Pancreatic islet -cell subtypes are derived from biochemically-distinct and nutritionally-regulated islet progenitors

Endocrine islet beta cells comprise heterogenous cell subsets. Yet the origin, stability, and physiological significance of these subsets remain largely unknown. Using combinatorial cell lineage tracing, scRNA-seq, and DNA methylation analysis, we show here that embryonic islet progenitors with differential gene expression and DNA methylation produce stable beta-cell subtypes of different function and viability in adult mice. Differentially expressed genes, including the Myt transcription factors, voltage-gated channels, and Ca2+-sensor synaptotagmins, contribute to the functional differences of these subtypes. Maternal overnutrition, a major diabetes risk factor, reduces the proportion of endocrine progenitors of the better-functionality beta-cell subtype. Intriguingly, the gene signature that defines mouse beta-cell subtypes can reliably divide human cells into two populations, with the proportion of better-functionality beta cells reduced in diabetic donors. These results establish that some beta-cell subtypes are determined via DNA methylation in embryonic islet progenitors, which is regulated by diabetes-causing maternal factors. The implication is that modulating DNA methylation in islet progenitors can be explored to improve beta-cell function in the prevention and therapy of diabetes. Mice with a genotype of Myt1cCre; Ngn3nCre; Ai9 (MNA) will be derived via routine crossing. This allows the activation of islet progenitors that co-express Ngn3 and Myt1 to be labeled with tdTomato permanently. When MNA mice were at postnatal day 2 (P2) and 2-months, they will be used for islet isolation. Islets were then washed with Ca2+/Mg2+ free HBSS and dissociated into single cells (~3-5 minutes). They were then used for InDrop RNAseq.内分泌胰岛β细胞存在异质性细胞亚群，然而这些亚群的起源、稳定性及生理学意义仍未得到充分阐明。 本研究采用组合式细胞谱系示踪、单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与DNA甲基化分析技术，证实携带差异化基因表达与DNA甲基化特征的胚胎胰岛祖细胞，可在成年小鼠体内形成功能与存活能力各异的稳定β细胞亚群。 包括Myt家族转录因子、电压门控离子通道及钙离子感受器突触结合蛋白在内的差异表达基因，是这些β细胞亚群功能差异的重要成因。 作为糖尿病主要风险因素之一的母体营养过剩，会降低功能更优的β细胞亚群对应的内分泌祖细胞比例。 值得注意的是，可区分小鼠β细胞亚群的基因特征标签，同样能可靠地将人类胰岛细胞分为两个亚群，且糖尿病供体体内功能更优的β细胞亚群比例显著降低。 上述结果证实，部分β细胞亚群的命运由胚胎胰岛祖细胞的DNA甲基化模式决定，而该过程可被诱发糖尿病的母体因素调控。 这一发现提示，可通过调控胰岛祖细胞的DNA甲基化水平改善β细胞功能，从而应用于糖尿病的预防与治疗。 本研究将通过常规杂交构建基因型为Myt1cCre; Ngn3nCre; Ai9（简称MNA）的小鼠模型。该模型可实现对共表达Ngn3与Myt1的胰岛祖细胞的永久tdTomato荧光标记。 分别在MNA小鼠出生后第2天（P2）及2月龄时，分离其胰岛组织。 随后将胰岛用不含钙离子与镁离子的汉克斯平衡盐溶液（Ca²⁺/Mg²⁺-free HBSS）洗涤，并解离为单细胞悬液（解离时长约3-5分钟），最后用于InDrop单细胞RNA测序（InDrop RNAseq）。