Project description:Formation of the hepato-pancreato-biliary organ system in mammals is a paradigm for organogenesis, whereby a small progenitor population of the ventral foregut gives rise to a multitude of different adult tissues, including liver, pancreas, gallbladder, and extra-hepatic bile ducts. The multipotent ventral foregut cell population undergoes an initial fate segregation into hepatic and pancreato-biliary progenitors in response to signaling cues from surrounding mesodermal tissues. Subsequently, pancreato-biliary progenitors give rise to ventral pancreatic and gallbladder progenitors. In this study, we used single-cell RNA sequencing to molecularly characterize progenitor populations in the ventral foregut that contribute to the formation of hepatic, pancreatic, and biliary organ rudiments throughout organogenesis.
Project description:Endoderm cells undergo a sequence of fate choices to generate insulin-secreting M-NM-2 cells. Studies of chromatin transitions during this process have been limited to the pancreatic progenitor stage that can be reconstituted from stem cells in vitro, with a gap in understanding the induction of endocrine cells. To address this, we established conditions for isolating endoderm cells, pancreatic progenitors, and endocrine cells from different staged embryos and performed genome wide analysis of the H3K27me3 mark of the repressive Polycomb complex. During the transition from endoderm to pancreas progenitors and during the transition from pancreas progenitors to endocrine cells, genes that lose the H3K27me3 mark typically encode transcriptional regulators, whereas genes that acquire the mark typically are involved in cell biology morphogenesis. Precocious depletion of the EZH2, a H3K27 methylase, at the pancreas progenitor stage enhanced the production of endocrine cells, leading to a later increase in pancreatic beta cells. Similarly, pharmacologic inhibition of EZH2 in embryonic pancreatic tissue explants and human embryonic stem cell cultures led to an increase in endocrine progenitors in vitro. These findings reveal a repeating target gene pattern in H3K27me3 dynamics and provide a means to modulate M-NM-2 cell development from stem cells. Analyzed five FACS-sorted tissues in early mouse embryo; for each tissue we sequenced H3K27me3 and input; no replicates
Project description:Pancreatic stellate cells are thought to be the predominant source of cancer-associated fibroblasts (CAFs) in pancreatic cancer. We developed a mouse model which allows us to track and analyze stellate cells and stellate cell-derived CAFs in vivo during pancreatic tumorigenesis for the first time. We find that stellate cells in fact give rise to a minority of all CAFs. Here, we have used lineage reporters to isolate stellate cell-derived and non-stellate cell-derived CAFs and compared them by RNA-seq.
Project description:Endoderm cells undergo a sequence of fate choices to generate insulin-secreting β cells. Studies of chromatin transitions during this process have been limited to the pancreatic progenitor stage that can be reconstituted from stem cells in vitro, with a gap in understanding the induction of endocrine cells. To address this, we established conditions for isolating endoderm cells, pancreatic progenitors, and endocrine cells from different staged embryos and performed genome wide analysis of the H3K27me3 mark of the repressive Polycomb complex. During the transition from endoderm to pancreas progenitors and during the transition from pancreas progenitors to endocrine cells, genes that lose the H3K27me3 mark typically encode transcriptional regulators, whereas genes that acquire the mark typically are involved in cell biology morphogenesis. Precocious depletion of the EZH2, a H3K27 methylase, at the pancreas progenitor stage enhanced the production of endocrine cells, leading to a later increase in pancreatic beta cells. Similarly, pharmacologic inhibition of EZH2 in embryonic pancreatic tissue explants and human embryonic stem cell cultures led to an increase in endocrine progenitors in vitro. These findings reveal a repeating target gene pattern in H3K27me3 dynamics and provide a means to modulate β cell development from stem cells.
Project description:To understand the pathophysiology of diabetes and to develop more effective treatments, we need to unravel how pancreatic endocrine lineages are generated. ISL1 transcription factor has important role in differentiation, maturation and maintenace of endocrine cells. Eliminating ISL1 in mouse pancreatic endocrine progenitors leads to the diabetic phenotype with altered islet architecture. To evaluate effects of lacking transcription factor on molecular level we performed single cell transcriptomics.
