx1 and MafA).
In one embodiment, the transcription factor is delivered into the first cell in a viral vector.
In one embodiment, the viral vector is an adenoviral vector.
In one embodiment, a plurality of the first cells of the first cell type are reprogrammed to a plurality of the cells of the second cell type.
In one embodiment, the method further comprises isolating a population of the cells of the second cell type (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater are of the subject cell type).
In one embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the cells of the first cell type that have been treated with one or more components are reprogrammed to the cells of the second cell type.
In one embodiment, the method further comprises implanting the cells of the second cell type into a subject (e.g., a subject suffering from a metabolic disorder (e.g., pre-diabetes, Type I diabetes or Type II diabetes)).
In one embodiment, the cells of the second cell type are from a donor different than the subject (e.g., a relative of the subject).
In one embodiment, the cells of the second cell type are surgically implanted.
In one embodiment, the first differentiated human cell of a first cell type is reprogrammed to a differentiated human cell of a second cell type in vivo.
In one embodiment, the first differentiated mouse cell of a first cell type is reprogrammed to a differentiated mouse cell of a second cell type in vivo.
In another aspect, the invention features a method of culturing a first differentiated cell of a first cell type in culture medium, wherein the medium contains one or more components to thereby reprogram the first cell to a cell of a second cell type.
In one embodiment, the second cell type is a differentiated cell, an adult cell or a somatic cell.
In one embodiment, the second cell type is a human cell or a mouse cell.
In one embodiment, the second cell type is not an iPS cell.
In one embodiment, the second cell type is reprogrammed directly from the first cell type.
In one embodiment, the reprogramming does not produce iPS cells.
In one embodiment, the first cell type is different from the second cell type.
In one embodiment, both the first cell type and the second cell type are derived from the endoderm.
In one embodiment, the expression of a hormone (e.g., glucagon, somatostatin, pancreatic polypeptide) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., Glut2, GCK, PC1/3, NeuroD, Nkx2.2, Nkx6.1 or C-peptide) is up-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, the expression of a marker (e.g., amylase, ptf1a, Ck19, Nestin, Vimentin, Tuji) is down-regulated by a statistically significant amount in the second cell type relative to the first cell type.
In one embodiment, both the first cell type and the second cell type are pancreatic cells.
In one embodiment, the first cell type is a pancreatic exocrine cell.
In one embodiment, the second cell has one or more properties of a β-cell (e.g., size, shape or secretion of insulin)
In one embodiment, the second cell type is a cell type that secretes insulin.
In one embodiment, the expression of a hormone (e.g., insulin) is up-regulated by a statistically significant amount in the second cell type relative to the first cell-type.
In one embodiment, the component is a transcription factor.
In one embodiment, the transcription factor is an embryonic transcription factor.
In one embodiment, the transcription factor is selected from the group consisting of Pdx1, Ngn3, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6, Isl1 and MafA.
In one embodiment, the transcription factor is selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type is reprogrammed to the cell of the second cell type by treating the first cell with at least one transcription factor selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type is reprogrammed to the cell of the second cell type by treating the first cell with at least two transcription factors selected from the group consisting of Ngn3, Pdx1 and MafA.
In one embodiment, the first cell of the first cell type is reprogrammed to the cell of the second cell type by treating the first cell with at least three transcription factors (e.g., Ngn3, Pdx1 and MafA).
In one embodiment, the transcription factor is delivered into the first cell in a viral vector.
In one embodiment, the viral vector is an adenoviral vector.
In one embodiment, a plurality of the first cells of the first cell type are reprogrammed to a plurality of the cells of the second cell type.
In one embodiment, the method further comprises isolating a population of the cells of the second cell type (e.g., wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 50%, 75% or greater are of the subject cell type).
In one embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the cells of the first cell type that have been treated with one or more components are reprogrammed to the cells of the second cell type.
An isolated cell population, isolated from a method described herein.
A non-human subject comprising a cell derived from a method described herein.
It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.
The present invention may be as defined in any one of the following numbered paragraphs.

