Generation of Insulin-secreting Islet-like Clusters from Human Skin Fibroblasts

Abstract

Increasing evidence suggests that islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors and the necessity for chronic immunosuppression has made it impossible for this treatment to be used for the general diabetic population. Recent success in generating insulin-secreting islet-like cells from human embryonic stem (ES) cells, in combination with the success in deriving human ES cell-like induced pluripotent stem (iPS) cells from human fibroblasts by defined factors, have raised the possibility that patient-specific insulin-secreting islet-like cells might be derived from somatic cells through cell fate reprogramming using defined factors. Here we confirm that human ES-like iPS cells can be derived from human skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free protocol, we successfully generated insulin-producing islet-like clusters (ILCs) from the iPS cells under feeder-free conditions. We demonstrate that, like human ES cells, skin fibroblast-derived iPS cells have the potential to be differentiated into islet-like clusters through definitive and pancreatic endoderm. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Thus, our study provides evidence that insulin-secreting ILCs can be generated from skin fibroblasts, raising the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future. Increasing evidence suggests that islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors and the necessity for chronic immunosuppression has made it impossible for this treatment to be used for the general diabetic population. Recent success in generating insulin-secreting islet-like cells from human embryonic stem (ES) cells, in combination with the success in deriving human ES cell-like induced pluripotent stem (iPS) cells from human fibroblasts by defined factors, have raised the possibility that patient-specific insulin-secreting islet-like cells might be derived from somatic cells through cell fate reprogramming using defined factors. Here we confirm that human ES-like iPS cells can be derived from human skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free protocol, we successfully generated insulin-producing islet-like clusters (ILCs) from the iPS cells under feeder-free conditions. We demonstrate that, like human ES cells, skin fibroblast-derived iPS cells have the potential to be differentiated into islet-like clusters through definitive and pancreatic endoderm. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Thus, our study provides evidence that insulin-secreting ILCs can be generated from skin fibroblasts, raising the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future. Diabetes is a life-long disease defined by hyperglycemia. Although type I diabetes is caused by autoimmune destruction of the pancreatic endocrine beta cells of the patient, type II diabetes results from insulin resistance. Although current treatment for type I diabetes is obliged to rely on insulin injection, due to the difficulties in adjusting the amounts of insulin administration to accommodate fluctuations in physiological conditions, this treatment often results in episodes of hypo- and hyperglycemia. Even under strictly controlled conditions, such treatment delays but does not always prevent development of multiple complications including microvascular disease, diabetic retinopathy, nephropathy, and neuropathy (1Efrat S. Adv. Drug Delivery Rev. 2008; 60: 114-123Crossref PubMed Scopus (47) Google Scholar). Regeneration of insulin-producing beta cell as well as islet transplantation are the most promising long term solutions for diabetes treatment (1Efrat S. Adv. Drug Delivery Rev. 2008; 60: 114-123Crossref PubMed Scopus (47) Google Scholar). For example, clinical trials with islet cell transplantation from cadaver donors have resulted in promising outcomes (2Lakey J.R. Mirbolooki M. Shapiro A.M. Methods Mol. Biol. 2006; 333: 47-104PubMed Google Scholar, 3Shapiro A.M. Ricordi C. Hering B.J. Auchincloss H. Lindblad R. Robertson R.P. Secchi A. Brendel M.D. Berney T. Brennan D.C. Cagliero E. Alejandro R. Ryan E.A. DiMercurio B. Morel P. Polonsky K.S. Reems J.A. Bretzel R.G. Bertuzzi F. Froud T. Kandaswamy R. Sutherland D.E. Eisenbarth G. Segal M. Preiksaitis J. Korbutt G.S. Barton F.B. Viviano L. Seyfert-Margolis V. Bluestone J. Lakey J.R. N. Engl. J. Med. 2006; 355: 1318-1330Crossref PubMed Scopus (1569) Google Scholar, 4Gangemi A. Salehi P. Hatipoglu B. Martellotto J. Barbaro B. Kuechle J.B. Qi M. Wang Y. Pallan P. Owens C. Bui J. West D. Kaplan B. Benedetti E. Oberholzer J. Am. J. Transplant. 