Patent Application: US-201113582963-A

Abstract:
diabetes mellitus is characterized by either the inability to produce insulin and or as insensitivity to insulin secreted by the body . in either case , the body is unable to efficiently move blood glucose across cell membranes to be utilized . this leads to a variety of local and systemic detrimental effects . current treatments for diabetes focus on exogenous insulin administration and dietary control . provided herein are treatments of diabetes using a cellular therapy to ameliorate symptoms associated with both reduced insulin secretion and insulin sensitivity . using induced - pluripotent stem cells , beta - like cells similar to the endogenous insulin secreting cells were derived . these β - like cells secreted insulin in response to glucose , and corrected a hyperglycemic phenotype in a mouse model of type 2 diabetes via an ips cell transplant . within the type 2 diabetes mouse model , a long term correction of hyperglycemia was achieved as measured by blood glucose and hemoglobin alc measurements . reduction of hyperglycemia was also seen in a chemically - induced mouse model for type 1 diabetes .

Description:
the ability of ips cells to differentiate in vitro into insulin - secreting cells is described herein , and these cells are further shown to be functional in vivo to correct a hyperglycemic phenotype in diabetic mouse models . the ips cells described herein have a number of advantages including , but not limited to 1 ) the ips cells were derived from fibroblasts of c57bl6 background mice , close relatives of the diabetic mouse models used here , and thus reduce the possibility of graft rejection upon transplantation , 2 ) insulin produced from the transplanted ips cell derived β - like cells can be considered endogenous insulin , thereby allowing maintenance of normal glucose homeostasis as the mice age , and 3 ) an unlimited supply of cells for differentiation can be produced from the ips cells . a “ stem - cell ” is a cell characterized by the ability of self - renewal through mitotic cell division and the potential to differentiate into a tissue or an organ . the term “ subject ” refers to any individual , human or animal , that belongs to a species that normally produces insulin . an “ induced pluripotent stem ( ips ) cell ” is any type of cell derived from a non - pluripotent cell , which can include , but is not limited to , a hematopoietic cell , a mesenchymal cell , an epithelial cell , a skin cell , a neural cell , or any type of non - pluripotent somatic stem cell . the term “ pluripotent ” or “ pluripotency ” refers to cells with the ability to give rise to progency that can undergo differentiation , under appropriate conditions , into cell types that collectively exhibit characteristics associated with cell lineages from endoderm , mesoderm , and ectoderm . ips cells can contribute to tissues of a prenatal , postnatal , or adult organism . the term “ somatic cell ” refers to a cell that is not a germline cell . an example of a somatic cell is a fibroblast cell , although one skilled in the art would appreciate that a multitude of different types of somatic cells exist . the term “ β - like cell ” refers to a cell derived from an ips cell that has the same characteristics as a naturally occurring β cell and is an insulin secreting cell . one of skill in the art would recognize that an ips cell has the potential to differentiate into a β - like cells , and the β - like cells as described herein are cells that have been differentiated from the ips cells of the present disclosure and have the ability to secrete insulin . an “ insulin - secreting cell ” is any type of cell that inter alma , secretes insulin . the term “ autologous ips ” refers to ips cells induced from cells derived from the subject into which the ips cells will be reintroduced . the term “ reprogramming ” refers to the process of dedifferentiating a non - pluripotent cell into a cell exhibiting pluripotent stem cell characteristics . the term “ vector ” refers to any type of genetic element , such as a plasmid , phage , transposon , cosmid , chromosome , virus , etc ., that is capable of replicating when provided with appropriate control and accessory elements and which can transfer exogenous gene sequences into cells , including all manner of cloning and expression vehicles , as well as viral vectors . a cell is “ transformed ” following introduction of said vector . the term “ homologous recombination ” refers to the exchange of dna fragments between two dna molecules ( during crossover ). the fragments that are exchanged are flanked by sites of identical nucleotide sequences between the two dna molecules ( i . e ., homology dna regions ). the term “ integrate ” or “ chromosomal integration ” refers to the process in which a dna segment is introduced into a cell and becomes congruent with the chromosome through recombination between homologous dna regions on the introduced dna segment and within the chromosome . the term “ heterologous dna ” refers to dna that is not endogenous to the cell into which it is introduced , but can include an endogenous dna sequence containing modification . the term “ suicide gene ” refers to a gene encoding a protein whose expression results in the death of cells expressing the gene . the term “ inducible promoter ” refers to a polynucleotide sequence that initiates and facilitates the transcription of a gene when the host cell comprising the inducible promoter is exposed to some particular external stimulus . the activity of an inducible promoter may be triggered , for example , by chemical , physical , nutritional or cell growth factors . the term “ engrafting ” or “ engraftment ” or “ engraft ” refers to the process of infused or transplanted donor stem cells to the host organism . as used herein , stem cells can engraft anywhere in the host organism . the term “ treating ” or “ treat ” or “ treatment ” refers to the relief or alleviation of