    1. A method for reprogramming a cell of endoderm origin, the method comprising increasing the protein expression of at least two transcription factors selected from Pdx1, Ngn3, or MafA in the cell of enodermal origin, wherein the cell of enodermal origin is reprogrammed to exhibit at least two characteristics of a pancreatic β-cell.
    2. The method of claim 1, wherein the protein expression of Pdx1, Ngn3, and MafA are increased in the cell of enodermal origin.
    3. The method of claim 1 or 2, wherein the cell of endoderm origin is a pancreatic cell. (includes amylase-producing pancreatic cells)
    4. The method of any of claims 1 to 3, wherein the pancreatic cell is a exocrine cell.
    5. The method of any of paragraphs 1 to 3, wherein the pancreatic cell is a pancreatic duct cell.
    6. The method of any of paragraphs 1 to 3, wherein the pancreatic cell is an acinar pancreatic cell.
    7. The method of any of paragraphs 1 to 3, wherein the cell of endoderm origin is a liver cell.
    8. The method of any of paragraphs 1 to 3, wherein the cell of endoderm origin is a gall bladder cell.
    9. The method of any of paragraphs 1 to 3, wherein a characteristic of a pancreatic β-cell phenotype is secreting insulin in response to glucose.
    10. The method of any of paragraphs 1 to 9, wherein a characteristic of a pancreatic β-cell phenotype is expression of at least one marker selected from the group consisting of: Ngn3−, Pdx1+ and MafA+.
    11. The method of any of paragraphs 1 to 9, wherein the protein expression of a transcription factor selected from Pdx1, Ngn3, or MafA is increased by contacting the cell of endoderm origin with an agent which increases the expression of the transcription factor.
    12. The method of paragraph 11, wherein the agent is selected from the group consisting of: a nucleotide sequence, a protein, an aptamer and small molecule, ribosome, RNAi agent and peptide-nucleic acid (PNA) and anologues or variants thereof.
    13. The method of any of paragraphs 1 to 12, wherein protein expression is increased by introducing at least one nucleic acid sequence encoding a transcription factor protein selected from Pdx1, Ngn3, or MafA, or encoding a functional fragment thereof, in the cell of endoderm origin.
    14. The method of paragraph 13, wherein the protein expression of Pdx1 is increased by introducing a nucleic acid sequence encoding a Pdx1 polypeptide comprising SEQ ID NO: 2 or 32 or a functional fragment of SEQ ID NO: 2 or 32 into the cell of endoderm origin.
    15. The method of paragraphs 13 or 14, wherein the protein expression of Ngn3 is increased by introducing a nucleic acid sequence encoding a Ngn3 polypeptide comprising SEQ ID NO: 2 or 32 or a functional fragment of SEQ ID NO: 2 or 32 into the cell of endoderm origin.
    16. The method of any of paragraphs 13 to 15, wherein the protein expression of MafA is increased by introducing a nucleic acid sequence encoding a MafA polypeptide comprising SEQ ID NO: 3 or 33 or a functional fragment of SEQ ID NO: 3 or 33 into the cell of endoderm origin.
    17. The method of any of paragraphs 13 to 16, wherein the nucleic acid sequence is in a vector.
    18. The method of paragraph 17, wherein the vector is a viral vector or a non-viral vector.
    19. The method of paragraph 18, wherein the vector is a viral vector comprising a genome that does not integrate into the host cell genome.
    20. The method of any of paragraphs 1 to 19, wherein the cell of endoderm origin is in vitro.
    21. The method of any of paragraphs 1 to 19, wherein the cell of endoderm origin is ex vivo.
    22. The method of any of paragraphs 1 to 19, wherein the cell of endoderm origin is in vivo or present in a subject.
    23. The method of paragraph 22, wherein the subject is a human subject.
    24. The method of paragraphs 22 and 23, wherein the subject has, or is at risk of developing, diabetes.
    25. The method of paragraph 24, wherein the diabetes is selected from the group consisting of: Type I diabetes, Type II diabetes and pre-diabetes.
    26. The method of paragraphs 22 and 23, wherein the subject has or is at risk of developing a metabolic disorder.
    27. The method of any of paragraphs 1 to 26, wherein the cell of endoderm origin is a mammalian cell.
    28. The method of paragraph 27, wherein the mammalian cell is a human cell.
    29. The method of any of paragraphs 1 to 28, wherein the method further comprises contacting the cell of endoderm origin with at least one agent which increases the protein expression of at least one of the transcription factors selected from the group consisting of: NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 or Isl1.
    30. The method of any of paragraphs 1 to 29, wherein the pancreatic β-cell expresses a marker selected from the group consisting of: he markers method further comprises contacting the cell of endoderm origin with at least one agent which increases the protein expression of at least one of the transcription factors selected from the group consisting of: NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 or Isl1.
    31. The method of any of paragraphs 1 to 30, wherein the pancreatic β-cell is a pancreatic β-like cell.
    32. The method of paragraph 31, wherein the pancreatic β-like cell has an increased expression of a marker selected from the group consisting of: c-peptide, glucose transporter 2 (Glut2), glucokinase (GCK), prohormone convertase 1/3 (PC1/3), β-cell transcription factors NeuroD, Nkx2.2 and Nkx6.1 by a statistically significant amount relative to the cell of endoderm origin from which the pancreatic β-like cell was derived.
    33. The method of paragraph 31, wherein the pancreatic β-like cell has a decreased expression of a marker selected from the group consisting of: Amylase (Amy), glucagon, somatostatin/pancreatic polypeptide (SomPP), Ck19, Nestin, Vimentin and Tuji by a statistically significant amount relative to the cell of endoderm origin from which the pancreatic β-like cell was derived.
    34. An isolated population of pancreatic β-like cells obtained from a population of non-insulin producing cells of endoderm origin by a process comprising increasing the protein expression of at least two transcription factors selected from Ngn3, Pdx1 or MafA, or a functional fragment thereof, in the non-insulin producing cells of endoderm origin.
    35. The isolated population of pancreatic β-like cells of paragraph 34, wherein the expression is transient expression.
    36. The isolated population of pancreatic β-like cells of paragraphs 34 or 35, wherein the pancreatic β-like cells secrete insulin in response to an increase in glucose.
    37. The isolated population of pancreatic β-like cells of paragraphs 34 and 36, wherein the pancreatic β-like cell have a distinct morphology and localization as compared to endogenous pancreatic β-cells.
    38. The isolated population of pancreatic β-like cells of paragraph 37, wherein the pancreatic β-like cells have at least one characteristic selected from the group consisting of: cobblestone cell morphology, a diameter of between 17-25 μm and an intercalated location within exocrine acincar rosettes.
    39. The isolated population of pancreatic β-like cells of paragraph 34, wherein the increase in the protein expression of at least two transcription factors is accomplished using a vector.
    40. The isolated population of pancreatic β-like cells of paragraph 39, wherein the vector is a viral vector or a non-viral vector.
    41. The isolated population of pancreatic β-like cells of paragraphs 39 or 40, wherein the vector comprises a nucleic acid sequence encoding a Ngn3 polypeptide or a functional fragment thereof.
    42. The isolated population of pancreatic β-like cells of paragraphs 39 or 40, wherein the vector comprises a nucleic acid sequence encoding a Pdx1 polypeptide or a functional fragment thereof.
    43. The isolated population of pancreatic β-like cells of paragraphs 39 or 40, wherein the vector comprises a nucleic acid sequence encoding a MafA polypeptide or a functional fragment thereof.
    44. The isolated population of pancreatic β-like cells of paragraphs 39, wherein the population of non-insulin producing cells of endoderm origin is a population of pancreatic cells.
    45. The isolated population of pancreatic β-like cells of paragraphs 44, wherein the pancreatic cells are selected from the group consisting of: exocrine cells, pancreatic duct cells and an acinar pancreatic cells or a heterogeneous population thereof.
    46. The isolated population of pancreatic β-like cells of any of paragraphs 34 to 45, wherein the population of non-insulin producing cells of endoderm origin is a population of liver cells.
    47. The isolated population of pancreatic β-like cells of any of paragraphs 34 to 46, wherein the population of non-insulin producing cells of endoderm origin is a population of gall bladder cells.
    48. The isolated population of pancreatic β-like cells of any of paragraphs 34 to 45, wherein the population of non-insulin producing cells of endoderm origin is a population of mammalian cells.
    49. The isolated population of pancreatic β-like cells of paragraphs 48, wherein the mammalian cells are human cells.
    50. The isolated population of pancreatic β-like cells of any of paragraphs 34 to 49, wherein the population of non-insulin producing cells of endoderm origin is obtained from a subject that has diabetes, or has an increased risk of developing diabetes.
    51. The isolated population of pancreatic β-like cells of paragraph 50, wherein the diabetes is selected from the group consisting of: Type I diabetes, Type II diabetes and pre-diabetes.
    52. The isolated population of pancreatic β-like cells of any of paragraphs 34 to 49, wherein the population of non-insulin producing cells of endoderm origin is obtained from a subject that has a metabolic disorder, or has an increased risk of developing a metabolic disorder.
    53. A method for the treatment of a subject with diabetes, the method comprising administering a composition comprising an isolated population of pancreatic β-like cells according to paragraphs 34 to 52.
    54. The method of paragraph 53, wherein the pancreatic β-like cells are produced from non-insulin producing endoderm cells obtained from the same subject as the composition is administered to.
    55. The method of paragraph 53, wherein the subject has, or has an increased risk of developing, diabetes.
    56. The method of any of paragraphs 53 to 55, wherein the diabetes is selected from the group consisting of: Type I diabetes, Type II diabetes and pre-diabetes.
    57. The method of any of paragraphs 53 to 55, wherein the subject has, or has an increased risk of developing, a metabolic disorder.
    58. Use of the isolated population of pancreatic β-like cells according to paragraphs 27 to 45 for administering to a subject in need thereof.
    59. The use of paragraph 58, wherein the subject has, or has an increased risk of developing, diabetes.
    60. The use of paragraph 59, wherein the diabetes is selected from the group consisting of: Type I diabetes, Type II diabetes and pre-diabetes.
    61. The use of paragraph 58, wherein the subject has, or has an increased risk of developing, a metabolic disorder.
    62. The use of any of paragraphs 58 to 61, wherein the pancreatic β-like cells are produced from non-insulin producing endoderm cell obtained from the same subject as the pancreatic β-like cells are administered to.
    63. A kit comprising:
    
        a. a nucleic acid sequence encoding a Ngn3 polypeptide or a functional fragment thereof; and/or
        b. a nucleic acid sequence encoding a Pdx1 polypeptide or a functional fragment thereof; and/or
        c. a nucleic acid sequence encoding a MafA polypeptide or a functional fragment thereof
    
    
    64. The kit of paragraph 63, further comprising instructions for reprogramming a cell of endoderm origin to a cell with at least two characteristics of a pancreatic β-cell using the methods according to paragraphs 1 to 33.
    65. A composition comprising at least one non-insulin producing endodermal cell and at least one agent which increases the protein expression of at least two transcription factors selected from Ngn3, Pdx1 or MafA.
    66. A method for identifying an agent for reprogramming a cell of endoderm origin to a pancreatic β-like cell, comprising contacting the cell of endoderm origin with one or more test agents, determining the level of one or more β-cell reprogramming genes, and identifying the one or more test agents as an agent for reprogramming a cell of endoderm origin to a pancreatic β-like cell if the expression level of the β-cell reprogramming gene is higher in the cell of endoderm origin following the contacting step than the cell of endoderm origin in the absence of the contacting step.
    67. The method of paragraph 66, wherein the one or more reprogramming genes comprise one or more of Pdx1, Ngn3, MafA, NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 or Isl1.
    68. The method of paragraphs 66 or 67, wherein the one or more reprogramming genes comprise at least two of one or more of Pdx1, Ngn3 and MafA.
    69. The method of any of paragraph 66 to 68, wherein the one or more reprogramming genes comprise Pdx1, Ngn3 and MafA.


