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Differentiation of ES cells to insulin-producing cells by expression of transcription factors

通过转录因子的表达将 ES 细胞分化为胰岛素产生细胞

基本信息

批准号：
15390092
负责人：
MIYAZAKI Jun-ichi
金额：
$ 8.77万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390092/
关键词：
cell transcription factor insulin regeneration β cell ES細胞糖尿病

项目摘要

We tried to establish an efficient method for inducing differentiation of embryonic stem (ES) cells into insulin-producing cells. As a tool of investigation, we established an ES cell line by introducing the expression unit, in which exogenous pdx-1 expression was precisely regulable by the Tet-off system, into the ROSA26 locus.Using this cell line, we examined the effect of pdx-1 expression during in vitro differentiation via embryoid body formation. The results showed that pdx-1 expression clearly enhanced the gene expression of the insulin 2, somatostatin, Kir6.2, glucokinase, neurogenin3, p48, Pax6, PC2, and HNF6 genes in the differentiated cells. However gene expression level of insulin gradually decreased as cell culture passaged. To solve this problem, we constructed adenoviral (AdV) vectors expressing several transcription factors relating the differentiation of pancreatic β cells. Induction of neuroD gene to this cell line by AdV vector showed up-regulation of gene expression of insulin 2, insulin 1, glucagon and somatostatin.To improve the efficiency of differentiation, we established ES cell line in which exogenous sox17 expression was regulated by tetracycline. Induction of pdx-1 and mafA genes by adenoviral vectors to sox17-expressing ES cells induced the expression of insulin 2 gene.Thus, drug-regulable system of gene expression in these ES cell lines may be a useful tool for investigating the genes for early development and differentiation of pancreatic β cells.

本研究试图建立一种有效的胚胎干细胞向胰岛素分泌细胞诱导分化的方法。作为研究工具，我们将外源性pdx-1的表达可通过Tet-off系统精确调控的表达单位导入ROSA 26位点，建立了ES细胞系，利用该细胞系，通过胚状体的形成，研究了pdx-1的表达对体外分化的影响。结果显示，pdx-1表达明显增强分化细胞中胰岛素2、生长抑素、Kir6.2、葡萄糖激酶、神经生成素3、p48、Pax 6、PC 2和HNF 6基因的表达。随着细胞的传代，胰岛素基因表达水平逐渐下降。为了解决这一问题，我们构建了腺病毒（AdV）载体，表达与胰腺β细胞分化相关的几种转录因子。将neuroD基因导入该细胞系后，发现胰岛素2、胰岛素1、胰高血糖素和生长抑素基因表达上调，为了提高分化效率，我们建立了四环素调控外源sox 17表达的ES细胞系。腺病毒介导的pdx-1和mafA基因转染表达sox 17的ES细胞后，可诱导胰岛素2基因的表达，因此，药物可调控的基因表达系统可能是研究胰岛β细胞早期发育和分化相关基因的有效工具。