Regulatory variants active in iPSC-derived pancreatic progenitor cells are associated with Type 2 Diabetes in adults

Pancreatic progenitor cells (PPC) are an early developmental multipotent cell type that give rise to mature endocrine, exocrine, and ductal cells. To investigate the extent to which regulatory variants active in PPC contribute to pancreatic complex traits and disease in the adult, we derived PPC from induced pluripotent stem cells (iPSCs) of nine unrelated individuals and generated single cell profiles of chromatin accessibility (snATAC-seq) and transcriptome (scRNA-seq). While iPSC-PPC differentiation was asynchronous and included cell types from early to late developmental stages, we found that the predominant cell type consisted of NKX6-1+ progenitors. Genetic characterization using snATAC-seq identified 86,261 regulatory variants that either displayed chromatin allelic bias and/or were predicted to affect active transcription factor (TF) binding sites. Integration of these regulatory variants with 380 fine-mapped type 2 diabetes (T2D) risk loci identified regulatory variants in 209 of these loci that are functional in iPSC-PPC, either by affecting transcription factor binding or through association with allelic effects on chromatin accessibility. The PPC active regulatory variants in 65 of these loci showed strong evidence of causally underlying the association with T2D. Our study shows that studying the functional associations of regulatory variation in iPSC-PPC enables the identification and characterization of causal SNPs for adult Type 2 Diabetes.

Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells Promote the Proliferation and Differentiation of Pancreatic Progenitor Cells and the Engraftment Function of Islet-Like Cell Clusters

Pancreatic progenitor cells (PPCs) are the primary source for all pancreatic cells, including beta-cells, and thus the proliferation and differentiation of PPCs into islet-like cell clusters (ICCs) opens an avenue to providing transplantable islets for diabetic patients. Meanwhile, mesenchymal stem cells (MSCs) can enhance the development and function of different cell types of interest, but their role on PPCs remains unknown. We aimed to explore the mechanism-of-action whereby MSCs induce the in vitro and in vivo PPC/ICC development by means of our established co-culture system of human PPCs with human fetal bone marrow-derived MSCs. We examined the effect of MSC-conditioned medium on PPC proliferation and survival. Meanwhile, we studied the effect of MSC co-culture enhanced PPC/ICC function in vitro and in vivo co-/transplantation. Furthermore, we identified IGF1 as a critical factor responsible for the MSC effects on PPC differentiation and proliferation via IGF1-PI3K/Akt and IGF1-MEK/ERK1/2, respectively. In conclusion, our data indicate that MSCs stimulated the differentiation and proliferation of human PPCs via IGF1 signaling, and more importantly, promoted the in vivo engraftment function of ICCs. Taken together, our protocol may provide a mechanism-driven basis for the proliferation and differentiation of PPCs into clinically transplantable islets.

Jag1 modulates an oscillatory Dll1-Notch-Hes1 signaling module to coordinate growth and fate of pancreatic progenitors

SummaryNotch signaling controls proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into bipotent progenitors (BPs) and unipotent pro-acinar cells (PACs). Here we uncover fast ultradian oscillations in the ligand Dll1, and the transcriptional effector Hes1, which proved crucial for MPC expansion. Conversely Jag1, a uniformly expressed ligand, curbed MPC growth, but as expression later segregated to PACs it proved critical for specifying all but the most proximal 5% of BPs, while BPs were entirely lost in Jag1, Dll1 double mutants. Moreover, experimentally induced changes in Hes1 oscillation parameters was associated with selective adoption of BP or PAC fates. Anatomically, ductal morphogenesis and organ architecture is minimally perturbed in Jag1 mutants until later stages, when ductal remodeling fails and signs of acinar-to-ductal metaplasia appear. Our study uncovers oscillating Notch activity in the developing pancreas, which along with modulation by Jag1 is required to coordinate MPC growth and fate.

Notch Controls Multiple Pancreatic Cell Fate Regulators Through Direct Hes1-mediated Repression

Notch signaling and its effector Hes1 regulate multiple cell fate choices in the developing pancreas, but few direct target genes are known. Here we use transcriptome analyses combined with chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) to identify direct target genes of Hes1. ChIP-seq analysis of endogenous Hes1 in 266-6 cells, a model of multipotent pancreatic progenitor cells, revealed high-confidence peaks associated with 354 genes. Among these were genes important for tip/trunk segregation such asPtf1aandNkx6-1, genes involved in endocrine differentiation such asInsm1andDll4, and genes encoding non-pancreatic basic-Helic-Loop-Helix (bHLH) factors such asNeurog2andAscl1. Surprisingly, we find that Hes1 binds a large number of loci previously reported to bind Ptf1a, including a site downstream of theNkx6-1gene. Notably, we find a number of Hes1 bound genes that are upregulated by γ-secretase inhibition in pancreas explants independently ofNeurog3function, including the tip progenitor/acinar genes;Ptf1a, Gata4, Bhlha15, andGfi1. Together, our data suggest that Notch signaling suppress the tip cell fate by Hes1-mediated repression of the tip-specific gene regulatory network module that includes transcriptional regulators such as Ptf1a, Gata4, Mist1, and Gfi1. Our data also uncover new molecular targets of Notch signaling that may be important for controlling cell fate choices in pancreas development.