The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof.
EXAMPLES
The examples presented herein relate to the methods and compositions for producing reprogrammed pancreatic β-cells, for example from cells of endoderm origin such as pancreatic exocrine cells and other endodermal cells, such as liver cells, by increasing the expression of at least two transcription factors selected from Ngn3, Pdx1 or MafA, for example by using nucleic acid sequences to encoding the transcription factors Ngn3, Pdx1 or MafA or functional fragments thereof. Throughout this application, various publications are referenced. The disclosures of all of the publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention.
Methods
Adenovirus Construction and Purification.
Genes of interest were first cloned into a shuttle vector containing an IRES-nGFP, then into the pAd/CMV/V5-DEST adenoviral vector (invitrogen). High titer virus (>1×1010 pfu/ml) was obtained by purification with the AdEasy Kit (Stratagene).
Animals, Surgery, Physiological Studies.
Rag1−/− and Rag1−/−; NOD animals were obtained from Jackson Labs. Adult animals (>2 month) were injected with 100 ul (>1×109 pfu) of purified adenovirus directly into the splenic lobe of the dorsal pancreas. Blood glucose was measured with Ascensia Elite Blood Glucose Meter. Insulin levels were determined with an Ultrasensitive Insulin ELISA kit (Alpco).
Immunohistochemistry, BrdU Labeling, TUNEL Analysis.
This was performed as previously described37. 1 mg/ml BrdU was provided in drinking water for BrdU labeling following surgery. Apoptotic cells were recognized by TUNEL labeling with a TMR red Cell Death kit (Roche).
Electron Microscopy.
Dissected pancreas was fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 2 hours at room temperature. For conventional transmission electron microscopy, samples were further fixed by osmium tetroxide, embedded in Epon resin and sectioned at 60-80 nm. For immuno-gold labeling, ultrathin sections were cut at −120° C. and stained with gold conjugated antibodies. Images were obtained with a TecnaiTM G2 Spirit BioTWIN transmission electron microscope.
Viral Injection, Tissue Harvest.
For adult pancreas,  ̃100 ul of purified virus was injected directly into 2-3 foci of the dorsal splenic lobe with a 3/10 cc Insulin Syringe (Becton Dickinson). For skeletal muscle,  ̃20 ul virus was injected into the upper thigh. At the time of tissue harvest, the infected portion of the tissue was visualized by GFP fluorescence and dissected out. For adult pancreas, typically  ̃50% of the dorsal pancreas was taken.
Immunohistochemistry.
Adult mouse pancreata were fixed by immersion in 4% paraformaldehyde for 2 hours at 4 C. Samples were subsequently incubated in 30% sucrose solution overnight and embedded with OCT compound.
The following primary antibodies were used: rat anti-E-cadherin (Zymed), rat anti-PECAM1 (Pharmingen), goat anti-Ngn3 (Santa Cruz), guinea pig anti-Insulin (Dako), guinea pig anti-Glucagon (Linco), guinea pig anti-Pancreatic polypeptide (Linco), rabbit anti-somatostatin (Dako), rabbit anti-pancreatic polypeptide (Dako), goat anti-Somatostatin (Santa Cruz), goat anti-Pdx1 (Santa Cruz), guinea pig anti-Pdx1 (gift of Dr. Chris Wright), goat anti-βgalactosidase (Biogenesis), goat anti-Amylase (Santa Cruz), mouse anti-BrdU (Amersham), rabbit anti-mafA (Bethyl), chick anti-Nestin (Ames), chick anti-Vimentin (Chemicon), goat anti-Glut2 (Santa Cruz), Goat anti-VEGF (R&D), rabbit anti-PC1/3 (Chemicon), goat anti-glucokinase (Santa Cruz), rabbit anti-Ck19 (Melton lab stock), rabbit anti-chromogranin A/B (RDI), rabbit anti-Ptf1a (gift of Dr. Helena Edlund), goat anti-NeuroD (Santa Cruz), rabbit anti-Nkx6.1 (BCBC), rabbit anti-Sox9 (Santa Cruz), goat anti-Nkx2.2 (Santa Cruz), and rabbit anti-c-peptide (Linco).
Rodamin-Red-X, FITC, Cy5 and Alexa dye conjugated donkey secondary antibodies were obtained from the Jackson Immunoresearch laboratories and the Molecular Probes Inc. Tyramide amplification system (PerkinElmer) was used for PC1/3 and glucokinase staining. Immunofluorescence pictures were taken with a Zeiss LSM 510 META confocal microscope.
Cpa1CreERT2 Labeling of Mature Exocrine Cells.
Cpa1CreERT2;R26R double heterozygous animals were generated by mating homozygous Cpa1CreERT2 males with R26R homozygous females (Jackson lab). Two month old Cpa1CreERT2;R26R adults were injected with Tamoxifen at 6 mg per animal every third day for 4 times to label mature exocrine cells.
Physiological Studies.
Diabetic animals were produced with Intraperineal injection of streptozotocin (120 ug/g body weight) after overnight fasting with 2 month old adult animals of the Rag1 strain (Jackson lab). Hyperglycemic animals that display >250 mg/dl fasting blood glucose levels for at least 2 consecutive days were used for experiments.
Fasting blood glucose was measured on tail vein blood with an Ascensia Elite Glucometer (Bayer, Elkhart, Ind.) after 6-8 hr fasting. Non-fasting insulin level was determined from tail vein blood collected around 9 to 10 AM with an Ultrasensitive Insulin ELISA kit (Alpco, Windham, N.H.).
Average β-cell number per section was determined by sectioning through the entire pancreas at 15 um and collecting every third sections. Twenty randomly selected sections were immunostained for Insulin and DAPI to visualize individual β-cells. Total number of β-cells were counted and averaged from 3 animals.
Glucose tolerance test was performed by fasting animals over night (12 hr), followed by intraperineal injection of glucose (3 g/kg body weight).
Electron Microscopy.
Small pieces of pancreatic samples (1-2 mm) were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde for 2 hours at room temperature. For conventional electron microscopy, samples were further refixed with a mixture of 1% Osmiumtetroxide (OsO4)+1.5% Potassiumferrocyanide (KFeCN6) for 2 hours, washed in water and stained in 1% aqueous uranyl acetate for 1 hour followed by dehydration in grades of alcohol (50%, 70%, 95%, 2×100%) and propyleneoxide (1 hr), then infiltrated in propyleneoxide:Epon 1:1 ON and embedded in TAAB Epon. (Marivac Canada Inc. St. Laurent, Canada). Ultrathin sections (about 60-80 nm) were cut on a Reichert Ultracut-S microtome, picked up on to coppergrids, stained with 0.2% lead citrate and examined in a “Tecnai G2 Spirit BioTWIN” Transmission electron microscope. Images were taken with a 2 k AMT CCD camera.
For immuno-electron microscopy, fixed samples were infiltrated with 2.3M sucrose in PBS for 30 minutes then frozen in liquid nitrogen. Frozen samples were sectioned at −120° C., the sections transferred to formvar-carbon coated copper grids and floated on PBS until the immunogold labeling is carried out. The gold labeling was carried out at room temperature on a piece of parafilm. All antibodies and protein A gold were diluted 1% BSA. The diluted antibody solution was centrifuged 1 minute at 14 000 rpm prior to labeling to avoid possible aggregates. Grids were floated on drops of 1% BSA for 10 minutes to block for unspecific labeling, transferred to 5 μl drops of primary antibody and incubated for 30 minutes. The grids were then washed in 4 drops of PBS for a total of 15 minutes, transferred to 5 μl drops of Protein-A gold (G. Posthuma, University of Utrecht, the Netherlands) for 20 minutes, washed in 4 drops of PBS for 15 minutes and 6 drops of double distilled water.
For double labeling, after the first Protein A gold incubation, grids were washed in 4 drops of PBS for a total of 15 minutes then transferred to a drop of 0.2% Glutaraldehyde in PBS for 5 minutes, washed in 4 drops of PBS/0.15M glycine (to quench free aldehyde groups) then the send primary antibody is applied, followed by PBS wash and different size Protein-A gold as above. Antibodies used were rabbit anti-GFP (invitrogen) and guinea pig anti-Insulin (Dako).
Contrasting/embedding of the labeled grids was carried out on ice in 0.3% uranyl acetete (Electron Microcopy Sciences) in 2% methyl cellulose (SIGMA) for 10 minutes. Grids were picked up with metal loops (diameter slightly larger than the grid) and the excess liquid was removed by streaking on a filterpaper (Whatman #1), leaving a thin coat of methyl cellulose (bluish interference color when dry).
The grids were examined in a Tecnai G2 Spirit BioTWIN transmission electron microscope and images were recorded with an AMT 2 k CCD camera.
FACS Analysis, Islet Isolation, Gene Profiling.
For FACS sorting of GFP+ cells, Pancreata infected by the M3 inducing factors for one month were perfused through the common bile duct, digested with liberase and Elastase (Roche), and further dissociated into single cells with EDTA incubation. GFP+ cells were isolated by fluorescent activated cell sorting (FACS) with FACSaria (BD Bioscience). Staining of sorted cells indicate that  ̃70% are GFP+ and  ̃22% of total sorted cell are insulin+.
Islets were isolated by Liberase digestion of pancreas of Pdx1-GFP animals. Islets picked manually under a fluorescent dissecting scope. Pancreatic cells devoid of GFP+ islets were collected as non-islet sample. RNA was extracted with TRIZOLTM reagent (Invitrogen). Biotin labeled cRNA probes synthesized with the ILLUMINA TOTAL PREP RNA AMPLIFICATION KIT® (Ambion). Gene profiling performed with Sentrix BeadChip Array MouseRef-8 v1.1 (illumina) that contains probes for  ̃19,000 genes. Data analyzed with the BeadStudio software. For identifying differentially enriched genes, the following parameters suggested by illumina were used: P value<0.05, Diff score>30, Average signal>100.
RT-PCR.
Pancreatic tissues were harvested and immediately frozen in liquid nitrogen (LN2). Total RNA was extracted with the RNeasy kit (Qiagen). First strand cDNA was synthesizes with Superscript III kit (invitrogen). 30 cycles of semi-quantitative RT-PCR was performed with standard protocol. The following primer pairs were used:







Ngn3 viral transgene: ~350 bp;


Ngn3.F:








CAG ACG CTG CGC ATA GCG GAC CAC;
(SEQ ID NO: 40)


 


IRES2.R:



GCG GCT TCG GCC AGT AAC GTT AG
(SEQ ID NO: 41)


 


Pdx1 viral transgene: ~1.2 kb;



Pdx1.F:



GGA GCA AGA TTG TGC GGT GAC CTC;
(SEQ ID NO: 42)


 


IRES2.R:



GCG GCT TCG GCC AGT AAC GTT AG
(SEQ ID NO: 41)


 


MafA viral transgene: ~300 bp;



MafA.F:



