Generation of Functional Beta-Like Cells from Human Exocrine Pancreas

Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant β-like cells expressed insulin protein levels at ~15–30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.

[1]  Ali Asadi,et al.  The Role of ARX in Human Pancreatic Endocrine Specification , 2015, PloS one.

[2]  K. Docherty,et al.  Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture , 2015, PloS one.

[3]  CorritoreElisa,et al.  β-Cell differentiation of human pancreatic duct-derived cells after in vitro expansion. , 2014 .

[4]  D. Melton,et al.  Generation of Functional Human Pancreatic β Cells In Vitro , 2014, Cell.

[5]  James D. Johnson,et al.  Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells , 2014, Nature Biotechnology.

[6]  R. Maehr,et al.  Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway , 2014, eLife.

[7]  Y. Dor,et al.  Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice , 2013, Nature Biotechnology.

[8]  Wai Leong Tam,et al.  The epigenetics of epithelial-mesenchymal plasticity in cancer , 2013, Nature Medicine.

[9]  G. Pan,et al.  Vitamin C modulates TET1 function during somatic cell reprogramming , 2013, Nature Genetics.

[10]  J. Hecksher-Sørensen,et al.  The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells , 2013, PLoS genetics.

[11]  Hans Clevers,et al.  Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis , 2013, The EMBO journal.

[12]  K. Docherty,et al.  Suppression of Epithelial-to-Mesenchymal Transitioning Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue Toward Functional Insulin-Producing β-Like Cells , 2013, Diabetes.

[13]  Mohammad Wahid Ansari,et al.  The legal status of in vitro embryos , 2014 .

[14]  J. Miyazaki,et al.  Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells , 2013, eLife.

[15]  T. Endo,et al.  Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells* , 2013, The Journal of Biological Chemistry.

[16]  P. Larsen,et al.  Thyroid Hormone Promotes Postnatal Rat Pancreatic β-Cell Development and Glucose-Responsive Insulin Secretion Through MAFA , 2013, Diabetes.

[17]  M. Trotter,et al.  Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells , 2012, Diabetologia.

[18]  K. Docherty,et al.  Efficient differentiation of AR42J cells towards insulin-producing cells using pancreatic transcription factors in combination with growth factors , 2012, Molecular and Cellular Endocrinology.

[19]  G. Korbutt,et al.  Human Mesenchymal Stem Cells Protect Human Islets from Pro-Inflammatory Cytokines , 2012, PloS one.

[20]  D. Pipeleers,et al.  Plasticity of Adult Human Pancreatic Duct Cells by Neurogenin3-Mediated Reprogramming , 2012, PloS one.

[21]  S. Efrat,et al.  Redifferentiation of Expanded Human Pancreatic β-Cell-derived Cells by Inhibition of the NOTCH Pathway* , 2012, The Journal of Biological Chemistry.

[22]  I. Rooman,et al.  Lineage tracing evidence for transdifferentiation of acinar to duct cells and plasticity of human pancreas. , 2011, Gastroenterology.

[23]  Guoping Fan,et al.  Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx. , 2011, Developmental cell.

[24]  F. Ashcroft,et al.  Control of pancreatic β cell regeneration by glucose metabolism. , 2011, Cell metabolism.

[25]  N. Nardi,et al.  Co-transplantation of mesenchymal stem cells maintains islet organisation and morphology in mice , 2011, Diabetologia.

[26]  V. Gmyr,et al.  Evidence for Epithelial-Mesenchymal Transition in Adult Human Pancreatic Exocrine Cells , 2010, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[27]  David H. O'Connor,et al.  Mesenchymal Stem Cells Enhance Allogeneic Islet Engraftment in Nonhuman Primates , 2010, Diabetes.

[28]  Milton Waner,et al.  Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic β-like cells , 2010, Proceedings of the National Academy of Sciences.

[29]  H. Redl,et al.  Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. , 2010, Cell stem cell.

[30]  F. Pattou,et al.  Epithelial-Mesenchymal Transition in Cells Expanded In Vitro from Lineage-Traced Adult Human Pancreatic Beta Cells , 2009, PloS one.

[31]  K. Anseth,et al.  Cell-matrix interactions improve beta-cell survival and insulin secretion in three-dimensional culture. , 2008, Tissue engineering. Part A.

[32]  Yi Zhang,et al.  Generation of Insulin-secreting Islet-like Clusters from Human Skin Fibroblasts*♦ , 2008, Journal of Biological Chemistry.

[33]  Douglas A. Melton,et al.  In vivo reprogramming of adult pancreatic exocrine cells to β-cells , 2008, Nature.

[34]  Susumu Seino,et al.  Role of Cadherin-mediated Cell-Cell Adhesion in Pancreatic Exocrine-to-Endocrine Transdifferentiation*♦ , 2008, Journal of Biological Chemistry.

[35]  E. Kroon,et al.  Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo , 2008, Nature Biotechnology.

[36]  G. Korbutt,et al.  Generation of Insulin‐Producing Islet‐Like Clusters from Human Embryonic Stem Cells , 2007, Stem cells.

[37]  H. Deng,et al.  In vitro derivation of functional insulin-producing cells from human embryonic stem cells , 2007, Cell Research.

[38]  M. Barcová,et al.  Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas , 2006, Nature Medicine.

[39]  E. Kroon,et al.  Efficient differentiation of human embryonic stem cells to definitive endoderm , 2005, Nature Biotechnology.

[40]  T. Iwanaga,et al.  Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[41]  V. Toscano,et al.  3,5,3′‐Triiodo‐L‐thyronine enhances the differentiation of a human pancreatic duct cell line (hPANC‐1) towards a β‐cell‐Like phenotype , 2005, Journal of cellular physiology.

[42]  A. Nolan,et al.  Redifferentiation of insulin-secreting cells after in vitro expansion of adult human pancreatic islet tissue. , 2005, Biochemical and biophysical research communications.

[43]  B. Raaka,et al.  Epithelial-to-Mesenchymal Transition Generates Proliferative Human Islet Precursor Cells , 2004, Science.

[44]  M. Imamura,et al.  Hepatic regeneration and enforced PDX-1 expression accelerate transdifferentiation in liver. , 2004, Surgery.

[45]  Ahmed Mansouri,et al.  Opposing actions of Arx and Pax4 in endocrine pancreas development. , 2003, Genes & development.

[46]  M. Fujimiya,et al.  NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice , 2003, Nature Medicine.

[47]  B. Petersen,et al.  In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[48]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[49]  S. Bonner-Weir,et al.  In vitro cultivation of human islets from expanded ductal tissue. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[50]  I. Rooman,et al.  Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro , 2000, Diabetologia.

[51]  I. Barshack,et al.  Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia , 2000, Nature Medicine.

[52]  L. Harrison,et al.  Laminin-1 promotes differentiation of fetal mouse pancreatic beta-cells. , 1999, Diabetes.

[53]  A. Smit,et al.  Identification of a Molluscan Homologue of the Neuroendocrine Polypeptide 7B2* , 1997, The Journal of Biological Chemistry.

[54]  I. Rooman,et al.  In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells , 2004, Diabetologia.

[55]  Ames,et al.  Islet Transplantation in Seven Patients with Type 1 Diabetes Mellitus Using a Glucocorticoid-Free Immunosuppressive Regimen , 2000 .

[56]  Development and Stem Cells Research Article , 2022 .