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

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。广泛采用一种细胞疗法 需要充足的胰岛供应和有效的免疫保护，而不需要长期的系统免疫 压制。我的实验室和其他实验室表明，提取胰岛素分泌细胞是可行的。 来自可再生和丰富的胃肠道(GI)干细胞。然而，它还不可能 从人胃肠道组织中大量生产胰岛样有机化合物用于详细评估其 翻译潜力。 在初步研究中，我们建立了培养人胃干细胞的方法。 活组织检查样本并将其扩大到数十亿。HGSC是为瞬时激活而设计的 NGN3、PDX1和MAFA的稳定表达(统称为NPM因子)，领先 形成数以千计的GINS(胃胰岛素分泌)类有机物。获得的杜松子酒有机化合物 葡萄糖刺激的胰岛素分泌(GSIS)在10天内和移植后迅速 小鼠糖尿病逆转，血糖维持正常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 使用可伸缩派生方法进行处理。 GINS类器官含有25%-30%的细胞，这些细胞非常类似于胰岛β细胞。在这个项目中，我们 将评估这样一种假设，即产生较高比例的Beta样细胞的克隆HGSC系可以 很容易从供体组织中辨认出来。我们将使用Genetic开发一个标准的派生协议 NPM的结节和克隆选择，目的是始终如一地产生高功能的Gins 捐赠者的移植。GINS细胞缺乏关键的自身抗原，天然的免疫原性可能低于 胰岛β细胞。我们将通过结构性表达两种有效的基因来进一步保护自身免疫。 免疫调节剂PD-L1和CD47。正常的和免疫逃避的有机化合物将在体外进行评估 体内有一组抗原特异性的细胞毒性CD8T细胞，它是β细胞死亡的主要效应者 在T1D。总之，这些研究将创造一种可靠地生产自体杜松子酒的技术 移植物具有较强的自身免疫保护作用，适用于无免疫的长期血糖控制 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.