ACA TTC TGG AGA GCG AGA AGT GCC;
(SEQ ID NO: 43)


 


IRES2.R:



GCG GCT TCG GCC AGT AAC GTT AG
(SEQ ID NO: 41)


 


GADPH: ~400 bp;



F:



ACC ACA GTC CAT GCC ATC AC;
(SEQ ID NO: 44)


 


R:



TCC ACC ACC CTG TTG CTG TA
(SEQ ID NO: 45)






Example 1
Induction of Insulin+ Cells in Adult Mice.
Adenovirus that co-expresses each transcription factor (TF) together with nuclear GFP (nGFP) was purified. All nine viruses were pooled and injected as a mixture (referred to herein as “M9”, for mixture of 9 different transcription factors) into the pancreata of 2 month old adult mice (FIG. 1a). The immune-deficient Rag1−/− strain was used to avoid complications associated with viral elicited immune response24. One month after viral delivery, immunohistochemistry revealed a modest increase of extra-islet insulin+ cells among viral infected cells (nGFP+) in 2 out of 3 animals (FIG. 1D). To determine which of the nine factors are required, individual factors were removed from the pool one at a time. Pools lacking Nkx2.2, Nkx6.1, or Pax4 continued to produce increased extra-islet insulin cells (data not shown), suggesting that these genes are dispensable. Results for the other six genes were inconclusive. The inventors conducted another round of factor withdrawal with mixtures of the remaining six genes (M6) and three of them, Ngn3, Pdx1, and MafA, proved to be absolutely required (FIG. 1D). The combination of these three factors (referred to as M3) converts >20% of infected cells to insulin cells (red cells with green nuclei, FIG. 1C, 1E). Notably, single factors or combinations of any two factors do not elicit this effect (FIG. 1E). NeuroD (also known as Neurod1) can functionally replace Ngn3 in M3, but the resulting cocktail has reduced induction efficiency (FIG. 1E).
The inventors performed antibody labeling to confirm that these three inducing factors are co-expressed in the induced insulin cells (data not shown). In particular, the inventors performed co-expression analysis of the reprogramming effect of M3 factors in different cell types. To differentiate viral transgenes from endogenous genes, a Flag tagged Pdx1 (at C-terminus) and a Myc tagged MafA (at N-terminus) were used together with untagged Ngn3 to infect pancreas, mouse embryonic fibroblasts (at a MOI: 50), or skeletal muscle. Expression of M3 induced insulin expression in pancreas in vivo, but not in embryonic mouse fibroblasts in vitro or skeletal muscle in vivo (data not shown). Samples were analyzed after 10 days, and quantification showed that 97.5%, >99%, and 97.7% of Insulin+ cells co-express Ngn3, Pdx1.flag, and myc.MafA, respectively, in the pancreas sample, demonstrating that the majority of pancreatic β-like cells induced are insulin+ cells which also co-express all three factors (data not shown). Immunohistological analysis of cultured fibroblasts demonstrated >98% of nGFP+ cells co-express all three factors (data not shown), whereas in skeletal muscle, 92%, 86.6% and 89% of nGFP+ cells express Ngn3, Pdx1-flag and myc.MafA, respectively, demonstrating that most of them co-express M3 factors (data not shown). >500 insulin+ or nGFP+ cells were counted per condition in the analysis.
The inventors identified that the percentage of insulin cells among infected cells increases with progressive removal of factors from the pool such that M3 induces more insulin+ cells than M6, whereas M6 is better than M9 (FIG. 1D, 1E). This is likely due to the fact that a constant volume of virus was injected into each animal, regardless of the viral combinations. The effective concentration of Ngn3, Pdx1 and MafA viruses in a cocktail, therefore, increases when fewer factors are included. New insulin+ cells are detected 3 days after injection, but the expression level is low.
The intensity of insulin staining increases gradually so that by day 10, the level is comparable to that of endogenous β-cells (data not shown). In particular, the inventors demonstrated by immunostaining a progressive increase of insulin expression levels during M3 induction, where immunostaining was performed on samples harvested at different time points after M3 induction. 2 μm confocal images were taken with identical settings at non-saturating levels, and insulin staining analysed. Insulin staining was first detected at 3 days post induction (data not shown), and increases at day 7 and appears to reach that of islet β-cell level at day 10 (data not shown). No further increases were seen at day 30 (data not shown).
These new insulin cells are still present after 3 months, the longest time point the inventors analyzed, and remain as scattered individual cells or small clusters and do not form islets (FIG. 1C). The reprogramming effect of the three factors appears to be rather specific for pancreatic exocrine cells: infection of skeletal muscle in vivo or fibroblasts in vitro with M3 did not induce insulin expression, despite extensive co-expression of the three factors in the target cells (data not shown).
Example 2
The New Insulin+ Cells Originate from Differentiated Exocrine Cells.
Lineage analysis was performed to determine the origin of the new insulin+ cells. The five major cell types in the adult pancreas can be detected with lineage-specific molecular markers: exocrine (Amylase), duct (Ck19), endocrine (insulin, glucagon, somatostatin, pancreatic polypeptide), vascular (PECAM), and mesenchymal cells (Nestin, Vimentin). Upon injection with a control nGFP virus, the vast majority of infected cells (>95%) were found to be mature amylase+ exocrine cells (FIG. 2A), consistent with prior reports23. Non-exocrine cells together account for approximately 5% of infected population. Since more than 20% of M3 infected cells become insulin+ 10 days after viral delivery, this suggests that non-exocrine cells can contribute, at most, to a minor fraction of these new insulin+ cells. As there is little cell death and no enhanced proliferation during this reprogramming (data not shown), the majority of insulin cells would thus appear to originate from mature exocrine cells.
The inventors analyzed proliferation and apoptosis of pancreatic β-like cells by immunostaining (data not shown). In particular, continuous BrdU labeling was performed for 10 days immediately after pAd-M3 viral injection and the BrdU cells identified. 3.2% of pancreatic β-like cells and 12.9% of islet β-cells in the same animals incorporated BrdU over this period (n=3 animals. >1,000 insulin+ cells/animal, data not shown). The inventors also performed TUNEL labeling for analysis of apoptosis. TUNEL labeling revealed a general lack of apoptosis among pancreatic cells during reprogramming (data not shown) or among reprogrammed new β-cells (data not shown). The inventors discovered that 2 days after infection, insulin expression is not yet detectable (data not shown). Apoptotic cells at this stage do not correlate with viral infection (nGFP+).
To confirm the exocrine origin of the new insulin+ cells, the inventors genetically labeled mature exocrine cells with a mouse line (Cpa1CreERT2) that expresses an inducible form of Cre recombinase (CreERT2) specifically in adult exocrine cells19 (FIG. 2B). When crossed with the R26R reporter line, tamoxifen induction in double heterozygous Cpa1CreERT2;R26R adults indelibly labels 5-10% of mature exocrine cells with β-galactosidase (βgal) (data not shown); no label is found in other cell types. In particular, 10 days after nGFP viral infection, the most of the β-gal positive cells are are Amylase+ (Amy) mature exocrine cells, not duct cells (Ck19+) (data not shown). Following pAd-M3 injection, many βgal+ cells become insulin+ (data not shown), demonstrating direct evidence that mature exocrine cells give rise to new insulin+ cells. The inventors demonstrated 10 days after infection, many βgal+ insulin+ cells are present, as detected by triple immunostaining with insulin/GFP/βgal (data not shown).
Example 3
Pancreatic β-Like Cells Closely Resemble Endogenous Pancreatic Islet β-Cells.
The inventors next examined the new insulin cells to determine the extent to which they have been reprogrammed. Morphologically, exocrine cells are large with a cobble stone appearance (data not shown) whereas islet β-cells are much smaller and spindle shaped (data not shown). In particular, a comparison of immunostaining with Insulin/E-cadherin and nGFP of islet β-cells and pancreatic β-like cells show they are similar in size and shape but distinctly different from exocrine cells (data not shown). E-cadherin staining was used to visualize cell boundaries.
