Patent Description:
Subjects with type <NUM> diabetes lose most of their beta cell mass and depend on exogenous insulin for control of blood glucose levels. The life-long management of diabetes is imperfect, can result in complications and is a tremendous burden to the affected. Because beta cells are generated very slowly in mature islets, an exogenous source of beta cells could be therapeutically useful, not only for type <NUM> diabetes, but also for all other forms of diabetes, including type <NUM> diabetes and monogenetic forms of diabetes. Transplantation of islets from pancreatic organ donors can restore physiological regulation of blood glucose. However, obtaining islets from organ donors is logistically complex, and limited by the number of donors. In general, only a small number of patients with diabetes are treated using this approach.

Human pluripotent stem cells provide a potentially unlimited source of beta cells to replace the missing beta cells and restore glucose homeostasis. In animal models, such proof-of-principle experiments have been successful: human beta cells differentiated from stem cells can protect mice from diabetes upon transplantation. See Sui et al. However, there is a need for stable fully functional pancreatic beta cells differentiated from stem cells, as well as other cell types differentiated from stem cells.

A major obstacle in stem cell differentiation of all cell types is the generation of terminally differentiated and fully functional differentiated cells. This is a major obstacle for the generation of cells suitable for basic research, drug testing, and cell replacement suitable for therapeutic use.

Cell cycle plays an active role in developmental decisions. The duplication of the DNA is a fundamental requirement for cell proliferation in both embryonic development and in adult organs. Cell proliferation during development determines the number of cells in the adult organ and is limited by the number of embryonic progenitors in the pancreas. Proliferation of beta cells in the developing human pancreas occurs primarily during embryogenesis, and declines after birth. Indeed proliferation in the adult beta cells is essentially absent (Jennings et al. , <NUM>; Kulkarni et al. During terminal differentiation, many cell types, including beta cells, neurons and muscle cells undergo cell cycle exit to reach full maturity and adopt full functionality (Ameri et al. , <NUM>; Hardwick and Philpott, <NUM>; Walsh and Perlman, <NUM>).

<CIT> discloses compositions and methods of producing mammalian cell populations that include a high proportion of definitive endoderm cells, posterior foregut-like progenitor cells, pancreatic progenitor cells and/or pancreatic beta cells.

<CIT> discloses compositions and methods of producing mammalian cell populations that include a high proportion of pancreatic beta cells. Such cell populations are useful for treatment of diabetes. Also discloses are materials and methods for the direct differentiation of stem cells, such as embryonic stem cells, into functional pancreatic beta cells.

Based upon this knowledge, the inventor published on the concept that cell types use changes in the cell type specific DNA replication program to activate DNA replication checkpoints, and enable cell cycle exit and terminal differentiation (Georgieva and Egli, <NUM>). Thus, it follows that these changes may be induced to enable terminal differentiation and functional maturation of stem cell derived cells.

The current invention is a novel method for providing mature differentiated cells suitable for transplantation and grafting, wherein the graft function is maintained, as well as cells suitable for basic research and testing.

The current invention is a method of inducing cell cycle exit and terminal differentiation of cells undergoing differentiation from stem cells into pancreatic beta cells as defined in claim <NUM>. The method comprises:.

In some examples, the cells are contacted or incubated with the agents at about day <NUM> of the differentiation protocol, for about five days to about one week to about two weeks. In some examples, the cells are contacted or incubated with the agents at about day <NUM> of the differentiation protocol, for about one week to about two weeks. In some examples, the cells are contacted or incubated with the agents at early stage of differentiation of progenitor cells to mature differentiated cells which is from about day <NUM> to about day <NUM> of the entire differentiation protocol. In some examples, the cells are contacted or incubated with the agents at the late stage of differentiation of progenitor cells to mature differentiated cells which is from about day <NUM> to about day <NUM> of the entire differentiation protocol. In some examples, the cells are contacted or incubated with the agents for the entire duration of differentiation of progenitor cells to mature differentiated cells which is from about day <NUM> to about day <NUM> of the entire differentiation protocol. In some examples, the cells are contacted or incubated with the agents until about day <NUM> of the entire differentiation protocol. In some examples, the cells are contacted or incubated with the agents until the time of use of the cells, e.g., transplantation or grafting into a subject.

In some embodiments, the contacting or incubating of the cells with the various agents is accomplished by culturing the cells in media comprising the agents.

Agents which can be used in the methods of the invention include but are not limited to the following classes of molecules:.

Specific agents that induce stalling of DNA replication in a site and cell type specific manner also include PNA oligos that stably bind to DNA at specific sites of the genome.

In some embodiments, the agent is chosen from the group consisting of aphidicolin, cisplatin, ciprofloxacin, pyridostatin, E2Fi, A485, RL5a and etoposide.

In some embodiments, the pancreatic endocrine progenitor cells are at the G1 phase of the cell cycle.

In some embodiments, these specific mature differentiated cells have markers characteristic of a specific mature differentiated cell. In some embodiments, the cells are positive for cell markers C-peptide and NKX6. <NUM> (mature pancreatic cells).

Further embodiment of the current invention is a method of obtaining a pancreatic cell or cells differentiated from stem cells suitable for administering, grafting or transplanting into a subject, comprising the differentiation protocol set forth in Table <NUM>.

An additional embodiment of the current invention is a method of obtaining a substantially homogenous population of pancreatic cells differentiated from stem cells suitable for administering, grafting or transplanting into a subject, comprising the differentiation protocol set forth in Table <NUM>.

Yet an additional embodiment of the current invention is a method of inducing differentiation of stem cells into pancreatic cells suitable for administering, grafting or transplanting into a subject, comprising the differentiation protocol set forth in Table <NUM>.

In additional embodiments, the invention includes a specific mature differentiated cell or cells obtained by the methods described herein. In some embodiments, the specific mature differentiated cell or cells is chosen from the group consisting of pancreatic endocrine cells, pancreatic cells, and endocrine cells. In some embodiments, the cells are positive for cell markers C-peptide and NKX6. <NUM> (mature pancreatic cells).

