HNF1A deficiency impairs ß-cell fate, granule maturation and function

Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3, the second most frequent form of diabetes caused by single gene mutation. We generated human pancreatic stem cell-derived endocrine cells with mutations in HNF1A and show that HNF1A deficiency impairs scß-cell fate, insulin granule maturation and the secretion of insulin in a glucose responsive manner. Single-cell RNA sequencing reveals that HNF1A orchestrates a network of genes involved in glucose metabolism, zinc transport, calcium ion binding and hormone exocytosis. Furthermore, in both patients and stem cell-derived ß-cells, HNF1A deficiency altered the stoichiometry of secreted c-peptide to insulin. Sulfonylurea, used in the treatment of these patients, restored both insulin secretion and stoichiometry. Significantly, uncoupling of c-peptide and insulin secretion as described here questions the common practice in using c-peptide as a proxy to evaluate ß-cell function. We also demonstrate that correction of the HNF1A locus restores function, providing a path to cell therapy. Overall design: Human embryonic stem cells with different HNF1A genotypes (WT, KO1, KO2, R200Q homozygous) were differentiated into islet-like clusters of endocrine cells for 27-28 days in vitro. First set of 6 samples, clusters of islet-like cells were dissociated into single cells and analyzed by single cell RNA sequencing. There are two WT, two KO and two R200Q samples. Second set of 5 samples, clusters of islet-like cells were dissociated into single cells and insulin producing cells were purified by FACS sorting for INS-GFP. Cells were analyzed by bulk RNA sequencing. There are three WT and two KO.

HNF1A基因突变可导致青少年型糖尿病3型（Maturity Onset Diabetes of the Young type 3, MODY3），这是第二常见的单基因遗传性糖尿病。本研究构建了携带HNF1A突变的人胰腺干细胞来源内分泌细胞系，证实HNF1A缺失会以葡萄糖应答依赖的方式损伤β细胞（β-cell）命运决定、胰岛素颗粒成熟以及胰岛素分泌功能。单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析显示，HNF1A调控参与葡萄糖代谢、锌转运、钙离子结合以及激素胞吐作用的基因表达网络。此外，在患者体内与干细胞来源的β细胞中，HNF1A缺失均会改变分泌型C肽（c-peptide）与胰岛素的化学计量比。用于治疗该类患者的磺脲类药物（Sulfonylurea）可同时恢复胰岛素分泌与该化学计量比。值得注意的是，本研究中观察到的C肽与胰岛素分泌解偶联现象，对以C肽作为β细胞功能评估替代指标的通用临床实践提出了质疑。本研究还证实，修复HNF1A基因位点可恢复细胞功能，为细胞治疗提供了可行路径。

实验设计概述：将携带不同HNF1A基因型（野生型WT、KO1、KO2、纯合R200Q突变型）的人胚胎干细胞体外定向分化为胰岛样内分泌细胞簇，分化周期为27~28天。第一组实验共6个样本：将胰岛样细胞簇解离为单细胞悬液，通过单细胞RNA测序进行分析，样本包含2份野生型、2份KO型以及2份R200Q型样本。第二组实验共5个样本：将胰岛样细胞簇解离为单细胞悬液后，通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）针对INS-GFP标记的胰岛素分泌细胞进行纯化，随后开展批量RNA测序（bulk RNA sequencing）分析，样本包含3份野生型与2份KO型样本。