When dissociated into single cells, the diameter of Amylase+ exocrine cells range from 25-17 um whereas insulin+ β-cells range from 9-15 um. The induced cells are indistinguishable from islet β-cells in size and shape (FIG. 3A).
At the ultrastructural level, the reprogrammed cells have all the hallmarks of islet β-cells (FIGS. 3B and 3C). They possess the small dense secretory granules characteristic of insulin granules, and lack the large zymogen granules and dense assemblies of endoplasmic reticulum (ER) that are characteristic of exocrine cells (FIGS. 3B and 3C). Immuno-electron microscopy further showed that the pancreatic β-like cells express both GFP in the nucleus and abundant insulin in the granules (FIGS. 5A-5F). Interestingly, the induced β-cells often appear on the electron micrograph as intercalated within exocrine acinar rosettes (FIG. 3D). In wild type pancreatic samples, rare single or small clusters of β-cells reside outside islets, but they often associate with duct but not exocrine cells. The unique position of induced cells likely reflects their exocrine origin.
Molecular marker analysis reveals that most of the insulin+ cells coexpress genes essential for β-cell endocrine function including glucose transporter 2 (Glut2, also known as Slc2a2, expressed in 92.8% of the new insulin+ cells), glucokinase (GCK, 96.7%), prohormone convertase (PC1/3, 86.7%) (data not shown), and key β-cell transcription factors NeuroD (88.9%), Nkx2.2 (85.3%), and Nkx6.1 (85.9%) (data not shown). The induced insulin cells express C-peptide (data not shown). In particular, the inventors discovered by immunostaining one month after infection with pAd-M3, that most Insulin+ pancreatic β-like cells coexpress molecular markers specific for endocrine genes: glucose transporter 2 (Glut2), glucokinase (GCK), prohormone convertase 1/3 (PC1/3), and β-cell transcription factors NeuroD, Nkx2.2, and Nkx6.1 (data not shown). Some reprogrammed cells express marker genes but not insulin. The pancreatic β-like cells do not express Amylase, glucagon, or somatostatin/pancreatic polypeptide (SomPP), but do express c-peptide (c-peptide) (data not shown).
Expression profile analysis of the reprogrammed cells further indicates a strong overlap of endocrine-enriched genes between reprogrammed cells and islet cells, demonstrating a high degree of similarity between their endocrine programs (FIGS. 6A and 6B).
The new β-cells do not express exocrine genes such as Amylase or Ptf1a, the duct marker Ck19 (also known as Krt19), mesenchymal markers Nestin and Vimentin, nor the neuronal marker Tuji (data not shown). Nor do the new β-cells express any other pancreatic hormones such as glucagon, somatostatin or pancreatic polypeptide (data not shown). Thus, the new β-cells do not exhibit a hybrid or mixed phenotype, indicating silencing of non-β-cell programs.
The primary function of β-cells is to synthesize and release insulin. To facilitate the release of insulin into the circulation, β-cells, unique among pancreatic cell types, synthesize vascular endothelial growth factor (VEGF) which promotes local angiogenic remodeling25. Notably, pancreatic β-like cells similarly synthesize VEGF and induce angiogenesis so that blood vessels form next to these new cells (data not shown). In particular, immunostaining analysis demonstrated new pancreatic β-like cells synthesize vascular endothelial growth factor (VEGF), and induce local angiogenic remodeling. Note the close proximity of blood vessels (PECAM+) with the pancreatic β-like cells versus control infected cells (data not shown). Quantification indicates that in nGFP controls, 32% of infected cells lie adjacent to blood vessels whereas 61% and 83% of pancreatic β-like cells are directly juxtaposed to blood vessels 10 days and 30 days after induction, respectively.
To test whether pancreatic β-like cells release insulin, mice were rendered diabetic by streptozotocin (STZ) injection which specifically ablates islet β-cells. When subsequently injected with pAd-M3, fasting blood glucose levels of hyperglycemic animals show a significant and long lasting improvement compared to animals injected with control (nGFP) virus (FIG. 4A). In addition, the pAd-M3 animals show increased glucose tolerance (FIG. 7), have increased insulin levels in the serum (non-fasting, P<0.01, FIG. 4B), and possess large numbers of pancreatic β-like cells (FIG. 5C). RT-PCR analysis and direct observation revealed that virus injected into the pancreas does not spread to other internal organs such as liver and intestine that, theoretically, could modulate insulin secretion and/or response (FIG. 8). In addition, the inventors found no evidence that STZ treated animals show spontaneous conversion of exocrine cells to β-cells (FIG. 9). As the data in FIG. 4 shows, the total number of pancreatic β-like cells is rather small compared to the number of β-cells in normal animals and this may account for the limitation to the effectiveness in restoring glucose homeostasis. Alternatively, as the new β-cells are not reorganized into islet structures, this may limit their effectiveness. All together, these data show that pancreatic β-like cells can produce and secrete insulin in vivo.
Transient expression of inducing factors results in a long-lasting β-cell phenotype. The inventors' results thus far support the contention that a combination of three transcription factors fully reprograms exocrine cells to β-cells in vivo. To determine whether continued presence of these factors is required to maintain the phenotype of reprogrammed cells, the inventors used RT-PCR and primers specific to viral transgenes to detect their presence. Transgene expression from all three viruses was substantially diminished after one month and was undetectable after 2 months (FIG. 9). Ngn3 protein was undetectable by antibody staining one month after infection (data not shown). In particular, immunostaining for Ngn3 at different time points after pAd-M3 infection demonstrated a strong Ngn3 expression at day 10, which largely disappears by day 30 (data not shown). Time course immunostaining for Pdx1 and MafA was also performed (data not shown), where expression of both Pdx1 and MafA remains strong over the 60 day period examined. Pdx1 and MafA protein expression in the pancreatic β-like cells, however, remains consistently strong even after 2 months, indicating the activation of endogenous genes (data not shown). These results are consistent with the fact that endogenous islet β-cells do not express Ngn3, but do express Pdx1 and MafA20,21. Thus, a transient expression of the inducing factors is sufficient to convert exocrine cells to a stable new β-cell state.
Example 4
B-Cell Reprogramming does not Involve Dedifferentiation.
In principle, the conversion of exocrine cells to β-cells could be direct or involve dedifferentiation to common progenitors which then redifferentiate into β-cells. Indeed, exocrine and β-cells share a common progenitor during embryogenesis that is characterized by rapid division and expression of genes including Sox9 and Hnf6 (also known as Onecut119). Continuous BrdU labeling over the first 10 days of reprogramming, however, shows that few pancreatic β-like cells (3.2%) have divided (data not shown). In comparison, 12.9% of endogenous islet β-cells in the same animals incorporated BrdU (supplemental FIG. 3). In addition, the inventors detected no induction of Sox9 or Hnf6 (data not shown). These results suggest that in vivo reprogramming of exocrine to β-cells is a direct conversion of cell types and does not involve dedifferentiation. The inventors can not formally exclude the possibility that a very transient or partial dedifferentiation may occur, but the inventors' results indicate that extensive replication and reversion to a dedifferentiated cell for an appreciable time does not occur.
The inventors' results provide evidence that fully differentiated exocrine cells can be reprogrammed into cells that closely resemble β-cells in adult animals by a combination of just three transcription factors.
Table 1.
Three classes of pancreatic transcription factors. The list of genes was compiled from reference 16 and other publications. Note that the endocrine progenitor genes and adult beta cell genes overlap extensively, but have little overlap with pancreatic progenitor genes. Nine genes that exhibit β-cells phenotypes when mutated were selected for further analysis (shown in bold italic).
