For the purpose of illustrating the invention, there are depicted in drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

It is shown herein that genetic instability during DNA replication provides the cellular signals that prevent cell type transitions, and thereby stabilize the differentiated state. Thus, to induce terminal differentiation and obtain stable differentiated cells, interference with DNA replication may be performed at specific sites in the genome, or through unspecific interference with the progression of DNA replication. The current invention utilizes various agents which interfere with DNA replication and induce the terminal differentiation of mature differentiated cells which have improved function and stability.

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms.

As used herein, the term "induced pluripotent stem cells" commonly abbreviated as iPS cells or iPSCs, refers to a type of pluripotent stem cell artificially generated from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like.

As used herein, the term the terms "differentiation", "cell differentiation" and the like refer to a process by which a less specialized cell (i.e., stem cell) develops or matures or differentiates to possess a more distinct form and/or function into a more specialized cell or differentiated cell, (i.e., pancreatic beta cell).

A cell that results from this process termed herein as a "differentiated cell" and can include pancreatic endocrine cells, pancreatic cells, endocrine cells, as well as neurons, astrocytes, oligodendrocytes, retinal epithelial cells (RPE), epidermal cells, hair cells, keratinocytes, hepatocytes, intestinal epithelial cells, lung alveolar cells, hematopoietic cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, cartilage cells, bone cells, renal cells, adipocytes, chondrocytes, and osteocytes.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

With respect to cells, the term "isolated" refers to a cell that has been isolated from its natural environment (e.g., from a tissue or subject). The term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. As used herein, the terms "recombinant cell" refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

As used herein, the terms "pancreatic endocrine cell", "pancreatic cell" or "endocrine cell" are used interchangeably and denote cells found in the pancreas that secrete hormones. "Pancreatic beta cells" secrete insulin and "pancreatic alpha cells" secrete glucagon.

The term "homogenous" as used herein means of all of the same kind.

The term "substantially" as used herein means almost completely.

The terms "treat", "treatment", and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease, or reverse the disease after its onset.

The terms "prevent", "prevention", and the like refer to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development.

The term "subject" as used in this application means an animal with an immune system such as avians and mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors. Thus, the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications.

The term "patient" as used in this application means a human subject.

The term "in need thereof" would be a subject known or suspected of having or being at risk of developing a disease including but not limited to diabetes including type <NUM>, type <NUM>, and monogenetic.

A subject in need of treatment would be one that has already developed the disease. A subject in need of prevention would be one with risk factors of the disease.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject.

The term "agent" as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, drugs, biologics, small molecules, antibodies, nucleic acids, peptides, and proteins.

Standard methods in molecular biology are described <NPL>; <NPL>; <NPL>). Standard methods also appear in <NPL>, which describes cloning in bacterial cells and DNA mutagenesis (Vol. <NUM>), cloning in mammalian cells and yeast (Vol. <NUM>), glycoconjugates and protein expression (Vol. <NUM>), and bioinformatics (Vol.

The current invention is a method interfering with DNA replication to induce cell exit and terminal differentiation and thus obtain differentiated cells which have increased stability and purity, function and homogeneity of the differentiated cells, which in turn increases maturation and functionality, and reduces teratoma formation.

As shown herein the method increased the differentiation potential of cells that normally differentiate poorly (see Example <NUM>).

In the method according to the present invention, the agents which interfere with DNA replication are used to induce cell exit and terminal differentiation and thus obtain differentiated cells which have increased stability and function is a method for differentiating hPSCs into pancreatic beta cells. This method is exemplified in Example <NUM> and summarized in Table <NUM>.

In step <NUM>, the cells are contacted or incubated with fibroblast growth factor, in an amount ranging from about <NUM> ng/ml to about <NUM> ng/ml, with <NUM> ng/ml being preferred.

In step <NUM>, the cells are contacted or incubated with an agent or agents which inhibit sonic hedgehog and/or bone morphogenetic protein (BMP4). LDN193189, which inhibits both is exemplified. However, other agents which inhibit either sonic hedgehog or BMP4 or both can be used. These agents include but are not limited to vismodegib, taladegib, jervine, cyclopamine, and sonidegib. In this step, the cells are also contacted or incubated with an agent which activates the retinoic acid pathway. Retinoic acid is exemplified activator of the pathway and can be used in an amount ranging from <NUM> to <NUM>. Other agents which activate the pathway include but are not limited to Isotretinoin, Tazarotene, TTNPB, and AM80.

In step <NUM>, the cells are contacted or incubated with epidermal growth factor (EGF) in an amount ranging from about <NUM> ng/ml to about <NUM> ng/ml, with <NUM> ng/ml being preferred. Other agents which activate the protein kinase C pathway can be used in the method and include but are not limited to Bryostatin <NUM>, <NUM>, <NUM>, FR236924, PEP <NUM>, Phorbol <NUM>,<NUM>-dibutyrate, Phorbol <NUM>-myristate <NUM>-acetate, Prostratin, Pseudo RACK1, SC9, and TPPB.

In step <NUM>, the cells are contacted or incubated with thyroid hormone in an amount ranging from about <NUM> to about <NUM>, with <NUM> being preferred. The cells are also contacted or incubated with an agent or agents which inhibit Notch signaling. Dibenzazepine (DBZ) is exemplified but other agents can be used in the method including but not limited to Avagacestat, Begacestat, BMS299897, Compound E, Compound W, DAPT, DBZ, Flurizan, JLK6, L-<NUM>,<NUM>, LY450139, MRK560, and PF <NUM> hydrobromide. The cells are also contacted or incubated with an agent or agents which inhibit TGF-beta signaling. Alk5 is exemplified but other agents can be used including but not limited to A77-<NUM>, A <NUM>-<NUM>, D <NUM>, GW <NUM>, IN <NUM>, LY <NUM>, R <NUM>, Repsox, SB <NUM>, SB <NUM>, SB525334, SD <NUM>, and SM <NUM>.

This exemplary protocol of differentiating stem cells to pancreatic endocrine or beta cells includes a step where the cells are contacted or incubated with an agent or agent which interfere with DNA replication to induce cell exit and terminal differentiation (step <NUM>). As discussed below, this step can be used in any differentiation protocol to obtain mature differentiated cells with increased function and stability.

In some embodiments, the contacting or incubating of the cells with the various agents is accomplished by culturing the cells in media comprising the agents. Any media that is used for differentiating and culturing the cells of choice can be used in the methods of the invention.

