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.

Cell cultureGeneration of stem cell derived beta-like cells from human embryonic stem cells and from induced pluripotent stem cells MEL1 human embryonic stem cells (hESC) containing the INS GFP/W reporter 18 (referred to as pINSGFP throughout the manuscript) and sub-clones thereof 19,31 , type 1 diabetes patient induced pluripotent stem cells (T1D-iPSC) 24 (Supp. Fig 8 .) and iPSC from a healthy donor 48 (Supp. Fig. 9), as well as pNKX6.1 GFP reporter iPSC 46 were employed in this study. All iPSC lines were maintained on hESC qualified Matrigel (Corning #354277) in mTeSR+ media (STEMCELL Technologies #05826). MEL1 Subclones were only used for bulk RNA sequencing analysis experiments. Differentiation to stem cellderived beta-like cells (sBCs) was carried out in suspension-based, low attachment suspension culture plates as described 19 or in a bioreactor magnetic stirring system (Reprocell #ABBWVS03A-6, #ABBWVDW-1013, #ABBWBP03N0S-6) as follows:Confluent hESC cultures were dissociated into single-cell suspensions by incubation with TrypLE (Gibco #12-604-021) for 6 min at 37C. Detached cells were quenched with mTESR media. Live cells were counted using a MoxiGo II cell counter (Orflow), followed by seeding 0.5 × 10 6 cells per ml in mTeSR media supplemented with 10 M ROCK inhibitor (Y-27632, R&D Systems #1254-50) (cluster media). Bioreactors were placed on a magnetic stirring system set at 60 RPM in a cell culture incubator at 5 % CO2 to induce sphere formation for 48 h. To induce definitive endoderm differentiation, spheres were collected in a 50 mL Falcon tube, allowed to settle by gravity, washed once with RPMI

Both type 1 and type 2 diabetes are characterized by a progressive loss of beta cell mass that contributes to impaired glucose homeostasis. Although an optimal treatment option would be to simply replace the lost cells, it is now well established that unlike many other organs, the adult pancreas has limited regenerative potential. For this reason, significant research efforts are focusing on methods to induce beta cell proliferation (replication of existing beta cells), promote beta cell formation from alternative endogenous cell sources (neogenesis), and/or generate beta cells from pluripotent stem cells. In this article, we will review (i) endogenous mechanisms of beta cell regeneration during steady state, stress and disease; (ii) efforts to stimulate endogenous regeneration and transdifferentiation; and (iii) exogenous methods of beta cell generation and transplantation.

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