Mouse




Gene Name
GeneBank
SEQ ID NO:
















Pancreatic Progenitor











Pdx1
NM_008814
SEQ ID NO: 1



Ptf1a
NM_018809
SEQ ID NO: 10



Sox9
NM_011448
SEQ ID NO: 11



Hnf6
NM_008262
SEQ ID NO: 12



Hnf1b
NM_009330
SEQ ID NO: 13



Hnf3b
NM_010446
SEQ ID NO: 14



Hnf4a
NM_008261
SEQ ID NO: 15



Hex
NM_008245
SEQ ID NO: 16



Prox1
NM_008937
SEQ ID NO: 17



Hb9
NM_019944
SEQ ID NO: 18



Nr5a2
NM_030676
SEQ ID NO: 19







Endocrine Progenitor











Ngn3
NM_009719
SEQ ID NO: 2



MafA
NM_194350
SEQ ID NO: 3



NeuroD
NM_010894
SEQ ID NO: 4



Nkx2.2
NM_010919
SEQ ID NO: 5



Nkx6.1
NM_144955
SEQ ID NO: 6



Pax4
NM_011038
SEQ ID NO: 7



Pax6
NM_013627
SEQ ID NO: 8



Isl1
NM_021459
SEQ ID NO: 9



MafB
NM_010658
SEQ ID NO: 20



Brn4
NM_008901
SEQ ID NO: 21



Arx
NM_007492
SEQ ID NO: 22



Myt1
NM_008665
SEQ ID NO: 23



Wbscr14
NM_021455
SEQ ID NO: 24



VDR
NM_009504
SEQ ID NO: 25



IA1

SEQ ID NO: 26







Adult Beta cell











Pdx1
NM_008814
SEQ ID NO: 1



NeuroD
NM_010894
SEQ ID NO: 4



MafA
NM_194350
SEQ ID NO: 3



Nkx2.2
NM_010919
SEQ ID NO: 5



Nkx6.1
NM_144955
SEQ ID NO: 6



Pax6
NM_013627
SEQ ID NO: 8



Isl1
NM_021459
SEQ ID NO: 9



Foxo1
NM_019739
SEQ ID NO: 27



Hnf1a
NM_009327
SEQ ID NO: 28



Hnf3a
NM_008259
SEQ ID NO: 29



Hnf4a
NM_008261
SEQ ID NO: 30










The practice of the present invention will employ, unless otherwise indicated, conventional techniques of mouse genetics, developmental biology, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Current Protocols in Cell Biology, ed. by Bonifacino, Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., New York, 1999; Manipulating the Mouse Embryos, A Laboratory Manual, 3rd Ed., by Hogan et al., Cold Spring Contain Laboratory Press, Cold Spring Contain, New York, 2003; Gene Targeting: A Practical Approach, IRL Press at Oxford University Press, Oxford, 1993; and Gene Targeting Protocols, Human Press, Totowa, N.J., 2000. All patents, patent applications and references cited herein are incorporated in their entirety by reference.
The present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, systems and kits are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses are also contemplated herein. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims. Varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The articles “a” and an as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth herein. It should also be understood that any embodiment of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any cell of endoderm origin, any agent, any somatic cell type, any β-cell reprogramming agent, etc., may be excluded.
Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”. “Approximately” or “about” is intended to encompass numbers that fall within a range of ±10% of a number, in some embodiments within ±5% of a number, in some embodiments within ±1%, in some embodiments within ±0.5% of a number, in some embodiments within ±0.1% of a number unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value).
Certain claims are presented in dependent form for the sake of convenience, but Applicant reserves the right to rewrite any dependent claim in independent form to include the limitations of the independent claim and any other claim(s) on which such claim depends, and such rewritten claim is to be considered equivalent in all respects to the dependent claim in whatever form it is in (either amended or unamended) prior to being rewritten in independent format. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited.
REFERENCES
All references cited herein are incorporated herein by reference in their entirety as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only in terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.
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SEQUENCE LISTING:















SEQ ID NO: 1-polypeptide sequence of mouse pdx1


(accession no NM_008814)


MNSEEQYYAATQLYKDPCAFQRGPVPEFSANPPACLYMGRQPPP


 


PPPPQFTSSLGSLEQGSPPDISPYEVPPLASDDPAGAHLHHHLPAQLGLAHPPPGPFP


 


NGTEPGGLEEPNRVQLPFPWMKSTKAHAWKGQWAGGAYTAEPEENKRTRTAYTRAQLL


 


ELEKEFLFNKYISRPRRVELAVMLNLTERHIKIWFQNRRMKWKKEEDKKRSSGTPSGG


 


GGGEEPEQDCAVTSGEELLAVPPLPPPGGAVPPGVPAAVREGLLPSGLSVSPQPSSIA


 


PLRPQEPR


 


SEQ ID NO: 2-polypeptide sequence of mouse Ngn3


(accession No NP_033849.3)


MAPHPLDALTIQVSPETQQPFPGASDHEVLSSNSTPPSPTLIPR


 


DCSEAEVGDCRGTSRKLRARRGGRNRPKSELALSKQRRSRRKKANDRERNRMHNLNSA


 


LDALRGVLPTFPDDAKLTKIETLRFAHNYIWALTQTLRIADHSFYGPEPPVPCGELGS


 


PGGGSNGDWGSIYSPVSQAGNLSPTASLEEFPGLQVPSSPSYLLPGALVFSDFL


 


SEQ ID NO: 3-polypeptide sequence of mouse MafA


MAAELAMGAELPSSPLAIEYVNDFDLMKFEVKKEPPEAERFCHR


 


LPPGSLSSTPLSTPCSSVPSSPSFCAPSPGTGGGAGGGGSAAQAGGAPGPPSGGPGTV


 


GGASGKAVLEDLYWMSGYQHHLNPEALNLTPEDAVEALIGSGHHGAHHGAHHPAAAAA


 


YEAFRGQSFAGGGGADDMGAGHHHGAHHTAHHHHSAHHHHHHHHHHGGSGHHGGGAGH


 


GGGGAGHHVRLEERFSDDQLVSMSVRELNRQLRGFSKEEVIRLKQKRRTLKNRGYAQS


 


CRFKRVQQRHILESEKCQLQSQVEQLKLEVGRLAKERDLYKEKYEKLAGRGGPGGAGG


 


AGFPREPSPAQAGPGAAKGAPDFFL


 


SEQ ID NO: 31-amino acid sequence of HUMAN pdx1


(accession no NP_000200.1)


MNGEEQYYAATQLYKDPCAFQRGPAPEFSASPPACLYMGRQPPP


 


PPPHPFPGALGALEQGSPPDISPYEVPPLADDPAVAHLHHHLPAQLALPHPPAGPFPE


 


GAEPGVLEEPNRVQLPFPWMKSTKAHAWKGQWAGGAYAAEPEENKRTRTAYTRAQLLE


 


LEKEFLFNKYISRPRRVELAVMLNLTERHIKIWFQNRRMKWKKEEDKKRGGGTAVGGG


 


GVAEPEQDCAVTSGEELLALPPPPPPGGAVPPAAPVAAREGRLPPGLSASPQPSSVAP


 


RRPQEPR


 


SEQ ID NO: 32-amino acid sequence of HUMAN (Homo sapiens)


neurogenin 3 (NEUROG3), Ngn3 (GeneBank No: NP_066279.2)


MTPQPSGAPTVQVTRETERSFPRASEDEVTCPTSAPPSPTRTRG


 


NCAEAEEGGCRGAPRKLRARRGGRSRPKSELALSKQRRSRRKKANDRERNRMHNLNSA


 


LDALRGVLPTFPDDAKLTKIETLRFAHNYIWALTQTLRIADHSLYALEPPAPHCGELG


 


SPGGSPGDWGSLYSPVSQAGSLSPAASLEERPGLLGATFSACLSPGSLAFSDFL


 


SEQ ID NO: 33-amino acid sequence of HUMAN MafA


(accession no: NP_963883.2)


MAAELAMGAELPSSPLAIEYVNDFDLMKFEVKKEPPEAERFCHR


 


LPPGSLSSTPLSTPCSSVPSSPSFCAPSPGTGGGGGAGGGGGSSQAGGAPGPPSGGPG


 


AVGGTSGKPALEDLYWMSGYQHHLNPEALNLTPEDAVEALIGSGHHGAHHGAHHPAAA


 


AAYEAFRGPGFAGGGGADDMGAGHHHGAHHAAHHHHAAHHHHHHHHHHGGAGHGGGAG


 


HHVRLEERFSDDQLVSMSVRELNRQLRGFSKEEVIRLKQKRRTLKNRGYAQSCRFKRV


 


QQRHILESEKCQLQSQVEQLKLEVGRLAKERDLYKEKYEKLAGRGGPGSAGGAGFPRE


 


PSPPQAGPGGAKGTADFFL


 


SEQ ID NO: 34-mRNA sequence of HUMAN pdx1 (accession no NM_000209)


   1 gggtggcgcc gggagtggga acgccacaca gtgccaaatc cccggctcca gctcccgact


 


  61 cccggctccc ggctcccggc tcccggtgcc caatcccggg ccgcagccat gaacggcgag


 


 121 gagcagtact acgcggccac gcagctttac aaggacccat gcgcgttcca gcgaggcccg


 


 181 gcgccggagt tcagcgccag cccccctgcg tgcctgtaca tgggccgcca gcccccgccg


 


 241 ccgccgccgc acccgttccc tggcgccctg ggcgcgctgg agcagggcag ccccccggac


 


 301 atctccccgt acgaggtgcc ccccctcgcc gacgaccccg cggtggcgca ccttcaccac


 


 361 cacctcccgg ctcagctcgc gctcccccac ccgcccgccg ggcccttccc ggagggagcc


 


 421 gagccgggcg tcctggagga gcccaaccgc gtccagctgc ctttcccatg gatgaagtct


 


 481 accaaagctc acgcgtggaa aggccagtgg gcaggcggcg cctacgctgc ggagccggag


 


 541 gagaacaagc ggacgcgcac ggcctacacg cgcgcacagc tgctagagct ggagaaggag


 


 601 ttcctattca acaagtacat ctcacggccg cgccgggtgg agctggctgt catgttgaac


 


 661 ttgaccgaga gacacatcaa gatctggttc caaaaccgcc gcatgaagtg gaaaaaggag


 


 721 gaggacaaga agcgcggcgg cgggacagct gtcgggggtg gcggggtcgc ggagcctgag


 


 781 caggactgcg ccgtgacctc cggcgaggag cttctggcgc tgccgccgcc gccgcccccc


 


 841 ggaggtgctg tgccgcccgc tgcccccgtt gccgcccgag agggccgcct gccgcctggc


 


 901 cttagcgcgt cgccacagcc ctccagcgtc gcgcctcggc ggccgcagga accacgatga


 


 961 gaggcaggag ctgctcctgg ctgaggggct tcaaccactc gccgaggagg agcagagggc


 


1021 ctaggaggac cccgggcgtg gaccacccgc cctggcagtt gaatggggcg gcaattgcgg


 


1081 ggcccacctt agaccgaagg ggaaaacccg ctctctcagg cgcatgtgcc agttggggcc


 


1141 ccgcgggtag atgccggcag gccttccgga agaaaaagag ccattggttt ttgtagtatt


 


1201 ggggccctct tttagtgata ctggattggc gttgtttgtg gctgttgcgc acatccctgc


 