at least one symptom of a disorder in a subject . the term “ administer ” or “ administering ” refers to the dispensing , supplying , applying , giving , apportioning , or contributing stem cells to a subject in need thereof . routes for administration can include , but are not limited to , oral , subcutaneous or parenteral , including intravenous , intraportal , intraarterial , intramuscular , intraperitoneal , intranasal , as well as by infusion . the term “ multi - lineage progenitors ” refers to stem cells or progenitor cells capable of differentiating to mesoderm , ectoderm and / or endoderm lineages . the term “ transcription factor ” refers to polynucleotides and / or polypeptides that are involved in gene regulation in both prokaryotic and eukaryotic organisms . during in vitro differentiation of the ips cells to β - like cells , the applicants determined that a refinement of the differentiation protocol ( 17 ) significantly improved the efficiency of the differentiation process . by eliminating a trypsinization step between stages 2 and 3 , a substantial increase of 80 % in the survival of the differentiating cells was seen , resulting in a more robust cell yield . the use of two different mouse models demonstrated that the ips cells can be used for treating both type 1 or type 2 diabetes via the cellular transplant route . in type 2 model mice , the mice maintained a hyperglycemic phenotype and had insulin production that was rapidly depleted as the mice aged . three key features are hallmarks of type 2 diabetes : hyperglycemia , progressive failure of insulin secretion and the development of insulin resistance . impairment in insulin secretion is likely caused by β - cell exhaustion due to a constant , unsuccessful attempt to compensate for the existing insulin resistance . in addition , β - cell function is adversely affected by glucotoxicity , generating a downward cycle of hyperglycemia leading to decreased insulin secretion , which further worsens hyperglycemia . transplantation of these animals with one dose containing as few as 200 , 000 ips derived β - like cells showed that the cells engrafted well and persistently in the liver and resulted in restored insulin secretion and normalization of glucose levels within two days post implantation . the injection of 1 × 10 6 cells provided no detectable improvement in serum insulin levels above the response obtained with the lower cell dose . the control of glycemia was maintained for approximately 20 - 30 % of the life expectency of these mice . no significant weight reduction was observed in the ips - derived cell transplanted mice as compared to the control mice , lepr db ( data not shown ). without wishing to be bound by theory , in type 2 diabetes , impairment in insulin secretion is likely caused by β - cell exhaustion due to a constant , unsuccessful attempt to compensate for the existing insulin resistance . in addition , β - cell function is adversely affected by glucotoxicity , generating a downward cycle of hyperglycemia leading to decreased insulin secretion , which further worsens hyperglycemia . applicants hypothesize that transplanted insulin producing ips cells are able to compensate for the progressive failure of endogenous islet cells by secreting insulin , thus controlling blood glucose levels so that endogenous β - cell damage and destruction by glucotoxicity can be avoided and the islet cells in the pancreas can replicate ( fig1 ). therefore , the progression of type 2 diabetes begins a loss of insulin secretion inducing a hyperglycemic condition with concomitant glucotoxicity . insulin resistance follows shortly thereafter , and feeds back into the worsening condition as described above . therefore , in some embodiments , insulin producing ips cells will be transplanted at an early stage ( 3 weeks in the case of mice ) of the disease . while in other embodiments , insulin producing ips cells will be transplanted later in the disease progression ( 5 months in the case of mice ) after resistance is well entrenched . in one aspect , cells are provided where the cells are induced pluripotent stem cells ( ips ) and secrete insulin . in some embodiments , the cells are human cells . in some embodiments , the ips cells are derived from fibroblasts . in some embodiments , the ips cells are derived from adult somatic cells wherein generation of multi - lineage progenitors is performed without trypsinization ( see fig1 . stage 2 ). in some embodiments , the ips cells can secrete insulin for at least about 20 days , at least about 30 days , at least about 8 weeks , at least about 3 months , at least about six months , at least about nine months , at least about one year or at least about two years . in another aspect , the ips cells can include microcarriers comprising a matrix being of a size to permit the aggregation and attachment of the ips cells to the matrix to assist in the engrafting of the ips cells . such matrices are known in the art , and can include but are limited to , charged and uncharged particles and can include any additional extracellular matrix components known in the art capable of supporting growth of stein cells . examples of components known in the art capable of supporting growth of stem cells can include , but are not limited to , polysaccharides , proteins , protoglycans , glycoproteins , glycosaminoglycans , fibrous proteins such as elastin , fibronectin , laminin , and collagen . in another aspect , methods of treating diabetes are provided comprising administering to a diabetic subject induced pluripotent stem cells ( ips ) that secrete insulin . in some embodiments , the diabetic subject is human . in some embodiments , the diabetic subject has type 1 diabetes . in some embodiments , the diabetic subject has type 2 diabetes . in some embodiments , the diabetic subject is still responsive to insulin . in some embodiments , the diabetic subject is resistant to insulin . in some embodiments , the ips cells