1. ¹Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, China
2. ²Beijing Laboratory Animals Research Center, Beijing 100012, China
3. ³Laboratory of Chemical Genomics, Shenzhen Graduate School of Peking University, the University Town, Shenzhen 518055, China
4. ⁴Provincial Key Laboratory of Embryonic Stem Cell Research, Tai-He Hospital Yunyang Medical College, 32 S. Renmin Rd., Shiyan 442000, China

Correspondence: Hongkui Deng, Tel: 86-10-6275-6954; Fax: 86-10-6275-6954 E-mail: hongkui_deng@pku.edu.cn; Dongsheng Li, Tel: 86-719-8801418; Fax: 86-719-8801418 E-mail: dsli@yymc.edu.cn

^*These two authors contributed equally to this work.

⁵Present address: The Scripps Research Institute, Chemistry Department, SP3130, 10550 North Torrey Pines Road, La Jolla, CA, USA.

Received 12 March 2007; Revised 13 March 2007; Accepted 13 March 2007.

Abstract

The capacity for self-renewal and differentiation of human embryonic stem (ES) cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid (RA) was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdx1 and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin (STZ)-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdx1, glucokinase, nkx6.1, IAPP, pax6 and Tcf1. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.