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

使用多能干细胞（PSC）（例如人胚胎干（ES）细胞和诱导多能干（iPS）细胞）开发用于1型糖尿病（T1 DM）的细胞替代疗法的最新进展已经聚集了势头。最近发表的数据显示，葡萄糖响应性辟田胞可以使用人ES细胞作为细胞来源通过单独的体外分化产生。这些分化的细胞可能适合于T1 DM的基于细胞的治疗。然而，T1 DM的特征在于自身免疫介导的胰岛中产生胰岛素的β细胞的破坏。这种凋亡破坏的介质是T效应细胞和促炎细胞因子，特别是IL-1 β、TNF-α和IFN-γ。这种自身免疫性在生命过程中持续存在，移植研究的有力证据表明，自身免疫性可再次发生并破坏移植到T1 DM患者体内的任何胰岛素产生细胞。因此，在基于ES细胞的疗法从实验研究转化为临床前测试之前，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着的敏感性。由细胞毒性T细胞和β细胞之间的细胞与细胞接触诱导的细胞死亡是另一种可以破坏它们的方式，但是使用ES细胞的基于细胞的糖尿病疗法设想包封，使得固体免疫系统与囊内的移植物分离。然而，值得注意的是，这些包封装置将不会阻止细胞因子，由于其大小和溶解度。这是本项目的目的是从悬浮和粘附的PSC产生葡萄糖响应性胰岛素产生细胞。将在体外和体内对终末分化的细胞进行功能表征。然后，将分析细胞细胞因子受体的表达以及它们是否可以通过自身趋化因子和细胞因子的表达来放大自身免疫。我们将讨论是否暴露于促炎性细胞因子的结果，特别是在胰岛素产生的替代细胞的活力和诱导凋亡的损失。这也包括主要的精氨酸激活的信号通路，促凋亡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的表征。从该项目收集的数据将产生答案，通过该保护措施替代细胞可以在未来免受可溶性免疫效应物的影响。这可以例如通过CRISP/Cas9介导的基因组编辑来工程化对精氨酸不敏感的PSC来实现。此外，该应用的结果可以作为未来基于PSC的T1 DM治疗成功的预测因素。

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.