Project description:Pancreatic endocrine cells arise from a NGN3+ population during pancreas organogenesis. To gain a more thorough understanding of this progenitor pool, we used a reporter mouse - NGN3-EGFP - and sorted EGFP+ cells from e15.5 pancreata of control animals. The data generated from this experiment will allow us to visualize gene expression levels in endocrine progenitors during normal development and can be used to compare against mutant animal gene expression.
Project description:Disruptions in pancreatic development lead to health issues such as pancreatic agenesis and congenital diabetes mellitus. Understanding pancreatic organogenesis is critical for identifying disease mechanisms and developing regenerative therapies. The pancreas consists of endocrine and exocrine cells, both of which are derived from multipotent progenitor cells (MPCs). MPC proliferation and differentiation are tightly controlled by multiple mechanisms, including post-transcriptional regulation by miRNAs. However, these regulatory factors are not fully understood. Here, we profiled miRNA expression in MPCs and identified that miR-302 was highly enriched during the earliest stages of pancreatic development. Loss of miR-302 resulted in reduced pancreatic size without altering the proportions of endocrine and exocrine cells at E17.5, suggesting that miR-302 regulates MPC number rather than differentiation. Transcriptomic analysis at E10.5 revealed that miR-302 modulates genes involved in the Wnt signaling pathway and cell cycle progression. Notably, miR-302 prolonged S phase in MPCs, leading to slower cell proliferation and a smaller MPC pool at E10.5. These findings provide the first comprehensive miRNA profile during early pancreatic development and establish miR-302 as a critical regulator of MPC number and pancreas size.
Project description:Disruptions in pancreatic development lead to health issues such as pancreatic agenesis and congenital diabetes mellitus. Understanding pancreatic organogenesis is critical for identifying disease mechanisms and developing regenerative therapies. The pancreas consists of endocrine and exocrine cells, both of which are derived from multipotent progenitor cells (MPCs). MPC proliferation and differentiation are tightly controlled by multiple mechanisms, including post-transcriptional regulation by miRNAs. However, these regulatory factors are not fully understood. Here, we profiled miRNA expression in MPCs and identified that miR-302 was highly enriched during the earliest stages of pancreatic development. Loss of miR-302 resulted in reduced pancreatic size without altering the proportions of endocrine and exocrine cells at E17.5, suggesting that miR-302 regulates MPC number rather than differentiation. Transcriptomic analysis at E10.5 revealed that miR-302 modulates genes involved in the Wnt signaling pathway and cell cycle progression. Notably, miR-302 prolonged S phase in MPCs, leading to slower cell proliferation and a smaller MPC pool at E10.5. These findings provide the first comprehensive miRNA profile during early pancreatic development and establish miR-302 as a critical regulator of MPC number and pancreas size.
Project description:The pancreatic islet contains multiple hormone+ endocrine lineages (alpha, beta, delta, PP and epsilon cells), but the developmental processes that underlie endocrinogenesis are poorly understood. Here, we generated novel mouse lines and combined them with various genetic tools to enrich all types of hormone+ cells for well-based deep single-cell RNA sequencing (scRNA-seq), and gene coexpression networks were extracted from the generated data for the optimization of high-throughput droplet-based scRNA-seq analyses. These analyses defined an entire endocrinogenesis pathway in which different states of endocrine progenitor (EP) cells sequentially differentiate into specific endocrine lineages in mice. Subpopulations of the EP cells at the final stage (EP4-early and EP4-late) show different potentials for distinct endocrine lineages. epsilon cells and an intermediate cell population were identified as distinct progenitors that independently generate both alpha and PP cells. Single-cell analyses were also performed to delineate the human pancreatic endocrinogenesis process. Although the developmental trajectory of pancreatic lineages is generally conserved between humans and mice, clear interspecies differences, including differences in the proportions of cell types and the regulatory networks associated with the differentiation of specific lineages, have been detected. Our findings support a model in which sequential transient progenitor cell states determine the differentiation of multiple cell lineages and provide a blueprint for directing the generation of pancreatic islets in vitro.
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:The pancreas is an endodermal organ with exocrine and endocrine cell compartments. The embryonic development of the pancreas relies on the the coordinated action of pancreatic progenitors and its surrounding microenvironments. The most abundant cell type of the pancreatic microenviroment are mesenchymal cells. These cells are known to contribute to the pancreas organ growth, morphogenesis and differentiation. In this study, we used single-cell RNA sequencing to molecularly characterize mesenchymal and pancreatic progenitor populations in the dorsal pancreas of the mouse embryos.
Project description:This experiment used RNA-Seq technology to explore gene expression in mouse Ngn3^GFP/+ [het] FACS sorted pancreatic cells at E15.5 (commited endocrine progenitor cells) and in Ngn3^GFP/GFP [null] at E15.5 (defective endocrine progenitor cells). This experiment is designed to understand the gene expression alteration in the endocrine lineage at different embryonic days. The aim is to understand both Ngn3 dependent and independent gene expression profiles so as to reveal the instructive signals that specfy the collective endocrine islet cell fate or specific islet cell type.
Project description:Primary human pancreatic ductal organoids (HPDO) have emerged as a model to study pancreas biology and model disease like pancreatitis and pancreatic cancer. Yet, donor material availability, genetic variability and a lack of extensive benchmarking to healthy and disease pancreas limits the range of applications. To address this gap, we established porcine pancreatic ductal organoids (PPDO) as a system from a reliable, genetically defined and easily obtainable source to model pancreatic ductal/progenitor biology. We benchmarked PPDO to HPDO and primary porcine pancreas using single-cell RNA sequencing (scRNA-Seq). We observed no overt phenotypic differences in PPDO derived from distinct developmental stages using extensive proteomics profiling, with a WNT/basal cell signaling enriched population characterizing PPDO. PPDO exhibited differentiation potential towards mature ductal cells and limited potential towards endocrine lineages. We used PPDO as a chemical screening platform to assess the safety of FDA-approved drugs and showed conserved toxicity of statins and α-adrenergic receptor inhibitors between PPDO and HPDO cultures. Overall, our results highlight the PPDO as a model for mammalian duct/progenitor applications.