1261 cctcctacag cactccacct tgggacctgt ttagagaagc cggctcttca aagacaatgg


 


1321 aaactgtacc atacacattg gaaggctccc taacacacac agcggggaag ctgggccgag


 


1381 taccttaatc tgccataaag ccattcttac tcgggcgacc cctttaagtt tagaaataat


 


1441 tgaaaggaaa tgtttgagtt ttcaaagatc ccgtgaaatt gatgccagtg gaatacagtg


 


1501 agtcctcctc ttcctcctcc tcctcttccc cctccccttc ctcctcctcc tcttcttttc


 


1561 cctcctcttc ctcttcctcc tgctctcctt tcctccccct cctcttttcc ctcctcttcc


 


1621 tcttcctcct gctctccttt cctccccctc ctctttctcc tcctcctcct cttcttcccc


 


1681 ctcctctccc tcctcctctt cttccccctc ctctccctcc tcctcttctt ctccctcctc


 


1741 ttcctcttcc tcctcttcca cgtgctctcc tttcctcccc ctcctcttgc tccccttctt


 


1801 ccccgtcctc ttcctcctcc tcctcttctt ctccctcctc ttcctcctcc tctttcttcc


 


1861 tgacctcttt ctttctcctc ctcctccttc tacctcccct tctcatccct cctcttcctc


 


1921 ttctctagct gcacacttca ctactgcaca tcttataact tgcacccctt tcttctgagg


 


1981 aagagaacat cttgcaaggc agggcgagca gcggcagggc tggcttagga gcagtgcaag


 


2041 agtccctgtg ctccagttcc acactgctgg cagggaaggc aaggggggac gggcctggat


 


2101 ctgggggtga gggagaaaga tggacccctg ggtgaccact aaaccaaaga tattcggaac


 


2161 tttctattta ggatgtggac gtaattcctg ttccgaggta gaggctgtgc tgaagacaag


 


2221 cacagtggcc tggtgcgcct tggaaaccaa caactattca cgagccagta tgaccttcac


 


2281 atctttagaa attatgaaaa cgtatgtgat tggagggttt ggaaaaccag ttatcttatt


 


2341 taacatttta aaaattacct aacagttatt tacaaacagg tctgtgcatc ccaggtctgt


 


2401 cttcttttca aggtctgggc cttgtgctcg ggttatgttt gtgggaaatg cttaataaat


 


2461 actgataata tgggaagaga tgaaaactga ttctcctcac tttgtttcaa acctttctgg


 


2521 cagtgggatg attcgaattc acttttaaaa ttaaattagc gtgttttgtt ttg


 


SEQ ID NO: 35 nucleotide mRNA sequence for human Ngn 3


(accession no NM_020999) 1167 bp mRNA


   1 cctcggaccc cattctctct tcttttctcc tttggggctg gggcaactcc caggcggggg


 


  61 cgcctgcagc tcagctgaac ttggcgacca gaagcccgct gagctcccca cggccctcgc


 


 121 tgctcatcgc tctctattct tttgcgccgg tagaaaggat gacgcctcaa ccctcgggtg


 


 181 cgcccactgt ccaagtgacc cgtgagacgg agcggtcctt ccccagagcc tcggaagacg


 


 241 aagtgacctg ccccacgtcc gccccgccca gccccactcg cacacggggg aactgcgcag


 


 301 aggcggaaga gggaggctgc cgaggggccc cgaggaagct ccgggcacgg cgcgggggac


 


 361 gcagccggcc taagagcgag ttggcactga gcaagcagcg acggagtcgg cgaaagaagg


 


 421 ccaacgaccg cgagcgcaat cgaatgcaca acctcaactc ggcactggac gccctgcgcg


 


 481 gtgtcctgcc caccttccca gacgacgcga agctcaccaa gatcgagacg ctgcgcttcg


 


 541 cccacaacta catctgggcg ctgactcaaa cgctgcgcat agcggaccac agcttgtacg


 


 601 cgctggagcc gccggcgccg cactgcgggg agctgggcag cccaggcggt tcccccgggg


 


 661 actgggggtc cctctactcc ccagtctccc aggctggcag cctgagtccc gccgcgtcgc


 


 721 tggaggagcg acccgggctg ctgggggcca ccttttccgc ctgcttgagc ccaggcagtc


 


 781 tggctttctc agattttctg tgaaaggacc tgtctgtcgc tgggctgtgg gtgctaaggg


 


 841 taagggagag ggagggagcc gggagccgta gagggtggcc gacggcggcg gccctcaaaa


 


 901 gcacttgttc cttctgcttc tccctggctg acccctggcc ggcccaggcc tccacggggg


 


 961 cggcaggctg ggttcattcc ccggccctcc gagccgcgcc aacgcacgca acccttgctg


 


1021 ctgcccgcgc gaagtgggca ttgcaaagtg cgctcatttt aggcctcctc tctgccacca


 


1081 ccccataatc tcattcaaag aatactagaa tggtagcact acccggccgg agccgcccac


 


1141 cgtcttgggt cgccctaccc tcactca


 


SEQ ID NO: 36 nucleotide mRNA sequence for HUMAN MafA


(accession no: NM_201589) 1062 bp mRNA


   1 atggccgcgg agctggcgat gggcgccgag ctgcccagca gcccgctggc catcgagtac


 


  61 gtcaacgact tcgacctgat gaagttcgag gtgaagaagg agcctcccga ggccgagcgc


 


 121 ttctgccacc gcctgccgcc aggctcgctg tcctcgacgc cgctcagcac gccctgctcc


 


 181 tccgtgccct cctcgcccag cttctgcgcg cccagcccgg gcaccggcgg cggcggcggc


 


 241 gcggggggcg gcggcggctc gtctcaggcc gggggcgccc ccgggccgcc gagcgggggc


 


 301 cccggcgccg tcgggggcac ctcggggaag ccggcgctgg aggatctgta ctggatgagc


 


 361 ggctaccagc atcacctcaa ccccgaggcg ctcaacctga cgcccgagga cgcggtggag


 


 421 gcgctcatcg gcagcggcca ccacggcgcg caccacggcg cgcaccaccc ggcggccgcc


 


 481 gcagcctacg aggctttccg cggcccgggc ttcgcgggcg gcggcggagc ggacgacatg


 


 541 ggcgccggcc accaccacgg cgcgcaccac gccgcccacc atcaccacgc cgcccaccac


 


 601 caccaccacc accaccacca ccatggcggc gcgggacacg gcggtggcgc gggccaccac


 


 661 gtgcgcctgg aggagcgctt ctccgacgac cagctggtgt ccatgtcggt gcgcgagctg


 


 721 aaccggcagc tccgcggctt cagcaaggag gaggtcatcc ggctcaagca gaagcggcgc


 


 781 acgctcaaga accgcggcta cgcgcagtcc tgccgcttca agcgggtgca gcagcggcac


 


 841 attctggaga gcgagaagtg ccaactccag agccaggtgg agcagctgaa gctggaggtg


 


 901 gggcgcctgg ccaaagagcg ggacctgtac aaggagaaat acgagaagct ggcgggccgg


 


 961 ggcggccccg ggagcgcggg cggggccggt ttcccgcggg agccttcgcc gccgcaggcc


 


1021 ggtcccggcg gggccaaggg cacggccgac ttcttcctgt ag


 


SEQ ID NO: 37-mRNA sequence of mouse pdx1 (accession no NM_008814)


   1 gtcaaagcga tctggggtgg cgtagagagt ccgcgagcca cccagcgcct aaggcctggc


 


  61 ttgtagctcc gacccggggc tgctggcccc caagtgccgg ctgccaccat gaacagtgag


 


 121 gagcagtact acgcggccac acagctctac aaggacccgt gcgcattcca gaggggcccg


 


 181 gtgccagagt tcagcgctaa cccccctgcg tgcctgtaca tgggccgcca gcccccacct


 


 241 ccgccgccac cccagtttac aagctcgctg ggatcactgg agcagggaag tcctccggac


 


 301 atctccccat acgaagtgcc cccgctcgcc tccgacgacc cggctggcgc tcacctccac


 


 361 caccaccttc cagctcagct cgggctcgcc catccacctc ccggaccttt cccgaatgga


 


 421 accgagcctg ggggcctgga agagcccaac cgcgtccagc tccctttccc gtggatgaaa


 


 481 tccaccaaag ctcacgcgtg gaaaggccag tgggcaggag gtgcttacac agcggaaccc


 


 541 gaggaaaaca agaggacccg tactgcctac acccgggcgc agctgctgga gctggagaag


 


 601 gaattcttat ttaacaaata catctcccgg ccccgccggg tggagctggc agtgatgttg


 


 661 aacttgaccg agagacacat caaaatctgg ttccaaaacc gtcgcatgaa gtggaaaaaa


 


 721 gaggaagata agaaacgtag tagcgggacc ccgagtgggg gcggtggggg cgaagagccg


 


