As the presence of NKX is

As the presence of NKX6-1 is one of the distinguishing features of these two lineages, expression of this transcription factor is used to monitor the emergence of the beta cell lineage in hPSC differentiation cultures. Populations enriched in NKX6-1+ monensin Supplier will give rise to functional beta cells following transplantation into immunodeficient mice, indicating that they do indeed represent progenitors of adult monohormonal lineages (Kelly et al., 2011; Kroon et al., 2008). Currently, the signaling pathways that regulate the generation of the NKX6-1+ progenitors are not well defined. hPSC-line heterogeneity appears to be one factor that can influence the generation of the beta cell progenitors, as Kelly et al. (2011) reported the development of populations consisting of 6%–45% NKX6-1+ cells from different cell lines with the same protocol. More recently, Rezania et al. (2012) showed that activation of PKC can enhance the generation of the NKX6-1+ progenitors, whereas inhibition of the transforming growth factor β (TGF-β) pathway blocks their development in H1 and ESI49 differentiation cultures. The effect of PKC activation, however, has not been tested on a broad range of hPSC lines. In this report, we show that the combination of epidermal growth factor (EGF) and nicotinamide signaling together with inhibition of the bone morphogenetic protein (BMP) pathways promotes the efficient development of NKX6-1+ progenitors from all of the hPSC lines tested. Additionally, we demonstrate that the duration of retinoic acid/fibroblast growth factor 10 (RA/FGF10) induction and inhibition of BMP and hedgehog signaling influences endocrine lineage development, with a short duration favoring the specification of NKX6-1+ progenitors, and an extended duration promoting the development of polyhormonal cells. Following transplantation into NOD scid gamma (NSG) recipient mice, the NKX6-1+ progenitors induced with NOGGIN, EGF, and nicotinamide (NENA) mature and give rise to the different endocrine lineages, including beta-like cells.
Results
Discussion The efficient generation of functional cell types from hPSCs in vitro is dependent on precise manipulation of the appropriate signaling pathways at key developmental stages within the lineage of interest. Efforts to identify the pathways that regulate pancreatic development in vitro are complicated by the existence of two developmental programs that give rise to distinct endocrine cell types. Our findings in this study uncover important differences in the regulation of the two programs and show that NKX6-1+ progenitors are specified within 24 hr from foregut patterned endoderm with the combination of NOGGIN, RA, and FGF10. Hedgehog signaling does not appear to play a role in this specification step. The generation of NKX6-1+ progenitors from this pancreatic endoderm is dependent on EGF and nicotinamide signaling (Figure 5). In contrast, specification of the polyhormonal program requires 72–96 hr of treatment with stage 3 factors and is dependent on inhibition of the hedgehog pathway. Expansion of the derivative lineages is not dependent on the addition of exogenous EGF and nicotinamide (Figure 5). By exploiting these differences, it is now possible to design differentiation strategies that selectively promote the generation of populations of NKX6-1+ progenitors, with minimal contamination of the polyhormonal lineage. Immunohistochemical analyses of fetal pancreata revealed that the very first hormone+ cells to arise in the human are insulin+ monohormonal cells (G7.5w). Their development is followed by the emergence of other monohormonal cells and a small population of polyhormonal cells (G8w–G9w). These observations suggest that human pancreatic development lacks the two waves of endocrine commitment found in the mouse (Jennings et al., 2013). It has been speculated that the absence of an early first-transition endocrine population in humans is due to the lack of direct contact of the developing pancreatic bud with the notochord (Jennings et al., 2013). In the mouse, expression of Pdx1 is induced by E8.5, when the endoderm is in contact with the notochord, whereas in humans it is first expressed when the gut tube has been separated from the notochord by the dorsal aorta. Given that the notochord is a known source of hedgehog inhibitors, these observations suggest that the duration of hedgehog inhibition may play a role in the development of the polyhormonal population in vivo. Our findings are consistent with this hypothesis and clearly demonstrate that the extended induction period with stage 3 media containing a hedgehog inhibitor promotes the development of polyhormonal cells.