administered are autologous ( i . e ., derived from the diabetic subject ). in some embodiments , at least about 200 , 000 ips cells are administered . in some embodiments , the ips cells are derived from adult somatic cells wherein generation of multi - lineage progenitors is performed without trypsinization ( see fig1 , stage 2 ). in some embodiments , the ips cells can secrete insulin for at least about 20 days , at least about 30 days , at least about 8 weeks , at least about 3 months , at least about six months , at least about nine months , at least about one year or at least about two years . in another embodiment , the ips cells are α - cells that synthesize and secrete glucagon . in some embodiments , the ips cells administered to a subject are a mixture of u - cells and β - cells . in some embodiments , the ips cells are administered by engrafting to liver parenchyma via intraportal vein injection . one of skill in the art will recognize that insulin producing ips cells can be administered , e . g ., by injection , at other sites . for example , insulin producing ips cells can be injected into the testicles or sub - renal capsule . in yet another aspect , methods of generating induced pluripotent stem cells ( ips ), as well as the ips cells produced by these methods , are provided that include transforming adult somatic cells with a vector that transiently expresses transcription factors that induce pluripotency . in some embodiments , the adult somatic cell is a human cell . in some embodiments , the adult somatic cell is a fibroblast . in some embodiments , the vector can not replicate in the transformed cell . in some embodiments , the vector can not integrate into the cell &# 39 ; s chromosome . in some embodiments , the vector is derived from a baculovirus . in some embodiments , the transcription factors are selected from oct4 , lin28 , nanog and / or sox2 . while not limited to , examples of said transcription factors can be found expressed as genbank accession numbers abf29403 . 1 or adw77327 . 1 ( oct4 ), aah28566 . 1 ( lin28 ), aap49529 . 1 ( nanog ) and np — 003097 . 1 ( sox2 ). one skilled in the art would appreciate that polynucleotides encoding said transcription factors can vary depending on the degenerate nature of the genetic code . one skilled in the art would further appreciate that homologs of said transcription factors having a percent identity , for example , 99 %, 98 %, 97 %, 96 %, 95 %, 90 %, 85 %, 80 %, 75 %, 70 %, can served as functional equivalents in the generation of ips cells . percent identity can be determined by aligning two sequences to be compared , determining the number of identical residues in the aligned portion , dividing that number by the total number of residues in the sequence that provides the basis for comparison , and by multiplying the result by 100 . a percentage identity can also be determined with reference to a specified region of a polypeptide against another polypeptide or region thereof . to determine percent identity , sequences can be aligned using methods and computer programs identifiable by a skilled person . “ sequence alignment ” indicates the process of lining up two or more sequences to achieve maximal levels of identity ( and , in the case of amino acid sequences , conservation ) for the purpose of assessing the degree of similarity . numerous methods for aligning sequences and assessing similarity / identity are known in the art such as , for example , the cluster method , wherein similarity is based on the megalign algorithm , as well as blastn , blastp , and fasta [ 9 , 10 ]. when using all of these programs , the preferred settings are those that results in the highest sequence similarity . ips cells as described herein can be generated in any way as known in the art , which can include , but is not limited to , transfection of exogenous stem cell - inducible genes and / or the channeling of the protein products of such genes into a non - pluripotent stem cell . introduction of suicide genes into stem cells to control the stem cell fate while stem cells , including ips cells , could be used to treat of a variety of diseases , including diabetes , or tissue repair , they carry oncogenic risks . therefore , it would be desirable to be able to control stem cell fate after they are introduced into the body . a fate - controllable stem cell as described herein refers to a stem cell whose fate can be controlled after transplantation of the stem cell after it is introduced into the body . in one aspect , embodiments of the invention relate generally to introduction of suicide genes into stem cells using a homologous recombination . in some embodiments , heterologous dna is integrated into the chromosome of the stem cell using homologous recombination to control stem cell fate after transplantation into the body . the heterologous dna can include , but is not limited to , promoter regions ( inducible ) that is integrated upstream of endogenous genes , coding sequences for one or more genes , drug selection cassettes , or the introduction of entire expression cassettes of suicide genes that include promoter regions and coding sequences for one or more genes . the concept of suicide genes is well known in the art . the suicide genes can be chosen from endogenous pro - apoptotic genes , for example , caspase - 9 , caspase - 8 , caspase - 2 , bh3 interacting domain death agonist ( bid ) or heterologous genes , for example , herpes simplex virus - 1 thymidine kinase ( hsv - 1tk ). one skilled in the art would understand that a variety of suicide genes can be used to control the fate of the stem cells after transplantation . the suicide genes can be controlled by any inducible promoter as is known in the art , including , but not limited to , the tet - on ( clonetech , mountain view , calif . ), which is induced by the tetracycline derivative doxycycline , or by a homodimerization system . the induced suicide genes can be introduced into any sites on the