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      Indolactam V/GLP-1-mediated Differentiation of Human iPS Cells into Glucose-Responsive Insulin-Secreting Progeny

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          Abstract

          Nuclear reprogramming of somatic tissue enables derivation of induced pluripotent stem (iPS) cells from an autologous, non-embryonic origin. The purpose of the current study was to establish efficient protocols for lineage-specification of human iPS cells into functional glucose-responsive, insulin-producing progeny. We generated human iPS cells, which were then guided with recombinant growth factors that mimic the essential signaling for pancreatic development. Reprogrammed with four stemness factors, human fibroblasts were here converted into authentic iPS cells. Under feeder-free conditions, fate-specification was initiated with activin A and Wnt3a that triggered engagement into definitive endoderm, followed by priming with FGF10 and KAAD-cyclopamine. Addition of retinoic acid, boosted by the pancreatic endoderm inducer indolactam V (ILV), yielded pancreatic progenitors expressing PDX1, NGN3 and NEUROD1 markers. Further guidance, under IGF-1, HGF and DAPT, was enhanced by glucagon like peptide-1 (GLP-1) to generate islet-like cells that expressed pancreas-specific markers including insulin and glucagon. Derived progeny demonstrated sustained expression of PDX1, and functional responsiveness to glucose challenge secreting up to 230 pM of C-peptide. A pancreatogenic cocktail enriched with ILV/GLP-1 offers a proficient means to specify human iPS cells into glucose-responsive hormone-producing progeny, refining the development of a personalized platform for islet-like cell generation.

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          Most cited references 49

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          In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.

          Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of 'customized' embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells-derived from mouse fibroblasts-can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.
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            Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.

            Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human beta-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implanted mice, human insulin and C-peptide are detected in sera at levels similar to those of mice transplanted with approximately 3,000 human islets. Moreover, the insulin-expressing cells generated after engraftment exhibit many properties of functional beta-cells, including expression of critical beta-cell transcription factors, appropriate processing of proinsulin and the presence of mature endocrine secretory granules. Finally, in a test of therapeutic potential, we demonstrate that implantation of hES cell-derived pancreatic endoderm protects against streptozotocin-induced hyperglycemia. Together, these data provide definitive evidence that hES cells are competent to generate glucose-responsive, insulin-secreting cells.
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              Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

              Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.
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                Author and article information

                Journal
                9421525
                8603
                Gene Ther
                Gene therapy
                0969-7128
                1476-5462
                27 September 2010
                4 November 2010
                March 2011
                1 September 2011
                : 18
                : 3
                : 283-293
                Affiliations
                [1 ]Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA
                [2 ]Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, and Medical Genetics, Mayo Clinic, Rochester, MN, USA
                [3 ]Department of Neurology, Mayo Clinic, Rochester, MN, USA
                [4 ]Division of Endocrinology, Mayo Clinic, Rochester, MN, USA
                Author notes
                Corresponding author: Dr. Yasuhiro Ikeda, Molecular Medicine Program, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, Tel: 507-538-1252; Fax: 507-266-2122. ikeda.yasuhiro@ 123456mayo.edu
                Article
                nihpa239556
                10.1038/gt.2010.145
                3060028
                21048796

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                Funding
                Funded by: National Heart, Lung, and Blood Institute : NHLBI
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: T32 HL007111-35 ||HL
                Funded by: National Heart, Lung, and Blood Institute : NHLBI
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: R56 AI074363-01A1 ||AI
                Funded by: National Heart, Lung, and Blood Institute : NHLBI
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: R01 HL083439-05 ||HL
                Categories
                Article

                Molecular medicine

                induced pluripotent stem cells, beta cells, islet, feeder-free ips culture

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