Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

作     者：Akira Shiroi Shigehiko Ueda Yukiteru Ouji Ko Saito Kei Moriya Yuko Sugie Hiroshi Fukui Shigeaki Ishizaka Masahide Yoshikawa 

作者机构：Division of Developmental Biology Department of Gastroenterology and Hepatology 

出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)

年 卷 期：2005年第11卷第27期

页      面：4161-4166页

核心收录：

学科分类：1002[医学-临床医学] 1001[医学-基础医学(可授医学、理学学位)] 100101[医学-人体解剖与组织胚胎学] 10[医学] 

主　　题：Animals Cell Differentiation Cell Line Cricetinae Gene Transfer Techniques Homeodomain Proteins Insulin Islets of Langerhans Mice Mice, Inbred Strains Stem Cells Transcription Factors 

摘      要：AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 ***： Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body （EB） culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone （DTZ）, a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing *** outgrowths were incubated in DTZ solution （final concentration, 100μg/mL） for 15 rain before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ***： DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells （Nkx-ES cells）, which was as much as 2 wk earlier, than those in the culture of parental ES cells （wt-ES）. The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 （PDX1）, proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular *** secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not ***： Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.