chromosome , but non - functional regions in the chromosomes are preferred . homologous recombination itself occurs commonly during the process of meiosis in eukaryotic systems . the process involves the alignment of highly similar dna sequences in chromosomes , and the exchange of dna sequences between the dna in each of the sister chromosomes . the complex series of molecular interactions is simply defined as “ cross - over ” ( fig2 a ). when these sequences are aligned , breaks in the double strand of dna facilitate the swapping of genetic material . two homologous sequences flanking a non - homologous sequence can be used to introduce a foreign dna fragment to the genomic dna ( fig2 b ). this strategy has been used extensively for gene knock - in or knock - out in mice . while there are several potential advantages of this system , the main one is the elimination of the foreign or heterologous dna randomly inserting into the dna . similarly , because homologous recombination requires highly similar stretches of dna sequence , one can be relatively certain of the location of the integrated insert and its copy number ( one or two as compared with 3 - 6 for retroviral delivery ). the present invention is further defined in the following examples . it should be understood that these examples , while indicating preferred embodiments of the invention , are given by way of illustration only . from the above discussion and these examples , one skilled in the art can ascertain the essential characteristics of this invention , and without departing from the spirit and scope thereof , can make various changes and modifications of the invention to adapt it to various uses and conditions . normal fibroblasts from green fluorescence protein ( gfp ) transgenic mice ( c57bl / 6 - tg ( ubc - gfp ) 30scha / j stock # 004353 ) ( jackson laboratory , bar harbor , me .) were transduced with 4 retroviral transcription factors as described by yamanaka et al . ( 12 , 13 ). the newly generated undifferentiated mouse ips cells were maintained on mitomycin - c treated mouse embryo fibroblast feeder layers in es medium containing knockout dmem ( kdmem , invitrogen , carlsbad , calif . ), 15 % es qualified fetal bovine serum ( fbs ) ( paa , ontario , canada ), 2 mm l - glutamine ( invitrogen ), 1 × 10 − 4 m nonessential amino acids ( invitrogen ), 1 × 10 − 4 m 2 - mercaptoethanol ( sigma , st . louis , mo . ), 1 × pen / strep ( invitrogen ) and 12 . 5 ng / ml lif ( chemicon , billerica , mass .). cultures were passaged using 0 . 25 % trypsin ( invitrogen ) at a 1 : 3 - 1 : 6 split ratio every 3 - 4 days . mouse embryo fibroblast feeder cells ( millipore , billerica , mass .) were maintained in dmem high glucose supplemented with 10 % certified fbs ( invitrogen ) and 1 × pen step . the 3 - stage differentiation protocol of ips cells into β - cells was adapted from the differentiation of embryonic stem ( es ) cells into insulin - producing cells by wobus et a with modification ( 17 ). at stage 1 , embryoid bodies ( eb ) were generated from ips cells . ips cells attached to feeder layers in 10 cm tissue culture dishes were washed one time with 1 × pbs ( invitrogen ) to remove residual serum . the cells were treated with 0 . 25 % trypsin for 30 seconds , trypsin was removed and cells were disrupted by manually scraping the culture dish with a sterile cell scraper ( bd biosciences ). eb medium ( es medium without lif ) was added to the cells and the cells were further disrupted into single cells and small pieces by pipetting up and down . the cell suspension was centrifuged at 300 g for 5 min . the supernatant was removed and cell pellet was resuspended into 10 ml of eb medium . to deplete ips cells of unwanted feeder cells , cells were plated onto a t75 tissue culture flask and the feeder cells were allowed to attach for 30 - 45 minutes at 37 ° c . in a humidified co 2 incubator . the feeder - depleted cells were then collected and centrifuged at 300 g for 5 min . the cell pellet was resuspended in eb media at 6000 cells / ml and plated as drops on 15 cm petri dishes at 18 - 22 rows of drops per plate . plates were gently flipped to invert drops and incubated at 37 ° c . incubator for 2 days . the ebs were collected and pooled by gently swirling plates with 1 × pbs and transferred to a 50 ml conical tube . pooled ebs were allowed to settle by gravity ( about 10 min . ), resuspended in 10 ml eb medium , transferred to 10 cm non - adherent petri plates and incubated for 3 more days . at stage 2 , ebs were collected and transferred to adherent culture at 10 - 15 ebs / 10 cm tissue culture dish to induce multi - lineage progenitors . for rt - pcr , immunofluorescence staining and elisa assay , 1 - 3 ebs were transferred into each well of 12 - well plates . the ebs cultured in eb medium were differentiated for 9 days , with media changes every 3 - 4 days . finally , at stage 3 , ebs were induced to further differentiate into β - like cells for 7 - 20 days . eb medium was replaced with β - cell selective differentiation medium consisting of dmem / f12 supplemented with 10 % knockout replacement serum , 20 nm progesterone ( sigma , st . louis , mo . ), 100 μm putrescine ( sigma ), 1 μg ml - 1 laminin ( sigma ), 10 mm nicotinamide ( sigma ), 1 × its premix containing insulin , transferrin and selenic acid ( bd biosciences ), 1327 media supplement ( invitrogen ), and 1 × pen / strep . after day 6 in selective medium , cells were trypsinized and replated into new culture dishes . again , media was changed every 3 - 4 days throughout stage 3 . stage 3 , day 7 , 13 , and 20 ips cell derived β - like cells grown to ˜ 80 - 90 % confluence in 12 - well multiwell plates were washed three times with 1 × pbs . the wash was removed , 500 μl of krbh solution ( krbh : 129 mm nacl , 4 . 8 mm kcl , 2 . 5 mm cacl 2 , 1 . 2 mm kh 2 po 4 , 1 . 2 mm mgso 4 , 5 mm nahco 3 , 10 mm hepes , 0 . 1 % ( wt / vol ) bsa ) with 2 . 