Deciphering Pancreatic Islet β Cell and α Cell Maturation Pathways and Characteristic Features at the Single-Cell Level. Mus musculus

Pancreatic β and α cells play essential roles in maintaining glucose homeostasis. However, the mechanisms by which these distinct cell populations are generated, expand, and mature during pancreas development remain unclear. In this study, we addressed this critical question by performing a single-cell transcriptomic analysis of mouse β and α cells sorted from fetal to adult stages. We discovered that β and α cells use different regulatory strategies for their maturation and that cell proliferation peaks at different developmental times. However, the quiescent and proliferative cells in both the β lineage and α lineage are synchronous in their maturation states. The heterogeneity of juvenile β cells reflects distinct cell-cycling phases, origins, and maturation states, whereas adult β cells are relatively homogeneous at the transcriptomic level. These analyses provide not only a high-resolution roadmap for islet lineage development but also insights into the mechanisms of cellular heterogeneity, cell number expansion, and maturation of both β and α cells. Overall design: The overall goal of this study was to define the roadmaps for pancreatic β- and α-cell development. Specifically, we performed single-cell RNA-seq at various developmental stages of E17.5, P0, P3, P9, P15, P18 and P60 of β- and α- cells (except P3), as well as endocrine progenitor cells at P0, which were fluorescence-activated cell sorting (FACS) sorted from Insulin-RFP, Gcg-Cre; Rosa-RFP or Ngn3-GFP mouse strains, respectively. To develop a workflow to decipher the maturation process through bulk-cell transcriptomic analysis, we performed RNA-seq using 3-5 × 10^4 sorted cells at various developmental time points as we have done in the single-cell study. The background strains of our mouse samples are: Ngn3-GFP mice: mixed background of C57BL/6 and C3H Ins1-RFP mice: mixed background of C57BL/6 and C3H Gcg-Cre, Rosa-RFP mice: mixed background of C57BL/6, CBA/J and C3H.

胰腺β细胞与α细胞在维持葡萄糖稳态（glucose homeostasis）中发挥关键作用。然而，在胰腺发育过程中，这些独特的细胞群体如何产生、扩增并成熟，其背后的分子机制仍未阐明。本研究通过对胎龄至成年阶段分选得到的小鼠β细胞与α细胞进行单细胞转录组分析（single-cell transcriptomic analysis），解答了这一关键科学问题。研究发现，β细胞与α细胞的成熟调控策略存在差异，且二者的细胞增殖峰值出现在不同的发育阶段。但β细胞系与α细胞系中的静息细胞与增殖细胞，其成熟状态却保持同步。幼年β细胞的异质性反映了其细胞周期时相、起源及成熟状态的差异，而成年β细胞在转录组层面相对均一。本研究不仅为胰岛谱系发育提供了高分辨率的发育轨迹图谱，还为阐明β细胞与α细胞的细胞异质性、细胞数量扩增及成熟机制提供了新视角。
研究设计：本研究的整体目标是明确胰腺β细胞与α细胞的发育轨迹。具体而言，我们针对不同发育阶段的β细胞与α细胞（除P3阶段外）以及P0阶段的内分泌祖细胞开展了单细胞RNA测序（single-cell RNA-seq）；上述细胞分别通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）从Insulin-RFP、Gcg-Cre; Rosa-RFP或Ngn3-GFP小鼠品系中分选得到。为建立通过批量细胞转录组分析（bulk-cell transcriptomic analysis）解析细胞成熟过程的研究流程，我们参照单细胞测序研究的实验方案，对不同发育时间点分选得到的3~5×10^4个细胞开展了RNA测序。本研究中小鼠样本的遗传背景如下：Ngn3-GFP小鼠为C57BL/6与C3H的混合背景；Ins1-RFP小鼠为C57BL/6与C3H的混合背景；Gcg-Cre; Rosa-RFP小鼠为C57BL/6、CBA/J及C3H的混合背景。