2008; 8: 1250-1261Crossref PubMed Scopus (144) Google Scholar). Insulin independence with good glycemic control was achieved and sustained in some patients for more than 2 years. Although promising, this approach faces multiple challenges. A major obstacle is the shortage of donors when compared with the large patient population. In addition, because the low yield of islet cells from cadaver tissue, it requires large number of donor cells to generate sufficient insulin-producing beta cells that are capable of producing and releasing adequate amounts of insulin in response to normal physiological signals (2Lakey J.R. Mirbolooki M. Shapiro A.M. Methods Mol. Biol. 2006; 333: 47-104PubMed Google Scholar, 3Shapiro A.M. Ricordi C. Hering B.J. Auchincloss H. Lindblad R. Robertson R.P. Secchi A. Brendel M.D. Berney T. Brennan D.C. Cagliero E. Alejandro R. Ryan E.A. DiMercurio B. Morel P. Polonsky K.S. Reems J.A. Bretzel R.G. Bertuzzi F. Froud T. Kandaswamy R. Sutherland D.E. Eisenbarth G. Segal M. Preiksaitis J. Korbutt G.S. Barton F.B. Viviano L. Seyfert-Margolis V. Bluestone J. Lakey J.R. N. Engl. J. Med. 2006; 355: 1318-1330Crossref PubMed Scopus (1569) Google Scholar, 4Gangemi A. Salehi P. Hatipoglu B. Martellotto J. Barbaro B. Kuechle J.B. Qi M. Wang Y. Pallan P. Owens C. Bui J. West D. Kaplan B. Benedetti E. Oberholzer J. Am. J. Transplant. 2008; 8: 1250-1261Crossref PubMed Scopus (144) Google Scholar). Furthermore, chronic immunosuppression is necessary after allograft transplantation. These factors have made it impossible for this treatment to be used for the general diabetic population. Although the recurring autoimmune response has to be prevented to avoid destruction of the transplanted cells, derivation of patient-specific islet-like cells from adult tissues may allow both shortage of organ donors and allograft rejection to be resolved. However, generation of functional islet-like clusters (ILCs) 4The abbreviations used are: ILC, islet-like clusters; ES, embryonic stem; hES, human ES; iPS, induced pluripotent stem; HESM, human embryonic stem cells medium; MEF, mouse embryonic fibroblast; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; DAPI, 4′,6-diamidino-2-phenylindole; RT-PCR, reverse transcription-PCR; H&E, hematoxylin and eosin. from adult progenitor cells including pancreatic progenitor cells has been tested with limited success (5Sapir T. Shternhall K. Meivar-Levy I. Blumenfeld T. Cohen H. Skutelsky E. Eventov-Friedman S. Barshack I. Goldberg I. Pri-Chen S. Ben-Dor L. Polak-Charcon S. Karasik A. Shimon I. Mor E. Ferber S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7964-7969Crossref PubMed Scopus (243) Google Scholar, 6Suzuki A. Nakauchi H. Taniguchi H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5034-5039Crossref PubMed Scopus (126) Google Scholar, 7Zalzman M. Anker-Kitai L. Efrat S. Diabetes. 2005; 54: 2568-2575Crossref PubMed Scopus (150) Google Scholar). When compared with adult progenitor cells, human ES cells remain the most promising source for ILC generation due to their unlimited replicative capacity and differentiation potentials. Recent studies have proved that human ES cells can be differentiated into functional islet-like clusters using an in vitro differentiation protocol, although the efficiency is relatively low (8Jiang J. Au M. Lu K. Eshpeter A. Korbutt G. Fisk G. Majumdar A.S. Stem Cells (Durham). 2007; 25: 1940-1953Crossref PubMed Scopus (421) Google Scholar). In addition, transplantation of pancreatic endoderm derived from human ES cells in vitro can rescue mice with induced insulin deficiency (9Kroon E. Martinson L.A. Kadoya K. Bang A.G. Kelly O.G. Eliazer S. Young H. Richardson M. Smart N.G. Cunningham J. Agulnick A.D. D'Amour K.A. Carpenter M.K. Baetge E.E. Nat. Biotechnol. 2008; 26: 443-452Crossref PubMed Scopus (1424) Google Scholar). However, the application of human ES cell-derived ILC was largely restricted by generation of patient specific ES cells. Recent demonstrations that human ES cell-like induced pluripotent stem (iPS) cells can be generated from adult human somatic cells by enforced expression of defined transcription factors (10Lowry W.E. Richter L. Yachechko R. Pyle A.D. Tchieu J. Sridharan R. Clark A.T. Plath K. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2883-2888Crossref PubMed Scopus (848) Google Scholar, 11Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2397) Google Scholar, 12Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15285) Google Scholar, 13Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin II Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8273) Google Scholar, 14Mali P. Ye Z. Hommond H.H. Yu X. Lin J. Chen G. Zou J. Cheng L. Stem Cells (Durham). 