 781 gagcaagatt gtgcggtgac ctcgggcgag gagctgctgg cagtgccacc gctgccacct


 


 841 cccggaggtg ccgtgccccc aggcgtccca gctgcagtcc gggagggcct actgccttcg


 


 901 ggccttagcg tgtcgccaca gccctccagc atcgcgccac tgcgaccgca ggaaccccgg


 


 961 tgaggacagc agtctgaggg tgagcgggtc tgggacccag agtgtggacg tgggagcggg


 


1021 cagctggata agggaactta acctaggcgt cgcacaagaa gaaaattctt gagggcacga


 


1081 gagccagttg ggtatagccg gagagatgct ggcagacttc tggaaaaaca gccctgagct


 


1141 tctgaaaact ttgaggctgc ttctgatgcc aagcgaatgg ccagatctgc ctctaggact


 


1201 ctttcctggg accaatttag acaacctggg ctccaaactg aggacaataa aaagggtaca


 


1261 aacttgagcg ttccaatacg gaccagc


 


SEQ ID NO: 38 nucleotide mRNA sequence for mouse Ngn 3


(accession No: NM_009719) 1540 bp mRNA linear


   1 atgcagctca gaaatccctc tgggtctcat cactgcagca gtggtcgagt acctcctcgg


 


  61 agcttttcta cgacttccag acgcaattta ctccaggcga gggcgcctgc agtttagcag


 


 121 aacttcagag ggagcagaga ggctcagcta tccactgctg cttgacactg accctatcca


 


 181 ctgctgcttg tcactgactg acctgctgct ctctattctt ttgagtcggg agaactagga


 


 241 tggcgcctca tcccttggat gcgctcacca tccaagtgtc cccagagaca caacaacctt


 


 301 ttcccggagc ctcggaccac gaagtgctca gttccaattc caccccacct agccccactc


 


 361 tcatacctag ggactgctcc gaagcagaag tgggtgactg ccgagggacc tcgaggaagc


 


 421 tccgcgcccg acgcggaggg cgcaacaggc ccaagagcga gttggcactc agcaaacagc


 


 481 gaagaagccg gcgcaagaag gccaatgatc gggagcgcaa tcgcatgcac aacctcaact


 


 541 cggcgctgga tgcgctgcgc ggtgtcctgc ccaccttccc ggatgacgcc aaacttacaa


 


 601 agatcgagac cctgcgcttc gcccacaact acatctgggc actgactcag acgctgcgca


 


 661 tagcggacca cagcttctat ggcccggagc cccctgtgcc ctgtggagag ctggggagcc


 


 721 ccggaggtgg ctccaacggg gactggggct ctatctactc cccagtctcc caagcgggta


 


 781 acctgagccc cacggcctca ttggaggaat tccctggcct gcaggtgccc agctccccat


 


 841 cctatctgct cccgggagca ctggtgttct cagacttctt gtgaagagac ctgtctggct


 


 901 ctgggtggtg ggtgctagtg gaaagggagg ggaccagagc cgtctggagt gggaggtagt


 


 961 ggaggctctc aagcatctcg cctcttctgg ctttcactac ttggatccct agccctctca


 


1021 cagggcttaa ctaggcttct catcggtacc cttgctgctg cgcacagcag acattggggg


 


1081 ctgctcttct cttaactctc ctcggtgcag ccacatcaaa ctctcgctcc aagcatttga


 


1141 gaatggtagc actacctagt tggagactcc catacttcct ggtgagtctg ccctcattca


 


1201 aatctgccgg cctccgacca tccatcactt tttccagggt gacctaatcc agtgttgcgt


 


1261 cttacctcac tggctcctcc atccagctct tggcccatag atgatgttcg tcgtctttac


 


1321 tgcccgctac atgcagggtt tctgagcttc tccattctgc cttagtccac gaaggtgatc


 


1381 tgccttcttc tgcacttttc aagtcgttac ccttccccca agggagacca ggctgtgaac


 


1441 cggaaagccc tagcctatgg ctagagcatc tctccaactt gtctcccgtg tctaaagtgt


 


1501 gagttgcagg gacggttcct gaagcactgt ttgtctccct


 


SEQ ID NO: 39 nucleotide mRNA sequence for mouse MafA


(GeneBank No: NM_194350) 1080 bp mRNA linear


   1 atggccgcgg agctggcgat gggcgcagag ctgcccagca gcccactggc catcgagtac


 


  61 gtcaacgact tcgacctgat gaagttcgag gtgaagaagg agccgcccga ggccgagcgc


 


 121 ttctgccacc gcctgccgcc cggctcgctg tcctcgacgc ccctcagcac gccctgctcc


 


 181 tcggtgccct cttcgcccag cttctgcgca cccagcccgg gcacaggcgg cggcgcgggc


 


 241 ggcgggggca gcgcggctca ggccgggggc gccccggggc cgccgagtgg aggccccggc


 


 301 actgtcgggg gcgcctcagg aaaagcggtg ctggaggatc tgtactggat gagcgggtac


 


 361 cagcaccacc tgaaccccga ggcgctcaac ctgacgccgg aggacgcggt ggaggcgctc


 


 421 atcggcagc ggccaccacgg cgcgcaccac ggcgcgcatc acccggcggc tgctgcggcc


 


 481 tatgaggcc ttccggggtca gagcttcgcg ggcggcggcg gcgcggacga catgggtgcc


 


 541 ggccaccac cacggcgcaca ccacactgcc caccatcatc actctgccca ccatcaccat


 


 601 caccaccat caccaccacgg aggctctggc caccacggcg gaggcgcggg tcacggcgga


 


 661 ggcggcgca ggccaccacgt gcgcttggag gagcgcttct ccgacgacca gctggtatcc


 


 721 atgtccgtg cgggagctgaa ccggcagctc cgcggcttca gcaaggagga ggtcatccga


 


 781 ctgaaacag aagcggcgcac gctcaagaac cgcggctacg cgcagtcgtg ccgcttcaag


 


 841 cgggtgcag cagcggcacat tctggagagc gagaagtgcc agctccagag ccaggtggag


 


 901 cagctgaag ctggaggtggg gcgtctggcc aaggagcggg acctgtacaa ggagaaatac


 


 961 gagaagttg gcgggccgggg cggccccggg ggcgcgggcg gggccggctt ccctcgggag


 


1021 ccctcgcca gcgcaggctgg ccccggggcg gccaaaggcg cacccgactt ctttctgtga

## Claims

1. A method for reprogramming a first differentiated cell of exocrine origin to a second differentiated cell that exhibits at least two characteristics of a pancreatic β-cell, does not express pancreatic hormones other than insulin and does not express amylase, the method comprising increasing protein expression of transcription factors Pdx-1, Ngn3 and MaFA in the first differentiated cell of exocrine origin, thereby reprogramming the first differentiated cell of exocrine origin to a second differentiated cell that exhibits at least two characteristics of a pancreatic β-cell, does not express pancreatic hormones other than insulin and does not express amylase, wherein the first differentiated cell of exocrine origin is isolated from a subject or located in vivo within the subject.

2. The method of claim 1, wherein a characteristic of a pancreatic β-cell is secreting insulin in response to glucose.

3. The method of claim 1, wherein a characteristic of a pancreatic β-cell is expression of at least one marker selected from the group consisting of: Ngn3−, Pdx1+ and MafA+.

4. The method of claim 1, wherein the protein expression of the transcription factors is increased by contacting the first differentiated cell of exocrine origin with agents which increase the expression of the transcription factors.

5. The method of claim 1, wherein the first differentiated cell of exocrine origin is in vitro.

6. The method of claim 1, wherein the first differentiated cell of exocrine origin is ex vivo.

7. The method of claim 1, wherein the first differentiated cell of exocrine origin is in vivo or present in a subject.

8. The method of claim 7, wherein the subject has, or is at risk of developing, diabetes or a metabolic disorder.

9. The method of claim 1, wherein the first differentiated cell of exocrine origin is a mammalian cell.

10. The method of claim 1, wherein the method further comprises contacting the first differentiated cell of exocrine origin with at least one agent which increases the protein expression of at least one of the transcription factors selected from the group consisting of: NeuroD, Nkx2.2, Nkx6.1, Pax4, Pax6 or Isl1.

11. The method of claim 1, wherein the second cell exhibiting at least two characteristics of a pancreatic β cell has an increased expression of a marker selected from the group consisting of: c-peptide, glucose transporter 2 (Glut2), glucokinase (GCK), prohormone convertase 1/3 (PC1/3), β-cell transcription factors NeuroD, Nkx2.2 and Nkx6.1 by a statistically significant amount relative to the first differentiated cell of ended exocrine origin from which the second cell that exhibits at least two characteristics of a pancreatic β cell was derived.

12. The method of claim 1, wherein the second cell that exhibits at least two characteristics of a pancreatic βcell has a decreased expression of a marker selected from the group consisting of: Ck19, Nestin, Vimentin and Tuji by a statistically significant amount relative to the first differentiated cell of exocrine origin from which the β cell was derived.