5 mm glucose was added and the cells were incubated at 37 ° c . in a humidified co 2 incubator for 90 min . the cells were then washed 3 × with pbs to remove residual insulin produced during the initial induction . krbh solution containing 5 mm , 10 mm , 20 mm , 30 mm or 40 mm glucose was added and the cells were returned to the incubator for 90 min . supernatants were collected and saved , the cells were again washed 3 × with 1 × pbs , and 500 μl of krbh solution ( no glucose ) was then added to the cells to test whether the β - like cells continue to produce insulin in the absence of glucose . the levels of insulin produced by in vitro derived cells were measured using an elisa based insulin assay ( mercodia , upsala sweden ) performed according to manufacturer &# 39 ; s instructions . prior to cellular transplantation , stage 3 , day 7 ips cell derived β - like cells from two 10 cm culture dishes were dissociated into single cell suspensions by trypsinization . the cells were washed once with 1 × pbs and stained with ssea - 1 antibody ( 1 : 200 antibody dilution in 5 ml 1 × pbs , see table 2 ) for 30 min . at rt . ssea - 1 is a surface antigen found on stem cells and is used extensively for excluding cells bearing stem cell markers to reduce the chance of teratoma formation after transplantation . the cells were pelleted by centrifugation at 300 g for 5 min . the cells were stained with secondary antibody ( 1 : 1000 antibody dilution in 5 ml 1 × pbs , table 2 ) and incubated for 15 min , at rt . the cells were washed three times with 1 × pbs and resuspended in β cell differentiation medium at a concentration of 2 . 5 - 5 × 10 6 cells / ml medium . gfp positive / ssea1 negative cells were sorted using an icyt reflection cell sorter . the sorted cells were washed once with 1 × pbs and resuspended into basal dmem / f12 at a concentration of about 600 cells / μl prior to transplantation . all animal procedures were approved by applicants &# 39 ; institutional animal committee . the type 2 diabetes mellitus mouse model , lepr db , d57blks ; dock7 m , dba / j was purchased from jackson laboratories ( stock # 000642 ). we used genotyped mice homozygous for the spontaneous diabetes mutation ( lepr db ), which causes these mice to become obese at age 3 - 4 weeks . the mice also become hyperglycemic at 3 - 4 weeks of age due to unregulated glucose metabolism and severe destruction of insulin - producing pancreatic β cells . as the mice continue to age , they become resistant to exogenous insulin therapy ( 21 , 22 ). for the type 1 diabetes mellitus mouse model , c57bl6 mice were given a single dose of 180 mg / kg of streptozotocin , stz , ( sigma ) via intraperitoneal injection . stz is a chemical toxic to pancreatic β cells . the diabetic phenotype was confirmed by hyperglycemic readings . this diabetic condition was allowed to stabilize for 10 days prior to cellular transplantation . for these studies , stz - treated mice with fasting glucose concentrations of & gt ; 400 mg / dl were used . insulin tolerance test was performed as previously described ( 23 ). untransplanted type 2 female diabetic mice ( fasted for 6 hours ) at 4 , 8 and 16 weeks of age ( n = 3 per age group ) were given 0 . 75 u / kg of human insulin ( novo nordisk , clayton , n . c .) via intraperitoneal injection . blood glucose readings were obtained at 30 , 60 , and 90 minutes post injection via the tail vein using a hand - held lifescan onetouch glucometer ( johnson & amp ;. johnson , new brunswick , n . j .). cellular transplantation was done by portal vein injection using previously established protocols ( 24 ). under anesthesia , 200 , 000 facs - sorted gfp positive / ssea1 negative stage 3 ips cell derived β - like cells in 300 μl basal dmem / f12 medium were injected into the hepatic portal vein using a 32 gauge hamilton syringe . intraportal vein injection of in vitro derived β - like cells is an efficient way to directly engraft the cells into the murine hepatic sinusoids . the engrafted cells stably expressed green fluorescence protein enabling us to recognize and distinguish the transplanted cells from other hepatic cell types . glucose readings were obtained every 2 - 3 days post transplantation from fasted mice via the tail vein using a hand held glucometer . 5 μl of blood were collected from three transplanted mice with normal glucose readings 4 weeks post transplantation ( n = 3 ) and the samples were sent to an independent company ( diabetes technologies , inc .) to obtain the hemoglobin alc readings . statistical differences between groups ( p & lt ; 0 . 05 ) were determined by excel &# 39 ; s one - tail student t - test . p value less than 0 . 05 was considered statistically significant . total rna was isolated from undifferentiated ips cells and differentiated β - like cells using trizol reagent ( invitrogen ) according to manufacturer &# 39 ; s instruction . 2 μg of total rna was used for reverse transcription with cdna archive kit ( applied biosystems , foster city , calif .) according to manufacturer &# 39 ; s instruction . pcr reactions were performed using platinum pcr supermix high fidelity ( invitrogen ) according to manufacturer &# 39 ; s instructions . pcr primer sequences for endogenous / total retroviral transcripts ( 8 ) and specific pancreatic markers ( 25 ) are provided table 1 . evaluation of proliferation and differentiation of ips cells was tested via teratoma formation in nude mice . ips cells were dissociated with 0 . 25 % trypsin for 2 minutes and then manually scraped using a cell scraper , collected and transferred into 15 ml conical tube , centrifuged at 1500 rpm for 5 minutes , and the cell pellet was resuspended in eb differentiation medium . 