As discussed, cell cycle exit is required and potentially sufficient for commitment to a specific cell lineage and also promotes functional maturation. To test this, the progression of the cell cycle was inhibited using compounds which interfere with DNA replication. The progression to S-phase can be inhibited at different stages of the cell cycle. DNA replication is licensed in the G1 phase of the cell cycle by binding to DNA with origin recognition complex and followed by recruitment of Cdc6, Cdt1 and MCM2-<NUM> sequentially to form pre-replication complex at origins. At the G1/S transition, the MCM complex with helicase activity is activated and unwinds the double strands with assistance of topoisomerase. DNA replication forks are established. After initiation, replication proceeds bi-directionally away from origins (Sclafani and Holzen, <NUM>). Inhibition of DNA replication can be achieved by blocking any of these sequential events occurring before and during replication, and by DNA damage.

As shown herein DNA replication inhibition in G1 halted cell cycle progression of pancreatic progenitors, leading to an increase in differentiation efficiency of beta cells and to greater functionality in vitro measured by glucose stimulated insulin secretion. Agents which inhibit cell cycle progression in the late G1 and inhibit entry into S-phase were the most effective in promoting beta cell differentiation, while agents that affect early G1 phase were effective but less so. See Example <NUM>.

Upon transplantation, DNA replication inhibitor treated beta cells demonstrated higher human C-peptide secretion, greater responsiveness to glucose level changes, and protected mice from diabetes without the formation of teratomas or cystic structures. See Examples <NUM> and <NUM>. Therefore, inhibition of DNA replication during stem cell differentiation toward beta cells is an efficient method to ensure consistency in the generation of beta cells, useful for cell replacement to treat diabetes.

The method of the invention has been applied to beta cells, but is not unique to beta cells, as the same cellular principles could be applied to other terminally differentiated cell types, including but not limited to neurons, astrocytes, oligodendrocytes, retinal epithelial cells (RPE), epidermal cells, hair cells, keratinocytes, hepatocytes, intestinal epithelial cells, lung alveolar cells, hematopoietic cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, cartilage cells, bone cells, renal cells, adipocytes, chondrocytes, and osteocytes.

The methods and systems set forth herein generate a defined and reproducible cell population that is fully functional upon transplantation. Furthermore, the methods and systems set forth herein provide a substantially homogenous population of differentiated cells.

Agents which inhibit cell cycle progression in the G1 phase are used in embodiments of the present invention. Agents which inhibit the cell cycle progression in the early G1 phase can be used in the embodiments of the present invention. Agents which inhibit the cell cycle progression in the late G1 and inhibit entry into the S-phase can be used in embodiments of the present invention.

It is within the skill of the art to recognize that any agent which interferes with DNA replication can be used in embodiments of the methods of the invention, in particular DNA replication inhibition in G1, and more particular inhibition in the late G1. Several agents representing many types agents of interference with DNA replication are exemplified herein and showed to an increase in differentiation efficiency of cells and to greater functionality in vitro and in vivo. These included the following:
Pyridostatin (PDS) stabilizes G-quadruplexes and arrests cell cycle (Moruno-Manchon et al. , <NUM>; Zimmer et al. , <NUM>); Cisplatin (Cis) induces DNA damage via DNA cross link and low dose arrests cells at S phase (Qin and Ng, <NUM>; Wagner and Karnitz, <NUM>); E2F inhibitor (E2Fi) inhibits the master transcription factors involved in S phase entry (Ma et al. , <NUM>; Pardee et al. , <NUM>; Rouaud et al. , <NUM>); Etoposide (Eto) is a topoisomerase inhibitor and stops the unwind of the DNA helix during replication (Cliby et al. , <NUM>; Korwek et al. , <NUM>; Nam et al. , <NUM>; Smith et al. , <NUM>); Ciprofloxacin (Cipro) inhibits MCM2-<NUM> replicative helicase at replication origin (Simon et al. , <NUM>); A485 inhibits p300, a histone acetylase, and arrests the cell cycle and inhibits p300 dependent transcription (Lasko et al. RL5a arrests the cell cycle by inhibiting replication origin licensing (Gardner et al.

It is also within the skill of the art to determine the amount of agent to be used in the methods of the invention. By way of example, aphidicolin was used in amounts of about <NUM>, <NUM> and <NUM>. However, amounts ranging from about <NUM> to about <NUM> can be used. Cisplatin was used in an amount of about <NUM>, however, a range of about <NUM> to about <NUM> can be used. Ciprofloxacin was used in an amount of about <NUM>, however, a range of about <NUM> to about <NUM> can be used. Pyridostatin was used in an amount of about <NUM>, however, a range of about <NUM> to about <NUM> can be used. E2F inhibitor was used in an amount of about <NUM>, however, a range of about <NUM> to about <NUM> can be used. A485 was used in an amount of about <NUM>, however, a range of about <NUM> to about <NUM> can be used. RL5a was used in an amount of about <NUM>, however a range of about <NUM> to about <NUM> can be used. Etoposide was used in an amount of about <NUM>, however a range of about <NUM> to about <NUM> can be used.

The cells are contacted or incubated with the agent or agents at the stage where the cells have differentiated into progenitor cells and are ready to differentiate into mature cells (step <NUM> in Table <NUM>), in particular pancreatic endocrine progenitor cells. This step can be from about day <NUM> to about day <NUM> up to about day <NUM> of a differentiation protocol.

In some embodiments, the cells are contacted or incubated with the agents at about day <NUM> of the differentiation protocol, for about five days to about one week to about two weeks. In some embodiments, the cells are contacted or incubated with the agents at about day <NUM> of the differentiation protocol, for about one week to about two weeks. In some embodiments, the cells are contacted or incubated with the agents at early stage of differentiation of progenitor cell to mature differentiated cell which can be from about day <NUM> to about day <NUM>. In some embodiments, the cells are contacted or incubated with the agents at the late stage of differentiation of progenitor cell to mature differentiated cell which can from about day <NUM> to about day <NUM>. In some embodiment, the cells are contacted or incubated with the agents for the entire duration of differentiation of progenitor cell to mature differentiated cell which can be from about day <NUM> to about day <NUM> up to about day <NUM>.

