人胚胎干细胞诱导体细胞重编程并定向分化为胰岛素分泌细胞的研究

Differentiation of human embryonic stem cells into insulin-producing cells is a leading issue in developmental biology and diabetes research. We successfully induced human embryonic stem cells into islet-like cells which improved hyperglycemia in diabetic mice, and our research was published by journal of Biomaterials. However, there are two important barriers, including immunologic rejection and immaturity of differentiated cells, need to be overcome for future diabetes treatment. We will explore in the two aspects. Firstly, human somatic cells will be reprogrammed to pluripotent stem cells with diploid karyotype by cell fusion to human embryonic stem cells or by their extracts. Secondly, phosphatidylinositol 3-kinase inhibitor will be used as a substitute for B27 else to induce the differentiation of human embryonic stem cells into mature insulin-producing cells at late stage. Thus, we will have a further understanding for the role of phosphatidylinositol 3-kinase (PI3K) pathway in the differentiation. Furthermore, we will perform whole-exome sequencing of human embryonic stem cells, pluripotent stem cells and insulin-producing cells to look for a safety and efficacy program for reprogramming of somatic cells or differentiation, and construct a platform for future diabetes treatment by the application of stem cell transplantation.

诱导人胚胎干细胞分化为胰岛素分泌细胞是目前发育生物学与糖尿病研究领域的前沿课题。申请人在前期工作中成功诱导人胚胎干细胞定向分化为胰岛样细胞并发挥了降低糖尿病鼠血糖的作用，研究成果已在国际生物材料领域 “Biomaterials”杂志中发表。然而将人胚胎干细胞技术应用于临床仍需克服免疫排斥和分化细胞不成熟性等两个重要问题，本课题将围绕上述两个方面展开探索。通过人胚胎干细胞与体细胞融合或者采用人胚胎干细胞提取物诱导实现体细胞重编程，获得具有正常二倍体核型的人多能干细胞系；然后在分化晚期采用磷脂酰肌醇3激酶（PI3Ks）抑制剂替代B27诱导分化，以获得功能成熟的胰岛素分泌细胞，对PI3Ks信号通路在分化过程中的作用也将获得新的认识。我们同时对人胚胎干细胞、人多能干细胞以及胰岛素分泌细胞进行外显子组测序，旨在探索一条安全高效的体细胞重编程及定向诱导分化方案，为远期临床干细胞移植治疗糖尿病构建平台。

将人胚胎干细胞分化而来的胰岛样细胞移植治疗糖尿病也许是一种有希望的糖尿病治疗方法。我们使用PI3Kp110 beta抑制剂能够诱导人胚胎干细胞定向分化为成熟的胰岛素分泌细胞。这些胰岛样细胞免疫荧光结果显示胰岛素单染阳性率高，定量PCR结果显示一些包括MAFA的胰腺内分泌细胞基因表达量明显升高。结果显示PI3K非选择性抑制剂LY294002和PI3Kp110 beta选择性抑制剂TGX-221均可以诱导人胚胎干细胞的分化和成熟，除了TGX-221诱导组胰岛素基因表达量明显高于LY294002诱导组以外，大多数胰腺内分泌细胞基因表达量并无差别。另外，这些胰岛样细胞在体外可发生葡萄酸依赖性胰岛素分泌反应。将胰岛样细胞移植到鼠肾脏被摸下可以纠正糖尿病鼠高血糖状态，并能改善其生活质量。在国际上首次提出PI3Kp110 beta通路与胰岛样细胞的分化与成熟有关。