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

从胃干细胞中改造胰岛样类器官，用于 T1D 细胞替代疗法

基本信息

批准号：
10512923
负责人：
Qiao Joe Zhou
金额：
$ 62.5万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-12 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10512923
关键词：
Adoption Anterior Antigens Attenuated Autoantigens Autoimmune Autoimmune Responses Autologous Autologous Transplantation Beta Cell Biological Assay Biological Response Modifiers Biopsy Biopsy Specimen CD47 gene CD8-Positive T-Lymphocytes Cell Communication Cell Line Cell Therapy Cells Cellular Assay Clone Cells Cytotoxic T-Lymphocytes Derivation procedure Diabetes Mellitus Doxycycline Engineering Future G6PC2 gene Generations Genetic Glucose Glucose tolerance test Human Human Cloning Immune Immune Evasion Immunosuppression In Vitro Innate Immune Response Insulin Insulin-Dependent Diabetes Mellitus Islets of Langerhans Transplantation Knock-in Laboratories Methods Molecular Mus NPM1 gene Names Organoids Persons Physiologic pulse Production Protocols documentation Stains Standardization Stomach Structure of beta Cell of islet T-Lymphocyte Technology Tissue Donors Tissue Grafts Tissues Transplantation Variant age group cell replacement therapy cell type clinical translation cytotoxic CD8 T cells diabetic eye chamber gastric organoids gastrointestinal glucose monitor glycemic control immunogenic improved in vivo in vivo imaging insulin secretion insulitis islet new technology preproinsulin progenitor programmed cell death ligand 1 single-cell RNA sequencing stem cells translational potential tumor

项目摘要

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.

项目概要胰岛移植为 1 型糖尿病 (T1D) 提供了潜在的治愈方法。细胞疗法的广泛采用需要丰富的胰岛供应和有效的免疫保护，无需长期的全身免疫抑制。我的实验室和其他人表明衍生胰岛素分泌细胞是可行的来自可再生且丰富的胃肠道 (GI) 干细胞。然而，还未能从人类胃肠道组织中大量生产胰岛样类器官，以对其进行详细评估翻译潜力。在初步研究中，我们建立了培养人胃干细胞（hGSC）的方法活检样本并将其扩大到数十亿。 hGSC 被设计用于瞬时激活 NGN3 的表达以及 PDX1 和 MAFA（统称为 NPM 因子）的稳定表达，导致形成数千个 GINS（胃胰岛素分泌）类器官。获得 GINS 类器官葡萄糖刺激胰岛素分泌 (GSIS) 在 10 天内以及移植后迅速逆转小鼠糖尿病并维持正常血糖超过 3 个月，且无肿瘤形成。因此，人类 GINS 类器官具有作为 T1D 潜在细胞产品的有利属性使用可扩展的推导方法进行处理。 GINS 类器官含有 25-30% 的细胞，与胰腺 β 细胞非常相似。在这个项目中，我们将评估以下假设：产生更高比例 Beta 样细胞的克隆 hGSC 系可以很容易从供体组织中识别出来。我们将开发一个使用遗传的标准衍生协议 NPM 敲入和克隆选择，旨在持续生产高功能 GINS 来自捐赠者的移植物。 GINS 细胞缺乏关键的自身抗原，并且天然的免疫原性可能低于胰岛β细胞。我们将通过两种有效的组成型表达来赋予进一步的自身免疫保护免疫调节剂 PD-L1 和 CD47。正常和免疫逃避类器官将在体外进行评估并在体内使用一组抗原特异性细胞毒性 CD8 T 细胞，这是 Beta 细胞死亡的主要效应器在 T1D 中。这些研究将共同创造一种可靠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，适合长期血糖控制而无需免疫隔离或压制。