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

项目摘要 胰岛移植为1型糖尿病（T1 D）提供了一种潜在的治疗方法。广泛采用这一有前途的 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 结果表明，从胃肠道（GI）组织中分离胰岛素分泌细胞是可行的。 然而，还不可能从人GI组织大规模生产胰岛样类器官， 详细评估其翻译潜力。 在初步研究中，我们建立了从人胃粘膜中培养人胃干细胞（hGSC）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NGN 3产生数千个GINS（胃胰岛素分泌）类器官 和MAFA（统称为NPM因子）的稳定表达。GINS类器官 在10天内获得葡萄糖刺激的胰岛素分泌（GSIS），并且在移植时， 在小鼠中迅速逆转糖尿病，并维持正常血糖超过3个月，无肿瘤 阵因此，人GINS类器官具有作为T1 D的潜在细胞产物的有利属性 治疗 GINS类器官含有25%的细胞，与胰腺E细胞非常相似，但缺乏GCG+ SST+细胞人类胰岛有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hGSC， 线和新的基于纳米颗粒的mRNA转导方法， 在细胞组成和功能上与人类胰岛非常相似的类器官。这些研究是基于 初步数据表明，hGSC克隆系明显不同，其中一些主要是 产生E样或D-/G样细胞。因此，它们的分化后代可以结合产生胰岛- 就像类器官。我们将进一步研究克隆系的染色质特征和PDX 1/MAFA基因组 结合以获得GINS形成的机制性见解。总之，这些研究构成了一个重大步骤， 在推进开发用于T1 D治疗的GINS类器官的长期目标方面。

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.