A novel progenitor population in pancreatic endocrine cell development

胰腺中的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，它们的缺失或功能障碍会导致糖尿病。组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而形成。虽然所有五种内分泌谱系都来自一个共同的内分泌前体细胞池，但这些内分泌前体细胞是如何与特定的内分泌细胞亚型结合的还知之甚少。需要对从内分泌前体到分化的内分泌细胞的精确分化阶段进行更详细的分子表征。在胰腺器官发生过程中，导管上皮中的一部分细胞瞬时表达神经生成素3(Ngn3)，这是一种内分泌前体转录因子，是内分泌谱系指定所必需的。谱系追踪表明，所有五种内分泌谱系都来自于这些表达Ngn3的前体，并且表达Ngn3的细胞不能产生内分泌以外的其他胰腺谱系。目前还不确定是否每个Ngn3()细胞都预先承诺了特定的内分泌谱系，或者是否在Ngn3表达后的较晚阶段决定了细胞的命运。利用小鼠胰腺的单细胞RNA测序(RNA-seq)，我们的实验室已经确定了一个新的细胞群体，该细胞群体表达内分泌谱系基因，并由转录因子Fev的差异表达来定义。这种FevHI群体的特征还包括在分化的内分泌细胞中检测到的Ngn3()或胰腺激素的缺乏。体内表达Ngn3的细胞的谱系追踪表明，Fev表达的细胞是Ngn3()前内分泌细胞的后代。因此，这个新的假定的内分泌前体群体似乎代表了短暂的Ngn3表达之后和激素获得之前的中间阶段。这项建议中概述的实验首先集中在描述这种新的FevHI群体在人类胎儿和成人胰腺中的空间和时间外观。此外，还将进行遗传谱系追踪研究，以确定这种假定的FevHI中间前体的下游谱系。最后，由于FEV全身基因敲除小鼠表现出葡萄糖稳态缺陷，将利用基于人类胚胎干细胞的平台和基因组工程策略来研究FEV本身在内分泌细胞规范和人β细胞功能成熟中的作用。这些研究的首要目标是为内分泌细胞命运分配的基本生物学提供新的见解，并利用这些知识产生功能性的人类胚胎干细胞衍生的β细胞，并再生用于治疗糖尿病的内源性β细胞。

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.