The specific mature cells differentiated from a pluripotent stem cell are pancreatic endocrine progenitor cells.

These cells have markers characteristic of a specific mature differentiated cell. In some embodiments, the cells are positive for cell markers C-peptide and NKX6. <NUM> (mature pancreatic cells).

The cells of the present invention include specific mature cells differentiated from a pluripotent stem cell, a substantially homogenous population of specific mature cells differentiated from a pluripotent stem cell, and compositions comprising any of these cells of the invention, including pharmaceutical compositions and cryopreserved compositions. The invention also includes in some embodiments, cell culture comprising any of the cells of the invention.

In another aspect, provided herein is a composition comprising the specific mature cells differentiated from a pluripotent stem cell suitable for administration, transplantation and grafting into a subject produced by any of the methods of the invention as described herein. In some embodiments, the specific cells differentiated from a pluripotent stem cell are chosen from the group consisting of pancreatic endocrine cells, pancreatic cells, and endocrine cells, neurons, astrocytes, oligodendrocytes, retinal epithelial cells (RPE), epidermal cells, hair cells, keratinocytes, hepatocytes, intestinal epithelial cells, lung alveolar cells, hematopoietic cells, endothelial cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, cartilage cells, bone cells, renal cells, adipocytes, chondrocytes, and osteocytes. In some embodiments, the composition is a pharmaceutical composition further comprising any pharmaceutically acceptable carrier or excipient.

The composition or pharmaceutical composition may comprise at least <NUM>,<NUM>, at least <NUM>,<NUM>, at least <NUM>,<NUM>, at least <NUM>,<NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, or at least <NUM> x <NUM><NUM> specific mature cells differentiated from a pluripotent stem cell suitable for administration, transplantation and grafting into a subject produced by any of the methods of the invention as described herein.

Herein there is also disclosed a cryopreserved composition of the specific mature cells differentiated from a pluripotent stem cell suitable for administration, transplantation and grafting into a subject produced by any of the methods of the invention as described herein. The cryopreserved composition may comprise at least <NUM>,<NUM>, at least <NUM>,<NUM>, at least <NUM>,<NUM>, at least <NUM>,<NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, or at least <NUM> x <NUM><NUM> cells differentiated from a pluripotent stem cell suitable for transplantation and grafting into a subject produced by the methods of the invention as described herein.

There is also disclosed a cell culture comprising the specific mature cells differentiated from a pluripotent stem cell produced by any of the methods of the invention as described herein. In some embodiments, the cell culture comprises at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, at least <NUM> x <NUM><NUM>, or at least <NUM> x <NUM><NUM> specific mature cells differentiated from a pluripotent stem cell suitable for administration, transplantation and grafting into a subject produced by the methods of the invention as described herein.

There is also disclosed a therapeutic use of the specific mature cells differentiated from a pluripotent stem cell suitable for administration, transplantation and grafting into a subject produced by any of the methods of the invention as described herein.

The invention provides for a population of substantially homogenous specific mature cells differentiated from a pluripotent stem cell suitable for administration, transplantation and grafting into a subject produced by any of the methods of the invention as described herein. The population of cells may comprise at least about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% differentiated cells.

The cells disclosed herein can be preserved, for example, cryopreserved for later use. Methods for cryopreservation of cells are well known in the art. For example, the pancreatic endocrine cells can be prepared in a form that is easily administrable to an individual. For example, provided herein are cells that are contained within a container that is suitable for medical use. Such a container can be, for example, a sterile plastic bag, flask, jar, or other container from which the cells can be easily dispensed.

The various cells of the invention can be cryopreserved, e.g., in cryopreservation medium in small containers, e.g., ampoules. Suitable cryopreservation medium includes, but is not limited to, culture medium including, e.g., growth medium, or cell freezing medium, for example commercially available cell freezing medium, e.g., C2695, C2639 or C6039 (Sigma). Cryopreservation medium preferably comprises DMSO (dimethylsulfoxide), at a concentration of, e.g., about <NUM>-<NUM>% (v/v). Cryopreservation medium may comprise additional agents, for example, methylcellulose and/or glycerol. HES-MSC are preferably cooled at about <NUM>. /min during cryopreservation. A preferred cryopreservation temperature is about -<NUM>. to about - <NUM>. , preferably about -<NUM>. to about -<NUM>. Cryopreserved cells can be transferred to liquid nitrogen prior to thawing for use. In some embodiments, for example, once the ampoules have reached about -<NUM>. , they are transferred to a liquid nitrogen storage area. Cryopreserved cells preferably are thawed at a temperature of about <NUM>. to about <NUM>. , preferably to a temperature of about <NUM>.

Pancreatic endocrine cells are exemplified herein and are particularly useful in treating and preventing diabetes. These cells can be positive for cell markers C-peptide and NKX6.

As discussed above, there is disclosed a pharmaceutical composition comprising a therapeutically effective amount of a cell, cells or population of cells and a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human, and approved by a regulatory agency of the Federal or a state government or listed in the U. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, cachets, troches, lozenges, dispersions, suppositories, ointments, cataplasms (poultices), pastes, powders, dressings, creams, plasters, patches, aerosols, gels, liquid dosage forms suitable for parenteral administration to a patient, and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable form of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

Pharmaceutical compositions adapted for nasal and pulmonary administration may comprise solid carriers such as powders which can be administered by rapid inhalation through the nose. Compositions for nasal administration may comprise liquid carriers, such as sprays or drops. Alternatively, inhalation directly through into the lungs may be accomplished by inhalation deeply or installation through a mouthpiece. These compositions may comprise aqueous or oil solutions of the active ingredient. Compositions for inhalation may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed so as to provide predetermined dosages of the active ingredient.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain anti-oxidants, buffers, baceriostats, and solutes that render the compositions substantially isotonic with the blood of the subject. Other components which may be present in such compositions include water, alcohols, polyols, glycerine, and vegetable oils. Compositions adapted for parental administration may be presented in unit-dose or multi-dose containers, such as sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile carrier, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Selection of a therapeutically effective amount will be determined by the skilled artisan considering several factors which will be known to one of ordinary skill in the art. Such factors include the particular form of the cells and other parameters such as bioavailability, which will have been established during the usual development procedures typically employed in obtaining regulatory approval for a pharmaceutical composition. Further factors in considering the amount include the condition or disease to be treated or the benefit to be achieved in a normal individual, the body mass of the patient, the route of administration, whether the administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus, the precise amount should be decided according to the judgment of the person of skill in the art, and each patient's circumstances, and according to standard clinical techniques.

