Engineering islet-like organoids from gastric stem cells for T1D cell replacement therapy

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。广泛采用细胞疗法 需要充足的胰岛供应和有效的免疫保护，而不需要长期的全身免疫 镇压我的实验室和其他人的实验表明，衍生胰岛素分泌细胞是可行的 从可再生和丰富的胃肠道（GI）干细胞。然而，一直未能 从人类胃肠道组织中大量生产胰岛样类器官，以详细评估其 平移势 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。 活检样本并将其扩大到数十亿。hGSC被工程化用于瞬时激活 NGN 3和PDX 1和MAFA（统称为NPM因子）的稳定表达， 形成数千个胃胰岛素分泌类器官。获得GINS类器官 葡萄糖刺激的胰岛素分泌（GSIS）在10天内，并在移植后，迅速 逆转了小鼠的糖尿病，并保持正常血糖超过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 用可扩展的衍生方法处理。 GINS类器官含有25-30%的细胞，与胰腺β细胞非常相似。本课题 将评估产生较高百分比的β样细胞的克隆hGSC系可以 很容易从供体组织中识别出来。我们将开发一个标准的衍生协议， NPM的敲入和克隆选择，目的是一致地产生高功能的GINS 捐赠者的移植物。GINS细胞缺乏关键的自身抗原，并且可能天然地比GINS细胞具有更低的免疫原性。 胰岛β细胞。我们将通过组成性表达两个有效的免疫保护因子来赋予进一步的自身免疫保护。 免疫调节剂PD-L1和CD 47。将在体外评价正常和免疫逃避类器官 在体内与一组抗原特异性细胞毒性CD 8 T细胞（β细胞死亡的主要效应子） 在T1 D。总之，这些研究将创造一种可靠的自体GINS生产技术 移植物具有较强的自身免疫保护作用，适用于长期血糖控制，无需免疫 孤立或压制。

PROJECT SUMMARY Islet transplantation offers a potential cure for Type 1 diabetes (T1D). Wide adoption of a cell therapy requires abundant islet supplies and effective immune protection without long-term systemic immune suppression. My laboratory and others showed that it was feasible to derive insulin-secreting cells from renewable and abundant gastrointestinal (GI) stem cells. 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 samples and expanded them to billions. The hGSCs were engineered for transient activation of NGN3 and stable expression of PDX1 and MAFA (collectively referred to as NPM factors), leading to formation of thousands of GINS (Gastric Insulin Secreting) organoids. 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 with a scalable derivation method. GINS organoids contain 25-30% of cells that closely resemble pancreatic Beta-cells. In this project, we will evaluate the hypothesis that clonal hGSC lines yielding a higher percentage of Beta-like cells can be readily identified from donor tissues. We will develop a standard derivation protocol using genetic knockin of NPM and clonal selection with the aim to consistently produce highly functional GINS grafts from donors. GINS cells lack key autoantigens and may be naturally less immunogenic than islet Beta-cells. We will confer further autoimmune protection by constitutive expression of two potent immune regulators PD-L1 an CD47. Normal and immune-evasive organoids will be evaluated in vitro and in vivo with a panel of antigen-specific cytotoxic CD8 T cells, the main effector of Beta-cell demise in T1D. Together, these studies will create a technology for reliable production of autologous GINS grafts with strong autoimmune protection, suitable for long-term glycemic control without immune isolation or suppression.