Derivation of pancreatic islet-like organoids from human gastric stem cells

项目总结 胰岛移植为1型糖尿病(T1D)提供了一种潜在的治疗方法。广泛采用这一前景看好的 治疗需要充足的胰岛供应和有效的免疫保护。我的实验室和其他人 结果表明，从胃肠道组织中分离出胰岛素分泌细胞是可行的。 然而，目前还不可能从人的胃肠道组织中大规模生产胰岛样有机化合物 对其翻译潜力的详细评估。 在初步研究中，我们建立了培养人胃干细胞的方法。 活组织检查或尸检样本可以扩大到数十亿。我们开发了一种可扩展的两步法 通过瞬时激活NGN3产生数千种胃胰岛素分泌类有机物 以及PDX1和MAFA的稳定表达(统称为NPM因子)。杜松子酒有机化合物 10天内获得葡萄糖刺激胰岛素分泌(GSIS)，并在移植后， 小鼠糖尿病迅速逆转，血糖维持在正常水平3个月以上，无肿瘤 队形。因此，人参皂苷类有机化合物具有作为T1D潜在细胞产物的有利属性 治疗。 GINS类有机化合物含有25%与胰腺E细胞相似的细胞，但缺乏GCG+ 和SST+细胞。人的胰岛有50%-75%的E细胞，25%-35%的D细胞和5%的G细胞。D-和G- 细胞在E细胞切片上发挥旁分泌作用。在本项目中，我们的目标是开发新的克隆性hGSC 适于大规模生产的线型和新型纳米颗粒信使核糖核酸转导方法 在细胞组成和功能上与人类胰岛非常相似的有机化合物。这些研究是基于 根据初步数据显示，HGSC克隆系明显不同，其中一些主要是 产生类E细胞或类D/G细胞。因此，它们分化出的后代可以组合成胰岛- 就像有机体一样。我们将进一步研究染色质特征和PDX1/MAFA基因组的克隆系 在杜松子酒形成过程中获得机械洞察力的约束。这些研究共同构成了重要的一步。 在推进开发用于T1D治疗的GINS有机化合物的长期目标方面。

PROJECT SUMMARY Islet transplantation offers a potential cure for Type 1 diabetes (T1D). Wide adoption of this promising therapy requires abundant islet supplies and effective immune protection. My laboratory and others showed that it was feasible to derive insulin-secreting cells from gastrointestinal (GI) tissues. However, it has not been possible to mass-produce islet-like organoids from human GI tissues for detailed assessment of their translational potential. In preliminary studies, we established methods to culture human gastric stem cells (hGSCs) from biopsy or autopsy samples that can be expanded to billions. We developed a scalable 2-step method to produce thousands of GINS (Gastric Insulin Secreting) organoids by transient activation of NGN3 and stable expression of PDX1 and MAFA (collectively referred to as NPM factors). GINS organoids acquired glucose-stimulated-insulin-secretion (GSIS) within 10 days, and upon transplantation, rapidly reversed diabetes in mice and maintained normoglycemia for over 3 months, with no tumor formation. Human GINS organoids thus have favorable attributes as a potential cell product for T1D treatment. GINS organoids contain 25% of cells that closely resemble pancreatic E-cells but a paucity of GCG+ and SST+ cells. Human islets have 50-75% E-cells, 25-35% D-cells, and 5% G-cells. Both D- and G- cells exert paracrine effects on E-cell section. In this project, we aim to develop new clonal hGSC lines and novel nanoparticle-based mRNA transduction method suitable for mass production of organoids that closely mimic human islets in cell composition and function. These studies are based on preliminary data indicating that hGSC clonal lines are markedly different, with some predominantly producing E-like or D-/G-like cells. Their differentiated progenies can thus be combined to yield islet- like organoids. We will further study the clonal lines for chromatin features and PDX1/MAFA genomic binding to gain mechanistic insight in GINS formation. Together, these studies constitute a major step in advancing the long-term goal of developing GINS organoids for T1D treatment.