2 × 10 6 dissociated ips cells in 100 μl dmem high glucose were mixed with an equal volume of geltrex ( invitrogen ) and injected subcutaneously into flanks of 2 nude mice ( taconic , hudson , n . y . ), thirty days post injection , teratomas were removed , dissected and fixed with formalin ( fisher scientific , pittsburgh , pa .). paraffin - embedded tissues were sectioned and stained with hematoxylin and eosin . cell cultures were washed three times with 1 × pbs and fixed for 15 - 30 minutes at room temperature ( rt ) in 4 % paraformaldehyde in pbs ( emd chemicals , gibbstown , n . j .). they were then washed 3 × in pbs , and permeabilized with 0 . 2 % triton x ( fisher scientific , pittsburgh , pa ) in pbs for 15 - 30 minutes at rt . for surface markers such as ssea - 1 , the permeabilization step was omitted . the cells were again washed 3 × with pbs and blocked for 15 - 30 min . in 5 % bovine serum albumin ( fisher scientific ) in pbs at rt . primary : and secondary antibodies are listed in table 2 . primary antibodies in 1 % bsa were added to the cells and incubated for 2 hrs . at 37 ° c . the cells were washed again 3 × with pbs , and secondary antibodies diluted in pbs were added and incubated for 1 hour at 37 ° c . the cells were washed again and the nuclei were counterstained with 1 mg / nil 4 ′, 6 - diamidino - 2 - phenylindole dapi ( sigma , st . louis , mo .) for 10 minutes followed by a final 3 washes in pbs . liver tissues were prepared by either of two methods , paraffin embedded tissues were used for the immunohistochemistry shown in fig6 , panels a , b , and c . frozen sections were prepared for the fluorescent images in in fig6 , panels d , e and f . livers were isolated and fixed in 10 % neutral - buffered formalin and embedded in paraffin . 5 micron sections were cut , placed on pre - cleaned slides , deparaffinized using xylene , and then hydrated using an alcohol gradient . ihc staining using rabbit anti - mouse gfp primary antibody was performed using rmr622g rabbit hrp polymer kit ( biocare , concord , calif .). deparaffinized slides were placed in 1 × rodent decloaker solution and heated to 125 ° c . for 30 min . using biocare &# 39 ; s decloaking chamber . the slides were removed and washed 3 × with deionized water . rodent block m was applied for 30 min . to reduce both non - specific background staining and endogenous mouse igg . the slides were washed 2 × with pbs , and gfp primary antibody in 1 % antibody dilution buffer ( 1 : 100 ) was applied and incubated overnight at 4 ° c . in a humidified chamber . the slides were washed with pbs and rabbit hrp polymer solution was applied for 20 nun . the slides were again washed in pbs and betazoid dab chromogen reagents ( 1 drop of betazoid dab chromogen added to 1 ml of betazoid dab substrate buffer ) was applied for 5 min . the slides were then rinsed with deionized water and treated with hematoxylin for 30 seconds . the slides were washed with warm alkaline tap water to blue the nuclei . the slides were then dehydrated , cleared and coverslipped . cytoplasm appears brown and nuclei appear blue . 5 μm frozen liver sections were placed on positively charged slides and allowed to air dry at rt for 5 min . before fixing with 3 % formaldehyde / methanol for 15 min . at rt , followed by 5 min . incubation in methanol at − 20 ° c . the sections were washed 2 × with pbs for 5 min . and incubated for 10 min . at rt in 3 % hydrogen peroxide diluted in methanol . the sections were washed 2 × with pbs for 5 min and then incubated with 5 % bsa blocking buffer for 1 hour at rt . the blocking buffer was removed and 400 μl of insulin monoclonal primary antibody ( 1 : 100 , see table 2 ) in 1 % bsa as added and incubated overnight at 4 ° c . the antibody solution was removed and the sections were washed 3 × with pbs for 5 min . each . secondary antibody diluted 1 : 1000 with pbs was then added and incubated for 30 min . at rt . the secondary antibody solution was removed and the sections were washed 3 × in pbs for 5 min . each . nuclei were stained with 1 mg / ml dapi for 10 min . and sections were washed 3 × with pbs for 5 min . each . images were immediately captured on a fluorescent microscope . reprogramming of normal mouse skin fibroblasts into induced pluripotent stem cells normal fibroblasts from green fluorescence protein (( t ) transgenic mice were transduced with 4 retroviral transcription factors ( 10 - 12 ), using methods that we have previously described ( 26 ). in this current study , two ips subclone colonies were picked and expanded at 20 days post transduction ( fig8 ). mrna expression profiles of these two ips subclones show upregulation of the endogenous stem cell markers oct4 , sox2 , klf4 , and c - myc similar to those of embryonic stein ( es ) cells ( fig9 ). there was minimal expression of exogenous viral transgenes exhibited . immunofluorescence staining , used to access surface and intracellular antigens present in these ips cells , was positive for the well established pluripotent markers , oct4 , sox2 , nanog and ssea - 1 ( fig1 ). to determine their capacity for proliferation and differentiation , roughly 2 × 10 6 dissociated ips cells were injected subcutaneously into the flanks of three nude mice . thirty days post injection , a teratoma had formed in all injected mice , suggesting full proliferative capacity of the injected cells . differentiation was established via hematoxylin and eosin staining of tissue from the teratomas , showing the presence of cell types belonging to the three germ layers : ectoderm ( skin ), endoderm ( gut - like ) and mesoderm ( cartilage ) ( fig1 ). the morphology , mrna expression profile , immunofluorescence staining and teratoma formation confirm the in vitro generation of es - like ips cells . ips cells were driven to undergo a 3 stage differentiation into β - like cells which can produce insulin using modification of a previously published protocol for es cells ( 17 ). the time line of this differentiation is shown in fig1 . at stage 1 , the es - like ips cell cultures were dissociated into single cells and placed in suspension culture where they form embryoid bodies ( fig1 , top panel ). after 5 days in suspension culture , the embryoid bodies were returned to adherent culture to undergo further