2008; 26: 1998-2005Crossref PubMed Scopus (249) Google Scholar) have raised the possibility that genetically matched insulin-secreting ILCs can be derived from somatic cells through differentiation of patient-specific iPS cells. Here we confirm that human ES-like iPS cells can be derived from skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free in vitro protocol, we successfully generated insulin-producing ILCs from the iPS cells. Our studies thus raise the possibility that patient-specific iPS cells could potentially provide an alternative source for drug screen, study for disease mechanisms, and cell therapy for diabetes in the future. Plasmid Construction and Retroviruses Production—The human Oct4, Sox2, Klf-4, and c-Myc cDNAs were PCR-amplified from expressed sequence tag clones and cloned into pMXs at blunted BstXI sites (15Kitamura T. Koshino Y. Shibata F. Oki T. Nakajima H. Nosaka T. Kumagai H. Exp. Hematol. 2003; 31: 1007-1014Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). All sequences were confirmed by DNA sequencing. To generate retroviruses expressing the four factors, pGag-pol, pVSVG, and pMXs were co-transfected into 293T cells on one 6-well plate at cell density of 8.0 × 105 cells/well using SuperFect (Qiagen) according to the manufacturer's instructions. Six hours after transfection, the medium was changed to complete Dulbecco's modified Eagle's medium medium. The viruses were harvest 48 and 72 h after The viruses were through and by and of iPS of × 105 human cells on one plate were with retroviruses with after the × cells were mouse embryonic cells. the the medium was changed to modified Eagle's medium with and with to after the and were on their on and after iPS cell and the human ES cell were as on in and on using iPS cells were and by with at and cells on were with at with for and in in for 2 Cells were with at with were with and for SOX2, and were from was according to the manufacturer's For of differentiated cells, cell clusters were in at for in and with at for The cells were with at The of the are: mouse mouse and was and were used for were under a by The of the islet-like clusters under glucose was using an and the clusters were with and from and iPS cells on under the differentiation were using the I reverse transcription was using the reverse transcription were with using in expression were to was as with J. M. PubMed Scopus Google Scholar). of DNA was to The were using the All and were in for The clones were using an reverse and was using an and 2 × iPS cells on were by in a of and of and into the skin into the of were after and after were in in and for hematoxylin and and The the were used for and In of vitro differentiation of insulin-secreting islet-like cells was using a with (8Jiang J. Au M. Lu K. Eshpeter A. Korbutt G. Fisk G. Majumdar A.S. Stem Cells (Durham). 2007; 25: 1940-1953Crossref PubMed Scopus (421) Google Scholar). iPS and cells were in with and A for was on at a of the cells were with treatment of at for and into The cell were in with 2 and for 2 cell clusters were in in with and for the cells were in medium with and II for and II for 2 Insulin was as (8Jiang J. Au M. Lu K. Eshpeter A. Korbutt G. Fisk G. Majumdar A.S. Stem Cells (Durham). 2007; 25: 1940-1953Crossref PubMed Scopus (421) Google Scholar). clusters after were to a plate and were with medium. The cells were with medium glucose for at the was The cells were with medium glucose for The C-peptide in the were using the human C-peptide and the of C-peptide in the was according to the DNA of islet-like of iPS Cells from studies that ES-like pluripotent iPS cells can be generated through expression of defined transcription factors in differentiated mouse and human cells (10Lowry W.E. Richter L. Yachechko R. Pyle A.D. Tchieu J. Sridharan R. Clark A.T. Plath K. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2883-2888Crossref PubMed Scopus (848) Google Scholar, 11Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2397) Google Scholar, 12Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15285) Google Scholar, 13Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin II Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8273) Google Scholar, 14Mali P. Ye Z. Hommond H.H. Yu X. Lin J. Chen G. Zou J. Cheng L. Stem Cells (Durham). 2008; 26: 1998-2005Crossref PubMed Scopus (249) Google Scholar, N. Sridharan R. J. S. K. M. Yachechko R. Tchieu J. R. Plath K. K. Stem Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Ichisaka T. Yamanaka S. Nature. 2007; PubMed Scopus Google Scholar, K. Yamanaka S. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. R. T. M. K. R. Nature. 