Project description:The goal of this study is to molecularly characterize regulatory variation in pancreatic progenitor cells (PPC). Here, we derived PPC from iPSCs of nine iPSCORE individuals (DeBoever et al., 2017; Panopoulos et al., 2017), and generated RNA-seq, ATAC-seq, scRNA-seq, and snATAC-seq. We strive to understand the role of functional genetic variation during fetal pancreatic development that later give rise to adult pancreatic diseases.
Project description:Little is known about the molecular mechanisms that underlie the allocation of human pluripotent stem cell-derived pancreatic progenitors (hPSC-PPs) into specific endocrine lineages. By systematically interrogating the components of pancreatic differentiation media, we identify methods to reliably generate hPSC-PPs with high-grade NKX6-1 expression, but find that this alone has a minimal effect on modulating the endocrine composition of hPSC-derived islets. We then test the effect of altering the signaling cues provided after progenitor specification and find that the BMP and FGF/MEK pathways can be manipulated to shift hPSC-PPs between islet and non-islet endocrine lineages. We further find that the method of hPSC-PP specification integrates with these later cues in a way that can be leveraged to selectively increase the frequencies of specific islet lineages or enterochromaffin (EC)-like cells. Using a model of disrupted murine islet development that gives rise to pancreatic EC-like cells, we identify prolonged NEUROG3 expression as a conserved feature associated with both human and murine pancreatic EC-like cell differentiation. In addition to expanding our understanding of pancreatic endocrine lineage allocation, these findings provide some of the first tools to fine-tune composition of hPSC-derived islets for research and therapeutic applications.
Project description:Retinoic acid (RA) signaling is essential for multiple developmental processes, including appropriate pancreas formation from the foregut endoderm. RA is also required to generate pancreatic progenitors from human pluripotent stem cells. However, the role of RA during the later stages of pancreas development is not well understood. In this study, we generated an inducible system to block RA signaling and demonstrate that disruption of the RA pathway within the Neurog3-expressing endocrine progenitor population is required for appropriate mouse b cell differentiation and repression of critical d cell genes, including Somatostatin. In addition, inhibition of the RA pathway in hESC-derived pancreatic progenitors downstream of NEUROG3 induction impairs INSULIN expression. We further determined that RA-regulation of endocrine cell differentiation is mediated through Wnt pathway components. Together, these data demonstrate the importance of RA signaling in endocrine specification and identify conserved mechanisms by which RA signaling directs endocrine cell fate.
Project description:During embryonic development, islet progenitors are specified from pancreatic duct cells by transient expression of Neurog3, a transcription factor necessary and sufficient for initiation of islet development. To understand the dynamics of Neurog3-dependent endocrine cell fate determination, in this study we used ATAC-Seq to identify accessible genomic regions of purified duct, endocrine progenitor, and endocrine cells isolated from mice with varying Neurog3 dosage
Project description:To improve β-like cell differentiation from human pluripotent stem cells (hPSCs), we aimed to substitute a mechanistically elusive compound – nicotinamide (NA), which promotes pancreatic progenitor (PP) commitment, with a pathway-specific compound. By performing a low-throughput chemical screening, we demonstrated that tankyrase inhibitors (TNKSi) can replace NA and efficiently generate PPs. Surprisingly, highly selective TNKSi, such as WIKI4, generate PPs that give rise to islet-like populations with improved β-like cell frequencies and glucose responsiveness compared to NA-derived PPs. Characterization of differentially expressed genes (DEG) between WIKI4 and NA treatment revealed that WIKI4-derived PPs expressed higher levels of genes associated with integrin signalling and actin cytoskeleton pathways. Our findings show that compared to NA, WIKI4 treatment promotes changes in cell shape and motility. Together, our results provide novel insights into pancreatic differentiation and a model that better recapitulates native events associated with pancreas and endocrine commitment.