differentiation for 9 days . during this time , cells migrated from the attached spheroids ( fig1 , center panel ). after 9 days , the media was replaced with selective media containing laminin , insulin , nicotinamide , selenic acid , transferrin , progesterone and knock - out replacement serum , marking the beginning of stage 3 . during the ensuing 20 days the cells continued to differentiate ( fig1 , bottom panel ). the prior protocol ( 17 ) had included enzymatic detachment of the differentiated cells at the end of stage 2 . this procedure was detrimental to the differentiating pancreatic cells , with only 10 % of the cells surviving and reattaching after trypsinization . therefore , after 9 days , replaced the stage 2 medium with stage 3 selective medium without trypsinizing , and cell survival increased by 80 %. during stage 3 differentiation , multi - lineage cells were seen to he present at day 6 of selective media treatment ( fig1 , column 2 ). at this time , the cultures were trypsinized and replated onto new dishes in fresh selective medium . on day 13 , numerous clusters began to form ( fig1 , column 3 ). by day 20 , large cell clusters were evident ( fig1 , colummn 4 ). gfp was highly expressed throughout the differentiation process . cellular levels of insulin and nestin were analyzed at stage 3 by immunofluorescence staining . at day 7 , both insulin and nestin are expressed ( fig1 ). expression of nestin peaked at day 13 ( fig1 ) and decreased by day 20 ( fig2 a ). insulin was expressed at low levels at days 13 and 20 consistent with previous reports ( 7 ). mrna analysis revealed expression of several specific markers of in vitro pancreatic β cell differentiation as shown in fig2 b . the definitive endoderm markers , sox17 and hnf3b ( foxa2 ) were co - expressed in stage 2 and were detectable up to stage 3 . co - expression of cytokeratin 19 and nestin , suggesting the presence of multi - lineage progenitors , is seen during stages 1 through 3 . co - expression of pdx1 and hnf3b , indicative of pancreatic endoderm or epithelium generation , is seen in stages 1 and 2 . ngn3 , highly expressed in all endocrine progenitors ( 18 ), is co - expressed with pdx1 in stages 1 and 2 , indicating further commitment of the differentiated cells to the endocrine lineage . pax4 , pax6 and islet - 1 are important transcription factors controlling endocrine cell differentiation and were detected in all 3 stages . islet - 1 is , however , downregulated early in stage 3 as the cells progressed into a mature , differentiated phenotype . there are 5 endocrine cell types , α , β , δ , pp and ε cells , which produce the hormones glucagon , insulin , somatostatin , pancreatic polypeptide ( pp ) and ghrelin , respectively . insulin expression was detected as early as stage 1 and remained upregulated until stage 3 . furthermore , islet amyloid polypeptide ( iapp ), secreted by pancreatic β - cells at the same time as insulin ( 19 , 20 ), was also highly expressed in stages 1 to 3 . expression of somatostatin and pp was only evident in stage 1 . glucagon was either undetectable or had low expression in all of the differentiation stages ( data not shown ). ghrelin was not tested . amylase positive - acinar cells were detected in stages 1 and 2 but were undetectable at the selective differentiation stage 3 . glucose responsiveness of ips derived β - like cells was tested in vitro by exposure of glucose starved stage 3 cells to 5 glucose concentrations for 90 minutes . as shown in fig3 , day 20 differentiated cells responded to glucose in a dose - dependent manner . at the lower glucose concentrations ( 5 mm and 10 mm ), the differentiated cells were marginally responsive . at 20 mm glucose , insulin production peaked , at ˜ 8 fold higher than readings from 5 mm and 10 mm glucose - treated cells . at 30 mm and 40 mm , the insulin readings begin to decrease again . following glucose induction , the cells were washed thoroughly to remove residual insulin , and then re - incubated with basal krbh solution for 90 minutes . as shown in the same graph , no insulin was produced when glucose is removed , suggesting that the in vitro derived β - like cells will only produce insulin in response to glucose treatment . insulin was also produced by day 7 and 13 differentiated cells when exposed to glucose treatment ( data not shown ). type 2 diabetic mouse model phenotypic tests reveal hyperglycemia , abnormal insulin levels , and resistance to exogenous insulin therapy the type 2 diabetes mouse model ( lepr db , c57blks ; dock7 m , dba / j ) used herein shows depleted insulin production as the mice age , while maintaining a hyperglycemic phenotype . by the age of 3 - 4 weeks diabetic mice demonstrated hyperglycemia after 6 - 8 hours of fasting , with glucose concentrations greater than 300 mg / dl as compared to the normal c57bl6 mouse strain used as a control ( fig4 a ). at 3 weeks of age , diabetic mouse blood glucose concentrations ( n = 20 ) increased ˜ 2 . 6 fold as compared to normal controls . at 4 - 5 weeks of age , the glucose concentration of the diabetic mice increased ˜ 3 . 8 fold . after 6 weeks of age , the hyperglycemia is very severe and glucose levels are dangerously high , at & gt ; 600 mg / dl . insulin levels in serum samples collected from 4 week old diabetic mice ( n = 20 ) ( fig4 b ), were highly variable , ranging from 0 . 64 to 15 . 32 the mean insulin level was ˜ 17 fold higher than that of normal control mice n = 3 ). as the diabetic mice continued to age , insulin levels decreased . diabetic mice at 8 weeks ( n = 3 ) had ˜ 2 . 6 fold lower insulin levels and at 13 weeks ( n = 3 ) had ˜ 3 . 