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

doi：

Abstract：

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 gene.METHODS: 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 cells.The outgrowths were incubated in DTZ solution (final concentration, 100 μg/mL) for 15 min 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 ELISA.RESULTS: 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 clusters.Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated.CONCLUSION: Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

作者：
Author：	Akira Shiroi[1]  Shigehiko Ueda[2]  Yukiteru Ouji[1]  Ko Saito[2]  Kei Moriya[1]  Yuko Sugie[1]  Hiroshi Fukui[2]  Shigeaki Ishizaka[1]  Masahide Yoshikawa[1]
作者單位：	Division of Developmental Biology,Department of Parasitology, Nara Medical University, Kashihara,Nara 634-8521, Japan Department of Gastroenterology and Hepatology, Nara Medical University, Kashihara, Nara634-8521, Japan
期 刊：	世界胃腸病學雜志（英文版）   ISTICSCI
Journal：	WORLD JOURNAL OF GASTROENTEROLOGY
年，卷(期)：	2005, 11(27)
分類號：	R3
Keywords：	Embryonic stem cells    Insulin    Nkx2.2    Dithizone
機標分類號：	S8 R9
機標關鍵詞：	embryonic stem cells    ES cells    cultured cells    transfer of    in vitro
基金項目：