2007; PubMed Scopus Google Scholar). Although human iPS cells are capable of generating in can into cells of pancreatic endocrine is not To this we to generate human iPS cells by retroviral expression of human Oct4, Sox2, and c-Myc in the human cells. To this we expressed the four transcription factors in the fibroblasts by retroviral were after the and were at from the × cells in A of on and after were in cell medium on and the we successfully iPS cell of iPS of the iPS cell clones and from the human fibroblasts and cells for at in and The iPS clones at a but in their of differentiation To confirm that iPS cells we a number of cell that ES transcription factors and Oct4, and and a we also a for differentiation of cells. in and demonstrate that the iPS clones including and for Sox2, Oct4, and The of the were for the with a cells on the of some for tested was to that with the well cell The cell was for To the expression of the iPS we from iPS cells that were under feeder-free for at All of the clones the for cell when on not confirmed expression of the for cell including and glucose of were expressed in the fibroblasts of such as and more general in cell was in both iPS cells and the fibroblasts using that only and confirmed of studies have a the efficiency of somatic cell reprogramming and the DNA of the S. J. Nat. Biol. PubMed Scopus Google Scholar). We the DNA of the by sequencing. in demonstrate that, to the cells, the of is in the iPS cells, is in to the of in the fibroblasts, To confirm the of the human iPS cells, we of iPS cells into after and of from after the of cell of including and and and and and and tissues the results demonstrate that the cell that we generated the of human ES cells and are to be human iPS cells. of iPS Cells into confirmed the of the iPS cells, we are capable of into the pancreatic endocrine as generation of insulin-secreting cells has great potential in the development of therapy for To this we a serum-free differentiation from cells have been successfully differentiated into insulin-secreting islet-like clusters (8Jiang J. Au M. Lu K. Eshpeter A. Korbutt G. Fisk G. Majumdar A.S. Stem Cells (Durham). 2007; 25: 1940-1953Crossref PubMed Scopus (421) Google Scholar). iPS cell and and the cell were to the differentiation in Although four iPS cell human ES cell and and only iPS and the differentiation that iPS and may not be A and treatment to of cells a and were also for the iPS cells and and and and cells, from iPS cells, under low the to and some clusters The results that iPS clones and to of cells in response to the pancreatic cell differentiation To differentiation of both and iPS cells into pancreatic cell we the expression of pancreatic at of the differentiation by in the definitive such as and are expressed at after of treatment with and expression of of pancreatic cells, is and insulin are and insulin the at Importantly, both iPS and iPS in general expression as that of the human ES cells, that iPS cells and cells have differentiation potential for endoderm pancreatic To confirm expression of the differentiation we expression of and in in and C-peptide and in by in demonstrate that A and induced the expression of and in the of iPS cells as well as cells In addition, was in cells derived from both and iPS cells after 2 both C-peptide and glucagon-positive cells were in the cells of with a (8Jiang J. Au M. Lu K. Eshpeter A. Korbutt G. Fisk G. Majumdar A.S. Stem Cells (Durham). 2007; 25: 1940-1953Crossref PubMed Scopus (421) Google the expression of pancreatic was in the we that, to cells, the iPS cells derived from fibroblasts can be differentiated into islet-like endocrine cells. we a functional for the differentiated islet-like To this we insulin upon glucose using C-peptide low glucose of insulin as by the of are islet-like cells derived from and iPS cells glucose cell-derived islet-like cells to glucose (8Jiang J. Au M. Lu K. Eshpeter A. Korbutt G. Fisk G. Majumdar A.S. Stem Cells (Durham). 2007; 25: 1940-1953Crossref PubMed Scopus (421) Google Scholar). Importantly, cells derived from iPS also to glucose by insulin although the response from iPS is studies that long to glucose can of human islet cells M. M. L. M. Z. C. L. R. P. Bertuzzi F. A.M. G. P. R. G. F. Diabetes. PubMed Scopus Google Scholar). To the possibility that the of C-peptide in the under glucose were caused by cell we of cells by in demonstrate that most C-peptide-positive cells were thus the possibility that the release of C-peptide after glucose treatment is to we that iPS cells, like cells, have the potential to into functional islet-like clusters in although We that although the iPS used in our differentiation from the cells in of expression and the islet-like clusters generated from in glucose is that in number their of might to the However, more studies are to the of the We that in the differentiation potential of cell have been K. L. M. Y. Nat. Biotechnol. 2008; 26: PubMed Scopus Google Scholar, H. J. B. Chen C. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). Recent success in generating ES cell-like pluripotent iPS cells from somatic cells using defined factors has a to somatic for reprogramming (10Lowry W.E. Richter L. Yachechko R. Pyle A.D. Tchieu J. Sridharan R. Clark A.T. Plath K. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2883-2888Crossref PubMed Scopus (848) Google Scholar, 11Park I.H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. Nature. 