一种诱导I型糖尿病患者来源的iPSCs分化为胰岛素分泌细胞的新方法：内质网应激的作用及其机制

Type I diabetes (known as insulin-dependent diabetes, T1D) is characterized by deficient insulin production and requires daily administration of insulin, which seriously influence the patients’ life quality. And more, long-term insulin treatment can cause late symptoms. So far, pancreatic islet cell transplantation is the only effective strategy to cure T1D. However, this therapy is not widely used because of the scarcity of donor islets and the risk of long-term immunosuppressive. Recently, insulin-producing beta-like cells derived from induced pluripotent stem cells (iPSCs) become a potential source of cells for the treatment of T1D patients. Previous studies have reported that embryonic stem cells (ESCs) and iPSCs have been successfully differentiated into insulin-producing beta-like cells. However, the efficiency of ESC or iPSC differentiation models remains low, and the transplanted mice produce teratomas, limits its clinic application. To search a novel approach to avoid the limitation is our key point. Our previous studies have shown that endoplasmic reticulum (ER) stress response (ESR) or unfolded protein response (UPR)-inducing agents, thapsigargin (TG) or tunicamycin (TM), in vitro to induce mouse ESCs to differentiate into definitive endoderm cells (DECs), which can successfully differentiate into insulin-secreting cells. We also found that the co-expression of pancreas-relative transcription factors, Pdx1 and MafA with either Ngn3 or NeuroD by adenovirus vectors at the final stage of the differentiation, significantly increased the differentiation efficiency. And more, ESR-inducing agents can reduce ESC tumorigenesis. Based on these data, we propose this study to establish type I diabetes specific iPSCs (T1D-iPSCs), and explore a novel approach to produce insulin secreting cells derived from T1D-iPSCs via modulation of ESR and later ectopic expression of Pdx1 and MafA with either Ngn3 or NeuroD by adenovirus vectors combined with small molecular compounds, and then analyze the function of the insulin secreting cells by insulin and C-peptide secretion and amelioration of streptozotocin (STZ)-induced hyperglycemia following transplantation. And more, the molecular mechanism of lower tumorigenesis of T1D-iPSCs via modulation of ESR will also be explored. In addition, we will compare the DNA methylation levels of islet beta cells-related genes of various T1D-iPS cell lines by arrays of DNA methylation and gene expression, in order to find out the correlation between DNA methylation levels of islet beta cells-related genes of the iPS cell lines and the differentiation efficiency. We predict that the DNA methylation levels of islet beta cells-related genes of iPS cell lines are related to their differentiation efficiency, which will give us a guide to select a more suit iPS cell lines to differentiate into insulin-producing cells. The study will provide more molecular basis and a new strategy for cell therapy of iPSCs to type I diabetes.

胰岛移植是目前治疗I型糖尿病（T1D）最有效的方法，但是由于供体的匮乏和长期免疫抑制限制了它的广泛应用。最近，iPSCs分化为胰岛素分泌细胞成为治疗T1D潜在的细胞来源；但iPSCs分化效率低和形成畸胎瘤等缺点限制了其临床应用；因此，寻找安全、有效的分化方法就成为研究重点。我们前期研究发现内质网应激介导胚胎干细胞分化为定型内胚层细胞，可进一步分化为有功能的胰岛素分泌细胞；并且在分化的后期，通过腺病毒转导相关转录因子可显著提高分化效率；而且这种分化方法降低了畸胎瘤形成率。本项目拟建立T1D-iPSCs，探索内质网应激介导iPSCs分化为定型内胚层细胞，然后通过过表达相关转录因子联合小分子化合物提高其分化为胰岛素分泌细胞效率的方法及减少致瘤率的机制；最后比较T1D-iPS细胞系间特异基因的甲基化水平来预测这与其分化效率的相关性。本项目为iPSCs在T1D细胞治疗应用方面提供研究基础和新方法。

糖尿病是全球最普遍的、危害性最为广泛的慢性疾病之一, 其中, 胰岛素依赖的I型糖尿病患者( T1DM)大约占总的糖尿病患者的10%。目前,临床主要依靠补充胰岛素来治疗T1MD,但是那会导致后期的综合症.目前,没有真正有限的治疗方法。本研究通过建立T1DM的iPS细胞系,探索T1DM的致病机理和治疗方法..在本研究中我们建立了胰岛素分泌的报告iPS系统,用来优化iPS分化为胰岛素分泌细胞的效率.同时,我们建立了内质网应激介导的胰岛素分泌的方案, 优化了内质网应激的激活剂联合小分子抑制剂来分化iPS为胰岛素分泌的方法. 而且,通过insluin和 C-peptide分泌测定了分化的细胞对高糖的反应,进一步通过体内移植streptozotocin (STZ糖尿病鼠的高汤的改善作用.此外, 在皮下移植瘤的模型中检测了分化的胰岛素分泌细胞的安全性.. 此外,我们建立了WFS1基因突变的Wolfram 综合症的iPS细胞系 (iPSwFS1). Wolfram 综合症的表现是胰岛素依赖的糖尿病,视神经萎缩和耳聋.这个疾病是致命的,尚无有效的治疗方法,如果通过iPSwFS1作为细胞模型来探索Wolfram 综合症的胰岛素分泌不足的治病机理,对于该疾病的研究有重要的指导意义.在人类, WFS1位于内质网上,参与调节内质网的Ca2+离子浓度. WFS1的突变可以激活过多的内质网应激反应,从而引起胰岛beta细胞的失调,从而分泌胰岛素不足.所以,我们通过小分子药物库来筛选治疗改善该iPS分化为有作用的胰岛素分泌细胞的不足, 这对于开发治疗Wolfram 综合症的小分子药物有巨大的应用前景,并对Wolfram 综合症的治疗有很重要的指导意义.同时,我们也可以通过甲基化和基因表达芯片上分析WFS1基因引起的下游基因表达产物的变化,这为寻找WFS1基因引起的Wolfram 综合症的治病机理提供理论依据..总的说来,我们首先探索来生理条件下的内质网应激介导的,联合小分子抑制剂的iPS分化为胰岛素分泌的方法.同时,建立了WFS1基因突变的Wolfram 综合症的iPS细胞系 (iPSwFS1).以iPS为细胞模型,研究Wolfram 综合症的治病机理和治疗方法, 对于探索新的治疗WFS1基因引起的I型糖尿病有重要的指导意义和广大的应用前景.