Aims/hypothesisHuman embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge.MethodsWe used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors. This approach avoids the use of feeders, stroma cells or serum, all of which can interfere with experimental outcomes and could preclude future clinical applications.ResultsThis study reveals, for the first time, that activin/TGF-β signalling blocks pancreatic specification induced by retinoic acid while promoting hepatic specification in combination with bone morphogenetic protein and fibroblast growth factor. Using this knowledge, we developed culture systems to differentiate human pluripotent stem cells into near homogenous population of pancreatic and hepatic progenitors displaying functional characteristics specific to their natural counterparts. Finally, functional experiments showed that activin/TGF-β signalling achieves this essential function by controlling the levels of transcription factors necessary for liver and pancreatic development, such as HEX and HLXB9.Conclusion/interpretationOur methods of differentiation provide an advantageous system to model early human endoderm development in vitro, and also represent an important step towards the generation of pancreatic and hepatic cells for clinical applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2687-x) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.
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.
Cell therapy in the form of human islet transplantation has been a successful form of treatment for patients with type 1 diabetes for over 10 years, but is significantly limited by lack of suitable donor material. A replenishable supply of insulin-producing cells has the potential to address this problem; however to date success has been limited to a few preclinical studies. Two of the most promising strategies include differentiation of embryonic stem cells and induced pluripotent stem cells towards insulin-producing cells and transdifferentiation of acinar or other closely related cell types towards β-cells. Here, we discuss recent progress and challenges that need to be overcome in taking cell therapy to the clinic.
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