FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells.

Considering their unlimited proliferation and pluripotency properties, human embryonic stem cells (hESCs) constitute a promising resource applicable for cell replacement therapy. To facilitate this clinical translation, it is critical to study and understand the early stage of hESCs differentiation wherein germ layers are defined. In this study, we examined the role of FGF signaling in Activin A-induced definitive endoderm (DE) differentiation in the absence of supplemented animal serum. We found that activated FGF/MAPK signaling is required at the early time point of Activin A-induced DE formation. In addition, FGF activation increased the number of DE cells compared to Activin A alone. These DE cells could further differentiate into PDX1 and NKX6.1 positive pancreatic progenitors in vitro. We conclude that Activin A combined with FGF/MAPK signaling efficiently induce DE cells in the absence of serum. These findings improve our understanding of human endoderm formation, and constitute a step forward in the generation of clinical grade hESCs progenies for cell therapy.

Expression of regulatory genes for pancreas development during murine embryonic stem cell differentiation.

Insulin-producing cells derived from embryonic stem cells could be surrogates for beta cells in diabetes therapy. However, their derivation remains hard to achieve with current protocols which rely on initial embryoid body formation. We assume that factors known to inhibit pancreas development contribute to this limitation in vitro. To evaluate this hypothesis, embryoid bodies were examined after different culture periods by real time RT-PCR to profile the expression of genes known to regulate embryonic pancreas development. Our data indicate that transcripts for pancreas markers (insulin, glucagon and amylase ) were expressed during differentiation, but the highest levels achieved were at least 10(5) times lower than in the adult mouse pancreas. Notch signalling was activated as suggested by Delta, Jagged, Ngn3 and NeuroD1 profiles. However, Sonic hedgehog, a known inhibitor of pancreas induction in vivo drastically increased in day 6 embryoid bodies, while Inhibin betaA and betaB were down-regulated and follistatin up-regulated. Members of the Fibroblast- and Transforming Growth Factor families which pattern the endoderm were expressed at low levels, while those that inhibit pancreas development were highly transcribed. The profile of pancreas regulators expressed in embryoid bodies is therefore not compatible with differentiation of pancreatic and insulin-producing cells. These findings provide an explanation for the limited derivation of such cells to date, in addition to basic information for establishing novel differentiation protocols.

Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of ß-cell neogenesis.

The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into ß-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of ß-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-ß-cell transdifferentiation.

Signaling pathways during maintenance and definitive endoderm differentiation of embryonic stem cells.

Embryonic stem cells (ESCs) have the potential to be used as unlimited resources for tissue replacement therapy, thereby compensating for organ donor shortage. To reach this goal, the molecular principles governing early differentiation events in the developing embryo need to be addressed, understood and properly implemented in vitro. Studies carried out in several vertebrate models have established that Nodal/Activin A, BMP, WNT and FGF signaling pathways regulate early embryo development and that these pathways are similarly used during germ layer formation by cultured ESCs. However, differences have also been identified in the way these pathways function or interact in mouse vs. human ESCs, making it sometimes difficult to extrapolate findings from one system to the other. In this review, we discuss and compare the role of the relevant signaling pathways and their crosstalk during undifferentiated growth and during the endoderm differentiation of mouse and human ESCs.

The use of stem cells for pancreatic regeneration in diabetes mellitus.

The endocrine pancreas represents an interesting arena for regenerative medicine and cell therapeutics. One of the major pancreatic diseases, diabetes mellitus is a metabolic disorder caused by having an insufficient number of insulin-producing ß cells. Replenishment of ß cells by cell transplantation can restore normal metabolic control. The shortage in donor pancreata has meant that the demand for transplantable ß cells has outstripped the supply, which could be met by using alternative sources of stem cells. This situation has opened up new areas of research, such as cellular reprogramming and in vivo ß-cell regeneration. Pluripotent stem cells seem to be the best option for clinical applications of ß-cell regeneration in the near future, as these cells have been demonstrated to represent an unlimited source of functional ß cells. Although compelling evidence shows that the adult pancreas retains regenerative capacity, it remains unclear whether this organ contains stem cells. Alternatively, specialized cell types within or outside the pancreas retain plasticity in proliferation and differentiation. Cellular reprogramming or transdifferentiation of exocrine cells or other types of endocrine cells in the pancreas could provide a long-term solution.

Transplantation of human embryonic stem cell-derived pancreatic endoderm reveals a site-specific survival, growth, and differentiation.

Development of ß-cells from human embryonic stem cells (hESCs) could compensate for the shortage of islet donors required for diabetes therapy. Although pancreatic progenitors have been derived from hESCs using various protocols, no fully functional b-cells could be generated in vitro. We evaluated the in vivo growth and differentiation of PDX1+ pancreatic endoderm cells obtained from hESCs. Here we show site-specific survival and differentiation when comparing cells grafted in the epididymal fat pad or the subcutaneous space of NOD/SCID mice after 12 weeks follow-up. Subcutaneous grafts persisted and expressed PDX1 at all time points analyzed, showed PDX1 and NKX6.1 coexpression after 6 weeks, and contained NGN3+ cells after 12 weeks.These findings suggest that further specification along the pancreatic lineage occured at the subcutaneous site.In sharp contrast, in the fat pad grafts only a minority of PDX1+ cells remained after 2 weeks, and no further pancreatic differentiation was observed later on. In addition, contaminating mesenchymal cells present in the implants further developed into cartilage tissue after 6 weeks implantation in the fat pad, but not in the subcutaneous space. These findings indicate that the in vivo microenvironment plays a critical role in the further differentiation of transplanted pancreatic endoderm cells.

Sonic hedgehog and other soluble factors from differentiating embryoid bodies inhibit pancreas development.

Success of cell-replacement therapy for diabetes will largely depend on the establishment of alternative sources of pancreatic islet grafts. Embryonic stem (ES) cell differentiation toward pancreatic insulin-producing cells offers such perspectives, but there are still many challenges to overcome. Our previous studies suggested that the limited amount of insulin-positive cells derived from ES cells is related to the activation of pancreas inhibitory signals. To confirm this hypothesis, we report here that exposure of mouse embryonic pancreas explants to soluble factors from embryoid bodies (EBs) inhibits growth, morphogenesis, and endocrine and exocrine differentiation as evaluated by explant size and mRNA and protein expression. Sonic Hedgehog (Shh), an established pancreas repressor both at early and late developmental stages, was produced and secreted by EBs, and participated in the inhibitory effect by inducing its target Gli1 in the explants. Inhibition of Hedgehog pathway rescued the differentiation of Insulin-positive cells in the explants. In contrast to pancreatic cells, hepatic progenitors exposed to EB-conditioned medium showed improved differentiation of albumin-positive cells. In a model system of ES cell differentiation in vitro, we found that definitive endoderm induction by serum removal or activin A treatment further increased Hedgehog production and activity in EBs. Concomitantly, downregulation of the pancreas marker Pdx1 was recorded in activin-treated EBs, a phenomenon that was prevented by antagonizing Hedgehog signaling with Hedgehog interacting protein. These data strongly suggest that Hedgehog production in EBs limits pancreatic fate acquisition and forms a major obstacle in the specification of pancreatic cells from ES-derived definitive endoderm. Disclosure of potential conflicts of interest is found at the end of this article.