2008; 451: 141-146Crossref PubMed Scopus (2397) Google Scholar, 12Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15285) Google Scholar, 13Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. Slukvin II Thomson J.A. Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8273) Google Scholar, N. Sridharan R. J. S. K. M. Yachechko R. Tchieu J. R. Plath K. K. Stem Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Ichisaka T. Yamanaka S. Nature. 2007; PubMed Scopus Google Scholar, K. Yamanaka S. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. R. T. M. K. R. Nature. 2007; PubMed Scopus Google Scholar). has for the with cell derivation from human In addition, it also the generation of patient-specific stem cells the that such as and has been with be iPS can be used in the of this in therapy is the that can be potentially by cell type I diabetes is one of the most as pancreatic transplantation islet has great promise in achieving independence from insulin (2Lakey J.R. Mirbolooki M. Shapiro A.M. Methods Mol. Biol. 2006; 333: 47-104PubMed Google Scholar, 3Shapiro A.M. Ricordi C. Hering B.J. Auchincloss H. Lindblad R. Robertson R.P. Secchi A. Brendel M.D. Berney T. Brennan D.C. Cagliero E. Alejandro R. Ryan E.A. DiMercurio B. Morel P. Polonsky K.S. Reems J.A. Bretzel R.G. Bertuzzi F. Froud T. Kandaswamy R. Sutherland D.E. Eisenbarth G. Segal M. Preiksaitis J. Korbutt G.S. Barton F.B. Viviano L. Seyfert-Margolis V. Bluestone J. Lakey J.R. N. Engl. J. Med. 2006; 355: 1318-1330Crossref PubMed Scopus (1569) Google Scholar, 4Gangemi A. Salehi P. Hatipoglu B. Martellotto J. Barbaro B. Kuechle J.B. Qi M. Wang Y. Pallan P. Owens C. Bui J. West D. Kaplan B. Benedetti E. Oberholzer J. Am. J. Transplant. 2008; 8: 1250-1261Crossref PubMed Scopus (144) Google Scholar). For this we tested the differentiation potential of human iPS cells into insulin-producing We that the iPS cells derived from human skin cells can be differentiated into pancreatic Our results evidence that functional endocrine cells can be generated in vitro from human somatic cells. that islet cell transplantation therapy has been to be to patients with type I diabetes (2Lakey J.R. Mirbolooki M. Shapiro A.M. Methods Mol. Biol. 2006; 333: 47-104PubMed Google Scholar, 3Shapiro A.M. Ricordi C. Hering B.J. Auchincloss H. Lindblad R. Robertson R.P. Secchi A. Brendel M.D. Berney T. Brennan D.C. Cagliero E. Alejandro R. Ryan E.A. DiMercurio B. Morel P. Polonsky K.S. Reems J.A. Bretzel R.G. Bertuzzi F. Froud T. Kandaswamy R. Sutherland D.E. Eisenbarth G. Segal M. Preiksaitis J. Korbutt G.S. Barton F.B. Viviano L. Seyfert-Margolis V. Bluestone J. Lakey J.R. N. Engl. J. Med. 2006; 355: 1318-1330Crossref PubMed Scopus (1569) Google generation of patient-specific iPS cells and differentiation of iPS cells into insulin-secreting cells may to be a potential cell source for transplantation However, this approach can be although the iPS cells and human ES cells have efficiency of differentiation into insulin-secreting islet the general efficiency is In addition, the of C-peptide by in vitro differentiated islet-like cells from both iPS cells and human ES cells are when compared with that of adult human The low efficiency of is caused by the of current in vitro but not the of the iPS cells. For this it is to differentiation that the of insulin-secreting cells and their response to approach that has great promise is in of endocrine cells through transplantation of in vitro differentiated pancreatic endoderm cells (9Kroon E. Martinson L.A. Kadoya K. Bang A.G. Kelly O.G. Eliazer S. Young H. Richardson M. Smart N.G. Cunningham J. Agulnick A.D. D'Amour K.A. Carpenter M.K. Baetge E.E. Nat. Biotechnol. 2008; 26: 443-452Crossref PubMed Scopus (1424) Google Scholar). type I diabetes is an autoimmune disease, the iPS-derived insulin-producing cells can be by the cells. Although the is the capacity of iPS cells might the of the disease by the of iPS-derived cells by donor is one of the major in of derived from multiple donors A. K. S. A. Ryan E.A. Shapiro A.M. Am. J. Transplant. 2007; PubMed Scopus Google Scholar). the for iPS therapy that are with the reprogramming such as the of the and due to the of also be the that has been made in the 2 we that it not be long are resolved. is that although iPS cell clones in cell expression of pluripotent DNA at the we in their potential to into the pancreatic cells. the four iPS cell clones that we tested in the differentiation iPS to islet-like cell not The in of differentiation potential that not human iPS clones are pluripotent although the the the for human iPS cells. the differentiation used in this study has defined we be to the specific of the differentiation for some of the in combination with factors, such as and might to the for human iPS cells with differentiation We for the for and E. for of the with