A novel progenitor population in pancreatic endocrine cell development

胰腺内分泌细胞发育中的新型祖细胞群

• 批准号: 10253286
• 负责人: Julie Beth Sneddon
• 金额: $ 9.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10253286
• 关键词: Adult; Appearance; Atlases; Beta Cell; Biology; Blood Glucose; CRISPR interference; Cell Differentiation process; Cells; ChIP-seq; Data Set; Defect; Development; Diabetes Mellitus; Down-Regulation; Ductal Epithelium; Endocrine; Engineering; Event; Functional disorder; Genes; Genetic; Genetic Transcription; Genomics; Goals; Homeostasis; Hormones; Human; Impairment; Insulin; Islet Cell; Islets of Langerhans; Knockout Mice; Knowledge; Laboratories; Maintenance; Maps; Mediating; Molecular; Mus; Natural regeneration; Organogenesis; Pancreas; Pancreatic Hormones; Pathway interactions; Population; Resolution; Role; Source; Structure of beta Cell of islet; Technology; Testing; Time; Tissue imaging; Tissues; Work; base; beta cell replacement; blood glucose regulation; body system; differential expression; endocrine pancreas development; experimental study; fetal; glucose metabolism; human embryonic stem cell; human pluripotent stem cell; in vivo; insight; islet; mouse model; novel; novel marker; nutrient metabolism; pancreatic islet function; postnatal human; progenitor; real-time images; recombinase; regenerative therapy; single-cell RNA sequencing; stem cell differentiation; stem cell technology; stem cells; transcription factor; transcriptome sequencing; transcriptomics

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.

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，产生胰岛素的β细胞是控制全身血糖水平所必需的，它们的丧失或功能障碍会导致糖尿病。构成胰岛的内分泌细胞在发育过程中由内分泌前体或前内分泌细胞分化形成。虽然所有的五种内分泌谱系都来源于一个共同的内分泌前体，但这些促内分泌细胞是如何变成一个特定的内分泌细胞亚型的，人们知之甚少。需要对从内分泌前体到分化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，导管上皮中的一组细胞短暂表达神经原蛋白3 (Ngn3)，这是一种促内分泌转录因子，是内分泌谱系规范所必需的。谱系追踪表明，所有五种内分泌谱系都来源于这些表达ngn3的前体，并且表达ngn3的细胞不会产生除内分泌外的其他胰腺谱系。目前尚不清楚是否每个Ngn3（+）细胞都是预先参与到一个特定的内分泌谱系中，还是在Ngn3表达后的后期才做出细胞命运的决定。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表达内分泌谱系基因的新细胞群，并通过转录因子Fev的差异表达来定义。FevHI人群的另一个特征是分化内分泌细胞中缺乏Ngn3（+）或胰腺激素的表达。体内表达Ngn3细胞的谱系追踪显示，表达fev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瞬时Ngn3表达之后和激素获取之前的中间阶段。本提案中概述的实验首先重点描述了这种新型FevHI在人类胎儿和成人胰腺中的空间和时间外观。此外，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全身敲除小鼠表现出葡萄糖稳态缺陷，我们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本身在人类β细胞内分泌细胞规范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，以及利用这些知识来产生功能性的人类胚胎干细胞来源的β细胞和再生内源性β细胞以治疗糖尿病。