A standard therapeutically effective amount of cells to be administered, transplanted or grafted into a subject is about <NUM> x <NUM><NUM>. More specifically for pancreatic cells, about <NUM>,<NUM> to about <NUM> x <NUM><NUM> stem cell derived islet clusters which contain about several million cells can be administered, transplanted or grafted into a subject.

The novel method described herein for the generation of mature stable differentiated cells from stem cells, and the cells and substantially homogenous population of cells generated from any method of the invention, provide new therapies for many diseases.

In particular, the novel methods described herein for the generation of pancreatic endocrine cells from stem cells, and the cells and substantially homogenous population of cells generated from this method, provide new therapies for diabetes.

Thus, a method of treating or preventing diabetes comprising the steps of administering, transplanting or grafting a therapeutically effective amount of the cells obtained by the method of the present invention, is disclosed. Diabetes would include type <NUM>, type <NUM> and monogenetic forms. The subject is preferably a mammal or avian, and most preferably human. The cells, compositions, cell culture or pharmaceutical compositions can comprise pancreatic endocrine cells made by the methods of the invention, or a population of substantially homogenous pancreatic endocrine cells made by the methods of the invention.

A method of treating or preventing disease comprising the steps of administering, transplanting or grafting a therapeutically effective amount of the cells obtained by the method of the present invention, is disclosed. The cells, compositions, cell culture or pharmaceutical compositions can comprise specific mature differentiated cells made by the methods of the invention, or a population of substantially homogenous specific mature differentiated cells made by the methods of the invention.

The present disclosure also provides kits comprising the components of the combinations of the invention in kit form.

The kit includes reagents for practicing any of the methods of the invention for obtaining stable differentiated cells from hPSCs including differentiation medium and agents which interfere with DNA replication to induce cell exit.

The kit can include the pancreatic cells obtained by the current methods of the invention. The kit can also comprise reagents for culturing the cells.

The kit can include a pharmaceutical composition comprising the pancreatic cells obtained by the current methods of the invention.

The kit can include a cryopreserved composition the pancreatic cells obtained by the current methods of the invention.

The kit can include the specific mature differentiated cells obtained by the current methods of the invention. The kit can also comprise reagents for culturing the cells.

The kit can include a pharmaceutical composition comprising the specific mature differentiated cells obtained by the current methods of the invention.

The kit can include a cryopreserved composition comprising the specific mature differentiated cells obtained by the current methods of the invention.

The kits can further include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. For example, the following information regarding a combination of the invention may be supplied in the insert: how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

The present invention may be better understood by reference to the following nonlimiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention.

Human pluripotent stem cells were cultured and maintained in feeder-free plates with stemflex medium as described. Three cell lines were involved in this study as shown in Table <NUM>: MEL1 is human embryonic stem cell line (used as reference example); 1023A is a human induced pluripotent stem cell line reprogrammed from a skin fibroblast biopsied of a healthy control; and 1018E is a human induced pluripotent stem cell line reprogrammed from a skin fibroblast biopsied from a female type <NUM> diabetes patient. All human subjects research was reviewed and approved by the Columbia University Institutional Review Board, and the Columbia University Embryonic Stem Cell Committee.

Beta cells differentiated from human pluripotent stem cell lines as described in Example <NUM> (Sui et al. Indicated compounds that function as cell cycle inhibitors were added individually from day <NUM> to day <NUM> with concentration listed below:
The DNA replication inhibitors used in this study: <NUM>, <NUM> and <NUM> Aphidicolin (A0781, Sigma-Aldrich); <NUM> Cisplatin (<NUM>, EMD Millipore); <NUM> Ciprofloxacin (S2027, Selleck Chemicals); <NUM> Pyridostatin (S7444, Selleck Chemicals); <NUM> E2F Inhibitor (<NUM>, EMD Millipore); <NUM> A485 (<NUM>, Tocris); <NUM> RL5a (SML2187, Sigma-Aldrich); and <NUM> Etoposide (E55500, Research Products International). Indicated compounds were added individually from day <NUM> to day <NUM> with indicated concentration.

Total RNA was extracted from the cells at beta cell stage (d27) from different conditions using a RNeasy Mini Kit (Cat. No. <NUM>, QIAGEN). The total RNA was reverse transcripted into cDNA using iScript™ Reverse Transcription Supermix (Cat. No. <NUM>-<NUM>, Bio-Rad), and the cDNA were sequentially used as template with SsoFast™ EvaGreen® Supermix (Cat. No. <NUM>-<NUM>, Bio-Rad) for quantitative realtime PCR. The primers used in PCR are listed in Table <NUM>.

Clusters at d27 were collected and fixed with <NUM>% paraformaldehyde (PFA). The following steps were performed according to published method (Sui et al. Primary antibodies are listed in Table <NUM> and secondary antibodies are listed in Table <NUM>. Pictures were taken under OLYMPUS 1X73 fluorescent microscope or ZEISS LSM <NUM> confocal microscope.

The beta cell clusters were dissociated using TrypLETM Express into single cells. Cells were fixed with <NUM>% PFA for <NUM> and followed by permeabilization at -<NUM> with cold methanol for <NUM>. Primary antibodies were added at a dilution of <NUM>:<NUM> in autoMACs Rinsing Solution (Cat. No. <NUM>-<NUM>-<NUM>, Miltenyi Biotec) containing <NUM>% BSA at <NUM> for <NUM> hour. Secondary antibodies were added at a dilution of <NUM>:<NUM> at room temperature for <NUM> hour. The cells were filtered with BD Falcon <NUM> x <NUM> tube with cell strainer cap (Cat. No. <NUM>, BD) and subsequently analyzed by flow cytometer. Data were analyzed using Flowjo software. Negative controls were performed by only adding secondary antibodies.