6 fold lower insulin levels than the normal controls . insulin resistance also developed as the mice aged ( fig4 c ). exogenous insulin , injected into 4 week old ( n = 3 ) diabetic mice led to a drop in fasting glucose , occurring between 0 to 30 minutes post injection . with increased age the mice , at 8 weeks ( n = 3 ) and 16 weeks ( n = 3 ), showed minimal response to the injection , indicating resistance to insulin . ips derived β - like cells were able to engraft in liver parenchyma and ameliorate hyperglycemia in the type 2 diabetes mouse model ips derived insulin - secreting β - like cells were isolated by fags sorting to yield gfp positive / ssea1 negative cells which were transplanted into diabetic mice ( n = 30 ) by intraportal vein injection . fasting glucose measurements , begun 2 days post - transplantation , are shown in fig5 a . all 30 transplanted mice ( exhibiting 100 % engraftment efficiency ) exhibited normal glucose concentrations with readings comparable to c57bl6 normal controls ( n = 3 ). three diabetic mice transplanted with gfp negative / ssea1 negative negative control cells , ( feeder cells ), remained hyperglycemic . the unengrafted diabetic counterparts ( n = 20 ) remained hyperglycemic as well . at 4 weeks post transplantation , 20 of the original 30 β □- like cell transplanted mice survived and maintained normal glycemic control . at 8 weeks post transplantation , 15 of these mice were still alive and normoglycemic . data to week 12 showed continued maintenance of normal glucose levels in the treated mice . the 50 % mortality rate in the transplanted mice ( fig1 ) appeared to be the result of complications ( i . e ., thrombosis ) resulting from the surgical access of the portal vein . however , all of the mice survived for at least 1 week post - transplantation and exhibited normal glucose levels during that time . two mice had relapsed into hyperglycemia after maintaining regulated glucose level for 8 weeks . these 2 mice appeared to have re - developed insulin resistance ( fig1 ). engrafted ips derived β - like cells were able to produce insulin in vivo , and normalized hemoglobin alc levels the amelioration of hyperglycemia in the type 2 diabetic mouse model transplanted with ips cell derived β - like cells occurred concomitantly with an increase in in - vivo insulin concentration measured by mouse insulin elisa assays . by 21 - 56 days post transplantation β - like cell transplanted mice had ˜ 4 . 35 fold increase in insulin levels as compared to untreated mice ( p & lt ; 0 . 05 ) ( fig5 b ). insulin levels in the treated mice were somewhat higher than those of the untreated controls . hemoglobin alc tests to measure the amount of glucose attached to the hemoglobin of red blood cells were done 4 weeks post transplantation ( fig5 c ) the hemoglobin alc levels of untreated diabetic mice ( n = 3 ) were ˜ 2 . 4 fold higher than normal c57bl6 controls ( n = 3 ). transplanted mice with normal glucose readings 4 weeks post transplantation ( n = 3 ) had significantly improved the hemoglobin alc readings ( a better than 70 % improvement over the untreated mice ). intraportal vein injection of in vitro derived β - like cells is an efficient way to directly engraft the cells into the murine hepatic sinusoids . at 7 days and 4 weeks post transplantation , 3 mice were sacrificed to obtain tissues for immunohistochemical and immunofluorescence analysis to assess the distribution of engrafted cells in the liver . the engrafted cells stably expressed green fluorescence protein allowing recognition and the ability to distinguish the transplanted cells from other hepatic cell types . as shown in fig6 , cells were able to engraft stably into the liver parenchyma of the diabetic mouse model . spindle - shaped gfp positive cells detected by anti - gfp antibodies ( brown ) were evenly distributed throughout the microscopic liver sections of 7 day treated mice ( panels b and c ). minimal gfp positive cells were detected in lungs and spleen ( data not shown ). liver sections from the c57bl6 normal mouse strain were used as negative controls ( panel a ). engrafted β - like cells co - express insulin and gfp 7 days post transplantation as detected by immunofluorescence ( panels d - f ). normoglycemia in stz treated mice after transplantation with ips cell derived β - like cells to test whether the in vitro derived β - like cells are functional in an environment where islet cells are severely depleted , a model of type 1 diabetes , in vitro derived , insulin - secreting β - like cells were transplanted via intraportal vein injection into stz - treated mice with glucose concentrations of & gt ; 400 mg / dl ( fig7 ). at 2 days post transplantation with β - like cells , the glucose levels of the stz - treated mice ( n = 2 ) became normal , with glucose concentrations of 160 +/− 5 mg / dl . untransplanted stz - treated mice ( n = 3 ) maintained hyperglycemia with glucose concentrations & gt ; 400 mg / ell . glucose readings of treated mice from day 2 up to day 30 post transplantation continued to be normal while the untransplanted mice remained hyperglycemic . one difficulty with the clinical use of conventionally generated ips cells is the introduction of a known oncogene , namely c - myc , to the genome . thus , ips cells were generated that was devoid of this potent transcription factor . without c - myc , the efficiency of faithful reprogramming is observed to be much lower , but we were still able to derive ips cells using retroviral infection using only oct4 , sox2 , and klf4 . further characterization of these cells revealed that they express typical stem cell markers , such as ssea - 1 , nanog , and oct4 , as well as novel markers such as sall4 ( fig1 ), suggesting that these are indeed true ips cells . to ensure that these 3 factor ips ( 3f - ips ) cells could give rise to all germ layers of the developing embryo , these cells were injected into the flanks of nod - scid mice . genuine stem cells , under these conditions , will give rise to a teratoma composed of endodermal , ectodermal , and mesodermal cell lineages . indeed , 3f - ips cells gave rise to teratomas composed of all three germ layers within two weeks following injection . these data clearly suggest that 3f - 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