A novel progenitor population in pancreatic endocrine cell development

胰腺内分泌细胞合成和分泌能量平衡和营养代谢所需的激素。特别是，需要产生胰岛素的β细胞来控制全身血糖水平，它们的丧失或功能障碍导致糖尿病。构成胰岛的内分泌细胞在发育过程中由内分泌前体细胞或前内分泌细胞分化形成。虽然所有五种内分泌谱系都来自一个共同的内分泌前体库，但这些前内分泌细胞如何致力于特定的内分泌细胞亚型却知之甚少。从内分泌前体细胞到分化的内分泌细胞的分化的精确阶段的更详细的分子表征是需要的。 在胰腺器官发生期间，导管上皮中的一部分细胞短暂表达神经生成素3（Ngn 3），这是一种内分泌谱系特化所需的促内分泌转录因子。谱系追踪表明，所有五种内分泌谱系都来源于这些Ngn 3表达前体，并且Ngn 3表达细胞不会产生除内分泌外的其他胰腺谱系。尚未确定每个Ngn 3（+）细胞是否预先定型为特定的内分泌谱系，或者细胞命运决定是否在Ngn 3表达后的后期阶段做出。 使用单细胞RNA测序（RNA-seq）的小鼠胰腺，我们的实验室已经确定了一种新的细胞群，表达内分泌谱系基因，并定义为差异表达的转录因子Fev。该FevHI群体的特征还在于缺乏Ngn 3（+）或在分化的内分泌细胞中检测到的胰腺激素的表达。体内Ngn 3表达细胞的谱系追踪揭示了Fev表达细胞是Ngn 3（+）促内分泌细胞的后代。因此，这种新的推定的内分泌前体人口似乎代表了一个中间阶段后，短暂Ngn 3的表达和激素收购之前。 该提议中概述的实验开始集中于表征人胎儿和成人胰腺中这种新型FevHI群体的空间和时间外观。此外，还将进行遗传谱系追踪研究，以确定这种推定的FevHI中间体前体的下游谱系。最后，由于Fev全身敲除小鼠显示葡萄糖稳态缺陷，因此将使用基于人胚胎干细胞的平台和基因组工程策略研究FEV本身在人β细胞的内分泌细胞特化和功能成熟中的作用。这些研究的总体目标是为内分泌细胞命运分配的基础生物学提供新的见解，并利用这些知识产生功能性人胚胎干细胞衍生的β细胞和再生内源性β细胞用于治疗糖尿病。

Endocrine cells in the pancreas synthesize and secrete hormones required for energy homeostasis and nutrient metabolism. In particular, insulin-producing beta cells are required to control systemic blood glucose levels, and their loss or dysfunction leads to diabetes. The endocrine cells that make up pancreatic islets form during development from the differentiation of endocrine precursors, or pro-endocrine cells. Although all five endocrine lineages derive from a common pool of endocrine precursors, how these pro-endocrine cells become committed to a particular endocrine cell subtype is poorly understood. A more detailed molecular characterization of the precise stages of differentiation from an endocrine precursor to a differentiated endocrine cell is needed. During pancreatic organogenesis, a subset of cells in the ductal epithelium transiently expresses Neurogenin3 (Ngn3), a pro-endocrine transcription factor that is required for endocrine lineage specification. Lineage tracing has demonstrated that all five endocrine lineages are derived from these Ngn3-expressing precursors, and that Ngn3-expressing cells do not give rise to other pancreatic lineages besides endocrine. It is not yet determined if each Ngn3(+) cell is pre-committed to a specific endocrine lineage or if cell fate decisions are made at a later stage, after Ngn3 expression. Using single-cell RNA sequencing (RNA-seq) of murine pancreata, our laboratory has identified a novel cell population that expresses endocrine lineage genes and is defined by differential expression of the transcription factor Fev. This FevHI population is also characterized by lack of expression of Ngn3(+) or of the pancreatic hormones detected in differentiated endocrine cells. Lineage tracing of Ngn3-expressing cells in vivo has revealed that Fev-expressing cells are descendants of Ngn3(+) pro-endocrine cells. Thus, this novel putative endocrine precursor population appears to represent an intermediate stage following transient Ngn3 expression and before hormone acquisition. The experiments outlined in this proposal begin with a focus on characterizing the spatial and temporal appearance of this novel FevHI population in human fetal and adult pancreata. In addition, genetic lineage tracing studies will be undertaken to define the downstream lineage of this putative FevHI intermediate precursor. Lastly, as Fev whole body knockout mice display defects in glucose homeostasis, the role of FEV itself in endocrine cell specification and functional maturation of human beta cells will be investigated using a human embryonic stem cell-based platform and genomic engineering strategies. The overarching goal of these studies is both to provide new insights into the basic biology underlying endocrine cell fate allocation, as well as to harness this knowledge for generating functional human embryonic stem cell-derived beta cells and regenerating endogenous beta cells for the treatment of diabetes.