Krebs Ringer buffer (KRB) was prepared by addition of <NUM> NaCl, <NUM> KCl, <NUM> CaCl<NUM>, <NUM> MgSO<NUM>, <NUM> Na<NUM>HPO<NUM>, <NUM> KH<NUM>PO<NUM>, <NUM> NaHCO<NUM>, <NUM> HEPES and <NUM>% BSA in deionized water and was sterilized using <NUM> filter. <NUM> and <NUM> glucose solution were prepared in KRB for low glucose and high glucose challenge of sc-beta cell clusters. <NUM>-<NUM> sc-beta cell clusters (about 5x105 cells) were collected from control and cells treated with cell cycle inhibitors including APH and treated conditions and preincubated in <NUM>µl low glucose solution for <NUM> hour. Clusters were then washed once with low glucose solution and sequentially incubated in <NUM>µl of low glucose and then high glucose solution for <NUM> minutes. <NUM>µl supernatant of each condition were collected. Supernatants containing secreted C-peptide stimulated by low and high glucose were processed using Mercodia Ultrasensitive Human C-peptide ELISA kit. Fold changes of C-peptide secretion before and after glucose stimulation were calculated.

<NUM>-<NUM> weeks old male immuno-compromised mice (NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) from Jackson laboratories, Cat. No. <NUM>) were used for transplantation. For intra leg muscle transplantation, <NUM>-<NUM> million cells were collected and settled in tube with <NUM>µl Matrigel. All animal protocols were approved by the Institutional Animal Care and Use Committee in Columbia University.

The human C-peptide levels in mouse serum were measured every two weeks in the fed state. Intraperitoneal glucose tolerance test was performed by fasting overnight and injecting <NUM>/kg D-glucose solution at <NUM> weeks after mouse beta cells were ablated with one high dose (<NUM>/kg) Streptozocin (Cat. No. S0130-<NUM>, Sigma-Aldrich). Blood was collected in heparin-coated tube at fed state, fasting and <NUM> after glucose injection. Plasma were collected by centrifuging tubes at <NUM> for <NUM> minutes at <NUM>. The supernatants were collected for C-peptide and insulin detection with Mercodia M-Plex™ ARRAY Chemiluminescent Mercodia Beta Kit. Blood glucose levels was measured at fed state, fasting and every <NUM> minutes after glucose injection for <NUM> hours.

The sorted GFP positive and negative cells with and without cell cycle inhibitor (e.g., APH) treatment were lysed with RIPA buffer. Protein was quantified with Pierce BCA protein assay kit. 20ug of protein was loaded on <NUM>-<NUM>% Mini-PROTEAN TGS Precast Protein Gels (Cat. No. <NUM>, Bio-Rad) from each condition and ran at <NUM> V for <NUM> hour followed by transferring protein on PVDF membrane. The membrane was blocked with <NUM>% BSA in TBST for 1hour at RT, incubate with primary antibodies diluted in TBST with <NUM>% BSA overnight on a shaker. After washing with TBST, the membrane was incubated with secondary antibodies diluted in TBST with <NUM>% non-fat dry milk powder for <NUM> hour at room temperature. The image was taken using ChemiDoc Imaging Systems (Bio-Rad).

Data were analyzed by unpaired t test and by one-way ANOVA followed by Tukey's multiple comparison test (GraphPad Prism <NUM>, GraphPad Software, Inc. , La Jolla, CA) and expressed as mean ± standard deviation (SD). The differences observed were considered statistically significant at the <NUM>% level and were displayed on the figures as follows: *p<<NUM>, **p<<NUM>, ***p<<NUM>, ****p<<NUM>.

After <NUM>%-<NUM>% confluence of hiPSCs was achieved, beta cell induction was initiated by using combinations of growth factors and small molecules. The differentiation steps are summarized in Table <NUM> (i.e., definitive endoderm induction, primitive gut tube induction, posterior foregut induction, pancreatic progenitor induction, pancreatic endocrine progenitor induction, and pancreatic beta cell induction), At the pancreatic progenitor stage, cells were cultured as organoids using AggreWell <NUM><NUM>-well plate to promote cell to cell interaction.

The following steps were followed to obtain the definitive endoderm stage:.

The following steps were followed to obtain the primitive gut tube stage:
At day <NUM> - day <NUM>.

The following steps were followed to obtain the posterior foregut stage:
At day <NUM>-day <NUM>.

The following steps were followed to obtain the pancreatic progenitor stage
At day <NUM> - day <NUM>.

The following steps were followed to make 3D cell clusters:
At day <NUM> - day <NUM>.

The following steps were followed to obtain the pancreatic endocrine progenitor stage:.

The following steps were followed to obtain the pancreatic beta cell stage:
At day <NUM> - day <NUM>.

This protocol is based on six key cellular induction steps in hPSCs: <NUM>. A commercial definitive endoderm differentiation kit was used to activate Activin A and Wnt3a signaling pathway to give rise to <NUM>% SOX17 and FOXA2-positve definitive endoderm cells; <NUM>. Further induction of definitive endoderm cells to primitive gut tube with FGF7 was performed (D' Amour et al. <NUM>); <NUM>. The posterior foregut tube was induced by inhibition of sonic hedgehog and the BMP4 signaling pathway and the activation of the retinoic acid pathway (D' Amour et al. <NUM>; Mfopou and Bouwens (<NUM>); Mfopou et al. <NUM>); <NUM>. Further committed cells to pancreatie progenitors expressing PDX1 and NKX6. <NUM> by activation of protein kinase C pathway with EGF (Nostro et al. <NUM>; Sui et al. <NUM>); <NUM>. Performed pancreatic endocrine lineage commitment by addition of thyroid hormone and upregulation of NGN3 expression through inhibition of Notch signaling together with blockage of the TGF beta signaling pathway with ALK5 inhibitor (Rezania et al.

Aphidicolin (APH), a DNA polymerase inhibitor, was used to inhibit DNA replication (Koundrioukoff et al. APH was added during the differentiation from day <NUM> (pancreatic progenitor cell (PPC) stage) until day <NUM> (pancreatic beta cell (PB). Different time points of treatment at early stage (d15-d20), late stages (d20-d27) and whole duration of differentiation (d15-d27) were evaluated by the percentage of C-peptide positive cells, and C-peptide and NKX6. <NUM> double positive cells derived at the end of differentiation at day <NUM>. Addition of APH at all indicated stages increased the proportion of C-peptide positive cells, and C-peptide and NKX6. <NUM> dual positive cells (<FIG>). The most effective differentiation toward C-peptide and NKX6. <NUM> positive cells occurred when APH was added from day <NUM> to day <NUM> (<FIG>). The insulin expressing cells were evenly distributed in the islet-like clusters with high percentage indicated by the expression of GFP, whereas some parts of the clusters in control remained GFP negative (<FIG>).

