HNF1A deficiency impairs β-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 scβ-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 β-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 β-cell function. We also demonstrate that correction of the HNF1A locus restores function, providing a path to cell therapy. 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.

肝细胞核因子1α（HNF1A）突变可导致青少年发病的成年型糖尿病3型（Maturity Onset Diabetes of the Young type 3），这是单基因突变所致糖尿病中第二常见的类型。
我们构建了携带HNF1A突变的人胰腺干细胞源性内分泌细胞，实验证实HNF1A缺陷会以葡萄糖应答依赖的方式损害单细胞β细胞（scβ-cell）命运、胰岛素颗粒成熟以及葡萄糖应答性胰岛素分泌过程。
单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）结果显示，HNF1A调控着参与葡萄糖代谢、锌转运、钙离子结合以及激素胞吐作用的基因网络。
此外，在患者及干细胞源性β细胞中，HNF1A缺陷会改变分泌型C肽（C-peptide）与胰岛素的化学计量比。
用于治疗此类患者的磺脲类药物（Sulfonylurea）可同时恢复胰岛素分泌功能及该化学计量比。
值得注意的是，本研究中揭示的C肽与胰岛素分泌解偶联现象，对以C肽作为评估β细胞功能替代指标的通用临床实践提出了质疑。
我们还证实，校正HNF1A基因座可恢复细胞功能，为细胞治疗提供了可行路径。
我们将携带不同HNF1A基因型（野生型Wild Type, WT、敲除型Knock Out, KO1、敲除型KO2、纯合子R200Q）的人胚胎干细胞在体外诱导分化为胰岛样内分泌细胞团，诱导时长为27~28天。
第一组共6份样本：将胰岛样细胞团解离为单细胞后，通过单细胞RNA测序进行分析，其中包含2份WT样本、2份KO样本及2份R200Q样本。
第二组共5份样本：将胰岛样细胞团解离为单细胞后，通过针对INS-GFP的荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）纯化胰岛素分泌细胞，随后通过批量RNA测序进行分析，其中包含3份WT样本及2份KO样本。