Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population

et al. (2011) Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population. PLoS ONE 6(9): e24058. doi:10.1371/journal.pone.0024058 Pdx1 and Ngn3 Overexpression Enhances Pancreatic Differentiation of Mouse ES Cell-Derived Endoderm Population Atsushi Kubo 0 Robert Stull 0 Mitsuaki Takeuchi 0 Kristina Bonham 0 Valerie Gouon-Evans 0 Masayuki Sho 0 Masayuki Iwano 0 Yoshihiko Saito 0 Gordon Keller 0 Ralph Snodgrass 0 Martin Pera, University of Southern California, United States of America 0 1 First Department of Internal Medicine, Nara Medical University , Kashihara, Nara, Japan, 2 VistaGen Therapeutics , Inc. , South San Francisco , California, United States of America, 3 The Department of Gene and Cell Medicine, Mount Sinai School of Medicine , New York , New York, United States of America, 4 First Department of Surgery, Nara Medical University , Kashihara, Nara , Japan , 5 McEwen Centre for Regenerative Medicine , Toronto , Canada In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic b-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the b-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the bTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing b-islet cells from ES cells. - Islet transplantation has been shown to be useful in the treatment of patients with type 1 diabetes, even resulting in insulin independence [1,2]. However, this therapeutic approach is limited by a shortage of transplantable islets. Consequently, other potential sources of b-islet cells are currently being sought. Two such alternatives are pancreatic duct cells and endocrine progenitor cells [35]. Another possible source of insulinproducing cells are pluripotent stem cells (ES or iPS), which are self-renewing and retain the potential to differentiate into all three germ layers [6,7]. This makes pluripotent stem cells a very useful experimental model to study pancreatic development, and the pancreatic cells that develop from these stem cells are a potential source of islet cells for diabetes therapy. Within the embryo, the pancreas derives from epithelium of the foregut endoderm, which forms dorsal and ventral buds on approximately day 9 of murine embryonic development [8,9]. During pancreas and b-islet cell development, the transcription factor Pdx1 is expressed in the embryonic gut epithelium that will later give rise to the dorsal and ventral pancreas [1012]. The fact that Pdx1 is a master gene for early pancreatic development is demonstrated by the pancreatic agenesis occurring after bud formation in mice lacking functional Pdx1 [13]. In addition, ectopic expression of Pdx1 induces pancreatic budding from the gut epithelium [14]. After pancreatic bud formation, another transcription factor, Neurogenin 3 (Ngn3), plays a critical role in the formation of pancreatic endocrine precursors. Notably, mice lacking Ngn3 also lack the four pancreatic endocrine cells, which produce insulin (Ins), glucagon (Gcg), somatostatin (Sst) and pancreatic polypeptide (Ppy) [15]. Moreover, lineage tracking studies using the CreER loxP system have shown that, after Pdx1 expression, Ngn3positive cells give rise to all four endocrine cell types [16], suggesting that Ngn3 is expressed in the early endocrine progenitor cells that give rise to, and presumably contributes to the differentiation of, the four endocrine cell types. In addition, targeted disruption in mice has shown that various other transcriptional factors, including Pax4 [17], NeuroD [18], Nkx2.2 [19] and Nkx6.1 [20], are also critical for differentiation of endocrine progenitors into insulin producing b-islet cells. These factors must be expressed in a correct temporal order for appropriate lineage specification and differentiation of gut endoderm, pancreatic progenitors, endocrine progenitors and, finally, pancreatic b-islet cells. We previously established a protocol for the activin-induced development of definitive endoderm during mouse ES cell differentiation [21,22]. Similarly, DAmour et al. reported that pancreatic hormone-expressing endocrine cells can be differentiated from human ES cell-derived endoderm through induction with activin [23,24]. They further showed that the soluble growth factors that participate in pancreatic development during human embryonic development can mimic that process during human ES cell differentiation in vitro [23,24]. In the present study, we evaluated the transcriptional regulation that is critical for induction of b-islet cell differentiation from mouse ES cell-derived endoderm. Previous study have demonstrated that biphasic induction of Pdx1 induce insulin producing cells in ES cell derived endoderm [25]. In this study, we show that temporally controlled expression of Pdx1 and Ngn3 induces pancreatic endocrine genes, various b-islet cell-related transcription factors, including Pax4, Pax6, Isl1 and Nkx2.2, and efficiently yields a high frequency of b-islet cells that express very high levels of insulin. Lastly, although these b-islet cells appropriately process and secrete insulin and C-peptide protein in response to various insulin secretagogues, they do not demonstrate adult levels of glucose-controlled insulin secretion. Pdx-1 induces insulin mRNA expression in activininduced endoderm embryoid bodies (EBs) It is well known that Pdx1 is a master gene for early pancreatic development from the gut tube, and previous protocols resulted in very modest levels of Pdx1. Therefore, we engineered ES cells with a doxycycline (Dox)-inducible Pdx1 (tet-pdx1 ES cells) expression vector to evaluate the effect of temporal control of Pdx1 upregulation. For thes (...truncated)