To determine the most effective concentration, cells were exposed to APH from d15-d27 at increasing concentrations, from <NUM>, <NUM> to <NUM>. Cells treated with <NUM> APH gave rise to the highest percentage of c-peptide and NKX6. <NUM> positive cells (<FIG>).

To evaluate if the positive effect of APH on the derivation of insulin expressing cells is generalized across different cell lines, two iPSC lines with different differentiation potentials were included in the study, 1018E and 1023A. 1018E was previously identified as a cell line that differentiates poorly (Sui et al. The percentage of C-peptide positive cells was significantly upregulated after APH treatment in both cell lines (<FIG>). Remarkably, the poor differentiation potential of 1018E increased to the range of a differentiation competent cell line, from an average of <NUM>% to <NUM>%. Therefore, APH increased the purity of stem cell derived beta cells after formation of pancreatic progenitors. Significantly, it reduced the variability of beta cell differentiation. All (n=<NUM> independent differentiation experiments) cultures contained more than <NUM>% C-peptide positive cells.

To investigate the mechanism underlying the increased purity of stem cell-derived C-peptide positive cells induced by APH, cell proliferation, apoptosis and differentiation efficiency were examined during differentiation. The cell cycle progression was profiled at day <NUM>, day <NUM>, day <NUM> and day <NUM> of beta cell differentiation. In untreated cells, C-peptide positive cells started to form from day <NUM> (about <NUM>%) (<FIG>) and reached a peak at day <NUM> (about <NUM>%) (<FIG>). About <NUM>% of total cells underwent proliferation during <NUM> hours of Edu incubation at each stage before day <NUM> and very few C-peptide positive cells were labeled positive for Edu (<FIG>). At day <NUM>, the percentage of proliferating cells increased to about <NUM>% and the percentage of C-peptide positive cells decreased due to proliferation of non-C-peptide cells in the clusters (<FIG>). When APH was added from day <NUM> to day <NUM>, the number of C-peptide positive cells was increased as early as day <NUM>, <NUM> days after addition of APH (<FIG>). At day <NUM>, about <NUM>% cells expressed C-peptide and the high expression of C-peptide was maintained until the day <NUM>. Very few cells, if any, were proliferating through the whole duration of differentiation (<FIG>).

To determine whether the increased percentage of beta cells was due to cell death of proliferating progenitors or due to increased differentiation from pancreatic progenitor cells, absolute cell numbers and cell death were quantified. Cells at day <NUM> and day <NUM> with and without APH treatment were stained for TUNEL. No significant increase in cell apoptosis after APH treatment was found indicated by the percentage of TUNEL positive cells both at early (<FIG>) and late stage of APH treatment (<FIG>). The percentage of C-peptide positive cells was significantly increased at day <NUM> after APH treatment compared to that of control (<FIG>). The number of total cells at day <NUM> was comparable between APH and control group (<FIG>). Therefore, the increased percentage of C-peptide positive cells was due to the increased differentiation, not the elimination of non-C-peptide positive cells from day <NUM> to day <NUM>. After day <NUM>, APH maintained the purity of beta cell clusters by preventing the growth of non-beta cells.

To determine the cell cycle phase at which aphidicolin arrested pancreatic progenitors and beta cells, flow cytometry combined with Hoechst staining for DNA content and EdU labeling was performed.

By the end of day <NUM>, <NUM>% of control cells were in cell cycle through G1 to M phase indicated by the expression of KI67 (a proliferation marker expressed in the cells within cell cycle) and amount of DNA content (<FIG>). About <NUM>% of APH-treated cells were in cell cycle and arrested in G1 phase (<FIG>). The cell cycle gene expression in insulin expressing cells was also examined. The expression of P21, a cell cycle progression inhibitor, was upregulated and the expressions of Cyclin D1 and CDK4, both involved in G1-phase progression, were downregulated in APH treated insulin positive cells (<FIG>). These results demonstrated that APH promotes the differentiation of pancreatic progenitors to endocrine cells by G1 arrest.

To determine whether other compounds able to induce G1 arrest similarly promoted differentiation to beta cells, pancreatic progenitors were exposed to a panel of compounds with anti-proliferative properties: Pyridostatin (PDS) stabilizes G-quadruplexes and arrests cell cycle (Moruno-Manchon et al. , <NUM>; Zimmer et al. , <NUM>); Cisplatin (Cis) induces DNA damage via DNA cross link and low dose arrests cells at S phase (Qin and Ng, <NUM>; Wagner and Karnitz, <NUM>); E2F inhibitor (E2Fi) inhibits the master transcription factors involved in S phase entry (Ma et al. , <NUM>; Pardee et al. , <NUM>; Rouaud et al. , <NUM>); Etoposide (Eto) is a topoisomerase inhibitor and stops the unwind of the DNA helix during replication (Cliby et al. , <NUM>; Korwek et al. , <NUM>; Nam et al. , <NUM>; Smith et al. , <NUM>); CDK4 inhibitor (Cdk4i) arrests cells at early G1 phase (Huang et al. , <NUM>); Ciprofloxacin (Cipro) inhibits MCM2-<NUM> replicative helicase at replication origin (Simon et al. , <NUM>); A485 inhibits p300, a histone acetylase, and arrests the cell cycle and inhibits p300 dependent transcription (Lasko et al. RL5a arrests the cell cycle by inhibiting replication origin licensing (Gardner et al.

All of the tested inhibitors ceased the cell cycle progression by arresting cells at G1 phase (<FIG>). The majority of tested DNA replication inhibitors increased the percentage of C-peptide positive cells at the end of differentiation on day <NUM> (<FIG>). However, the increase was not equal for all. Etoposide, cisplatin and aphidicolin were the most effective compounds to induce differentiation of pancreatic progenitors to C-peptide positive cells (<FIG>). RL5a failed to increase the number of C-peptide positive compared to control and P300 inhibitor also showed no significant increase, possibly because the inhibition of transcriptional activity impairs insulin expression. Therefore, compounds that inhibit cell cycle progression in late G1, and inhibit entry into S-phase were the most effective in promoting beta cell differentiation, while compounds that affect early G1-phase (CDK4, RL5a) were less effective.

