Generation of insulin-producing islet-like clusters from human embryonic stem cells
Generation of insulin-producing islet-like clusters from human embryonic stem cells
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DOI：
10.1634/stemcells.2006-0761
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发表时间：
2007-08-01
期刊：
影响因子：
5.2
通讯作者：
Majumdar, Anish S.
中科院分区：
文献类型：
--
作者：
Jiang, Jianjie;Au, Melinda;Majumdar, Anish S.
最近在胰岛移植中的成功激发了该领域发现具有分化潜能的干细胞的替代来源。从自我更新的多能人类胚胎干细胞（hESC）中产生葡萄糖响应性、产生胰岛素的P细胞对于糖尿病治疗具有巨大的潜力。我们在这里报告了一种新的无血清协议的发展，以产生胰岛素生产胰岛样细胞簇（ILC）的人胚胎干细胞生长在无饲养条件下。在该36天方案中，用丁酸钠和激活素A处理hESC以产生共表达CXCR 4和Sox 17以及CXCR 4和Foxa 2的定形内胚层。然后将内胚层群体转化为细胞聚集体，并在表皮生长因子、碱性成纤维细胞生长因子和头蛋白的存在下进一步分化为表达Pdx 1的胰腺内胚层。此后不久，检测到Pft 1a和Ngn 3的表达，表明胰腺进一步分化。聚集体最终在胰岛素样生长因子II和烟酰胺的存在下成熟。胰腺特异性基因表达的hESC-衍生的ILC的时间模式显示出相当大的相似性，在体内胰腺发育，和最终的人口包含代表的导管，外分泌，和内分泌胰腺。hESC衍生的ILC含有2%-8%的人C肽阳性细胞，以及胰高血糖素和生长抑素阳性细胞。在ILC中测得的胰岛素含量高达70 ng胰岛素/μ g DNA，代表高于人胎儿胰岛的水平。此外，hESC衍生的ILC含有许多分泌颗粒，如通过电子显微镜测定的，并且以葡萄糖依赖性方式分泌人C肽。
Recent success in pancreatic islet transplantation has energized the field to discover an alternative source of stem cells with differentiation potential to 13 cells. Generation of glucose-responsive, insulin-producing P cells from self-renewing, pluripotent human ESCs (hESCs) has immense potential for diabetes treatment. We report here the development of a novel serum-free protocol to generate insulin-producing islet-like clusters (ILCs) from hESCs grown under feeder-free conditions. In this 36-day protocol, hESCs were treated with sodium butyrate and activin A to generate definitive endoderm coexpressing CXCR4 and Sox17, and CXCR4 and Foxa2. The endoderm population was then converted into cellular aggregates and further differentiated to Pdx1-expressing pancreatic endoderm in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin. Soon thereafter, expression of Pft1a and Ngn3 was detected, indicative of further pancreatic differentiation. The aggregates were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal pattern of pancreas-specific gene expression in the hESC-derived ILCs showed considerable similarity to in vivo pancreas development, and the final population contained representatives of the ductal, exocrine, and endocrine pancreas. The hESC-derived ILCs contained 2%-8% human C-peptide-positive cells, as well as glucagon- and somatostatin-positive cells. Insulin content as high as 70 ng of insulin/mu g of DNA was measured in the ILCs, representing levels higher than that of human fetal islets. In addition, the hESC-derived ILCs contained numerous secretory granules, as determined by electron microscopy, and secreted human C-peptide in a glucose-dependent manner.