Toxicity of proinflammatory cytokines towards surrogate insulin-producing cells generated from human pluripotent stem cells

促炎细胞因子对人多能干细胞产生的替代胰岛素产生细胞的毒性

• 批准号: 329435715
• 负责人: Privatdozent Dr. Ortwin Naujok
• 金额: --
• 依托单位: Institut für Klinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/329435715?language=en
• 关键词: Toxicity; proinflammatory; cytokines; towards; surrogate

Recent advances in the development of a cell replacement therapy for type 1 diabetes mellitus (T1DM) using pluripotent stem cells (PSC), such as human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, have gathered momentum. Lately data have been published showing that glucose-responsive beta cells can be produced by in vitro differentiation alone using human ES cells as the cell source. These differentiated cells are potentially suitable for a cell-based therapy of T1DM. However, T1DM is characterized by an autoimmune-mediated destruction of the insulin-producing beta cells in the islets of Langerhans. The mediators of this apoptotic destruction are T-effector cells and pro-inflammatory cytokines, especially IL-1beta, TNF-alpha, and IFN-gamma. This autoimmunity persists during life and there is strong evidence from transplantation studies that autoimmunity can reoccur and destroy any insulin-producing cell transplanted into a patient with T1DM. Thus, before ES cell-based therapies can be translated from experimental studies towards pre-clinical tests it must be determined whether surrogate cells made from ES cells harbor a significant sensitivity towards pro-inflammatory cytokines. Cell death induced by cell-to-cell contacts between cytotoxic T-cells and beta cells is another way by which they can be destroyed but cell-based therapies of diabetes using ES cells envisage encapsulation so that the solid immune system is separated from the graft inside the capsule. Of note, however, those encapsulation devices will not keep out cytokines due to their size and solubility.It is the aim of this project to generate glucose-responsive insulin-producing cells from PSCs in suspension and adherence. Terminally differentiated cells will be functionally characterized in vitro and in vivo. Then, the cells will be analyzed with respect to the expression of cytokine receptors and whether they can amplify autoimmunity by their own expression of chemokines and cytokines. We will address whether the exposure to pro-inflammatory cytokines results in a loss of viability and induction of apoptosis specifically in insulin-producing surrogate cells. This includes also the characterization of the main cytokine-activated signaling pathways, pro- and anti-apoptotic effector protein expression, and the possible interaction between the endoplasmic reticulum and mitochondria. Data gathered from this project will yield the answer by which protective measure surrogate cells may be protected in the future against soluble immune effectors. This could be achieved e.g. by CRISP/Cas9 mediated genomic editing to engineer cytokine-insensitive PSCs. Moreover, the results from this application can serve as a predictor for the success of PSC-based therapies of T1DM in the future.

利用多能干细胞(PSC)，如人胚胎干细胞(ES细胞)和诱导多能干细胞(IPS)治疗1型糖尿病(T1 DM)的细胞替代疗法的最新进展已形成势头。最近公布的数据表明，仅使用人类ES细胞作为细胞来源，仅通过体外分化就可以产生葡萄糖反应性β细胞。这些分化的细胞可能适合于基于细胞的T1 DM治疗。然而，T1 DM的特征是自身免疫介导的朗格汉斯胰岛中产生胰岛素的β细胞的破坏。这种凋亡破坏的媒介是T效应细胞和促炎细胞因子，特别是IL-1β、肿瘤坏死因子-α和干扰素-γ。这种自身免疫在生命中持续存在，来自移植研究的有力证据表明，自身免疫可以重复发生并摧毁任何移植到T1 DM患者体内的胰岛素产生细胞。因此，在基于ES细胞的治疗可以从实验研究转化为临床前试验之前，必须确定由ES细胞制成的替代细胞是否对促炎细胞因子具有显著的敏感性。通过细胞毒性T细胞和β细胞之间的细胞间接触而导致的细胞死亡是另一种破坏它们的方法，但使用ES细胞的基于细胞的糖尿病治疗设想的是封装，以便将固体免疫系统与胶囊内的移植物分开。然而，值得注意的是，由于细胞因子的大小和溶解性，这些封装设备不能将其拒之门外。本项目的目标是从悬浮和贴壁的PSC中产生葡萄糖响应性胰岛素产生细胞。终末分化细胞将在体外和体内进行功能鉴定。然后，将分析这些细胞的细胞因子受体的表达，以及它们是否可以通过自身表达的趋化因子和细胞因子来增强自身免疫。我们将讨论暴露在促炎细胞因子下是否会导致活性丧失和诱导凋亡，特别是在产生胰岛素的代理细胞中。这也包括细胞因子激活的主要信号通路的特征，促和抗凋亡效应蛋白的表达，以及内质网和线粒体之间可能的相互作用。从该项目收集的数据将给出答案，通过保护措施，代理细胞可能在未来保护免受可溶性免疫效应器的影响。这可以通过CRISP/Cas9介导的基因组编辑来实现，以设计细胞因子不敏感的PSCs。此外，这项应用的结果可以作为未来基于PSC的T1 DM治疗成功的预测因子。