Robust differentiation of human enteroendocrine cells from intestinal stem cells

Enteroendocrine (EE) cells are the most abundant hormone-producing cells in humans and are critical regulators of energy homeostasis and gastrointestinal function. Challenges in converting human intestinal stem cells (ISCs) into functional EE cells, ex vivo, have limited progress in elucidating their role in disease pathogenesis and in harnessing their therapeutic potential. To address this, we employed small molecule targeting of key transcriptional regulators, GATA4, JNK and FOXO1, known to mediate endodermal development and hormone production, together with directed differentiation of human ISCs from the duodenum and rectum. We observed marked induction of EE cell differentiation and gut-derived expression and secretion of SST, 5HT, GIP, GLP-1 and PYY upon treatment with various combinations of three small molecules: rimonabant, SP600125 and AS1842856. Robust differentiation strategies capable of driving human EE cell differentiation is a critical step towards understanding these essential cells and the development of cell-based therapeutics. Overall design: mRNA isolated from enteroids exposed to three different conditions, G2D12, AS and RSP, from three different enteroid lines (9 samples total, detailed in the 'metadata_9samples.txt' including the hashtag sequences for each sample). Samples were mixed using hashtagged antibodies prior to scRNA-Seq

肠内分泌细胞（Enteroendocrine，EE）是人体内数量最多的激素产生细胞，也是能量稳态与胃肠功能的关键调控因子。目前将人类肠干细胞（intestinal stem cells，ISCs）体外转化为功能性肠内分泌细胞仍存在诸多技术难题，这极大限制了学界对其在疾病发病机制中作用的阐明，也阻碍了其治疗潜力的开发与利用。为解决这一问题，本研究针对已知介导内胚层发育与激素生成的关键转录调控因子GATA4、JNK及FOXO1，采用小分子靶向干预手段，并结合取自十二指肠与直肠的人类肠干细胞定向分化策略。经三种小分子——利莫那班（rimonabant）、SP600125与AS1842856——的不同组合处理后，我们观察到肠内分泌细胞分化被显著诱导，同时肠道来源的SST、5HT、GIP、GLP-1与PYY的表达及分泌水平均显著上调。能够高效诱导人类肠内分泌细胞分化的稳定分化策略，是阐明这类关键细胞功能、开发细胞治疗手段的重要一环。实验整体设计：从3株不同的肠类器官（enteroid）系中，分别提取经G2D12、AS与RSP三种不同条件处理的肠类器官的mRNA，总计9个样本（详细信息见'metadata_9samples.txt'，包含每个样本的标签序列）。在进行单细胞RNA测序（single-cell RNA sequencing，scRNA-Seq）前，使用带有分子标签的抗体对样本进行混合。