With APH, a high proportion of cells in the islet-like clusters were positive for C-peptide and NKX6. <NUM>, a transcription factor for maintaining the beta cell identity and functional maturation (Schaffer et al. It was further investigated if APH had an effect on the preservation of beta cell identity after long-term culture. Aphidicolin was removed on day <NUM> to determine if the treatment had an effect on the stability of beta cells, and whether beta cell identity would be stable in the absence of the compound. Beta cells were cultured for additional <NUM> days until about day <NUM> and <NUM> days until about day <NUM> upon removal of these compounds at day <NUM>. In untreated control cells, the insulin-GFP expression was gradually decreased (<FIG>). In contrast, in APH treated cells, there was no decrease in C-peptide or NKX6. <NUM> on day <NUM> (<FIG>). After <NUM> days of releasing cells from APH, the percentage of C-peptide positive cells, and C-peptide and NKX6. <NUM> double positive cells was comparable to that of day <NUM> before releasing, whereas the percentage of C-peptide positive cells was significantly reduced in control group from day <NUM> to day <NUM> (<FIG>). After further culturing of the cells for extra <NUM> days, very few of cells in control group remained GFP expression and were positive for C-peptide. The number of GFP and C-peptide positive cells in APH treated condition was also decreased at day <NUM> compared to day <NUM> before removal of APH, but the percentage compared to control remained high, at about <NUM>% versus about <NUM>% in controls (<FIG>, <FIG>). This indicates that transient APH treatment was able to stabilize the beta cell identity in the in vitro culture condition even after release from the compound.

The ability of other DNA replication inhibitors to stabilize the beta cell identity was investigated. The expressions of GFP were maintained after releasing cells from these inhibitors for <NUM> days until day <NUM>. With additional <NUM> days of culture, the expressions of GFP were reduced in all conditions at certain levels, but the cells treated with Cis and Eto expressed higher GFP than the cells treated with APH compared to the other conditions (<FIG>, <FIG>).

APH increased the differentiation efficiency of stem cell derived beta cells. The next question was if the maturity and function of stem cell derived beta cells were improved after APH treatment.

Insulin positive cells were isolated based on GFP expression and the expressions of genes related to the maturation of beta cells including PDX <NUM>, NKX6. <NUM>, MAFA and insulin were evaluated (<FIG>). The expressions of PDX1, NKX6. <NUM>, MAFA and insulin were significantly upregulated in GFP positive cells isolated from APH treated clusters at transcription level compared to GFP positive cells in control clusters and no expression of beta cell markers was detected in GFP negative cells (<FIG>). The increased expression of PDX1, NKX6. <NUM>, MAFA and insulin was further confirmed at the translational level by Western Blot (<FIG>). In addition, the C-peptide secretion was increased <NUM>-<NUM>-fold, with an average of <NUM>-fold, in response to high levels of glucose (<FIG>).

To test the ability of aphidicolin treated islet-like clusters to regulate blood glucose levels, treated and untreated cells were grafted into NSG mice on day <NUM> of differentiation. After transplantation in the immunodeficient mice, the mice transplanted with APH treated cells developed higher human C-peptide starting from <NUM> weeks after engraftment compared to that of control mice (<FIG>). The increase became significant from <NUM> weeks after transplantation (<FIG>). In addition, the secretion of human C-peptide in mice was downregulated when mice were fasted (<FIG>, <FIG>) and increased after glucose injection (<FIG>), indicating the engrafted beta cells were able to respond to changes in blood glucose levels.

The ability of APH treated cells to protect mice from diabetes was determined after eliminating endogenous mouse beta cells by streptozotocin (STZ). STZ ablates mouse beta cells but is not toxic to human beta cells at the concentrations used. After STZ treatment, the blood glucose levels were monitored and grafted beta cells were challenged with high glucose to check their function. The blood glucose levels remained in the normal range over time in <NUM> out of <NUM> mice (<FIG>). Mice were tolerant to glucose and normalized blood glucose levels within <NUM> of glucose injection (<FIG>). The secretion of human C-peptide and insulin decreased after fasting and increased after glucose injection (<FIG>).

A major obstacle for the therapeutic translation of stem cell products is the formation of growths, in the form of a teratoma or of a cyst. To determine growth potential of the grafted cells, growth was evaluated by monitoring the graft with a luciferase reporter using in vivo imaging. Mice were transplanted with <NUM>-<NUM> million APH treated cells or untreated cells. After <NUM> weeks of transplantation, the graft size of APH mice was small, while controls were modestly larger (<FIG>). Eleven weeks later, the grafted cells in control mice displayed large growths, whereas the mice transplanted with APH treated cells remained the similar size as graft at <NUM> weeks of grafting (<FIG>). The different growth trend of grafted cells between control and APH treated cells was evident in the bioluminescence intensity (<FIG>). At <NUM> weeks of engraftment, the size of graft in APH group was an average of <NUM>-fold larger than that of <NUM> weeks compare to the control group which had <NUM>-fold increase (<FIG>). Graft growth occurred in controls even in cultures with very high differentiation efficiency (greater than <NUM>%). The increase of graft size was slower in the <NUM> mice transplanted with control cells with high differentiation efficiency but cystic structure still formed in three out of the three (<NUM>/<NUM>) mice (<FIG>, <FIG>). No cysts were observed in mice grafted with APH treated cells in four out of the four (<NUM>/<NUM>) mice (<FIG>).

Claim 1:
A method of inducing cell cycle exit and terminal differentiation of cells undergoing differentiation from stem cells into pancreatic beta cells, comprising the steps of:
a. sequentially differentiating the stem cells to obtain pancreatic endocrine progenitor cells; and
b. contacting or incubating the pancreatic endocrine progenitor cells with an agent which interferes with DNA replication, wherein the contact or incubation inhibits entry of the pancreatic endocrine progenitor cells into the S-phase of the cell cycle and arrests the cells at G1 phase