Differentiation of mouse embryonic stem cells into insulin producing cells induced by various growth factors / 中国组织工程研究

ABSTRACT

BACKGROUND: Previous studies have confirmed that embryonic stem cells call be induced to differentiate into insulin-producing cells, but the induction process takes a long time. Most of the processes take about one month.OBJECTIVE: Activin A, all-trans retinoic acid (ATRA), basic fibroblast growth factor (bFGF) and nicotinamide were applied in vitro in combination to observe whether mouse embryonic stem cells could be induct to differentiate into insulin-producing cell in a relatively short time.DESIGN: Cell observation experiment.SETTING: Shanghai Institute of Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.MATERIALS This study was performed at Shanghai Institute of Endocrine and Metabolic Diseases from October 2004 to February 2006. Two mice of clean grade and of 12.5-14.5 days of gestational age were provided by Shanghai SLAC Laboratory Animal Co., Ltd (Permission No. 2004A034). The protocol was performed in accordance with ethical guidelines for the use and care of animals. Mouse embryonic stem cell lines were supplied by Dr Changxian Zhang (CNRS UMR5641, France). Activin A was the product of the R & D Corporation. ATRA and nicotinamide were supplied by the Sigma Corporation, USA. BFGF was supplied by Gibco Corporaion. METHODS: Head and viscera were removed from embryos of the pregnant mouse. The remaining tissues were cut into pieces and digested with trypsin. Cell suspension was centrifuged and inoculated at 3×108L-1. The cells could be used as mouse feeder layer after 2-3 times of passage. The mouse embryonic stem cells (ESCs) were inoculated onto the feeder layer in knockout Dulbecco's modified Eagle medium (DMEM) supplemented with leukemia inhibitory factors (LIF). ESCs were passaged at 13-16 after 2-3 days of culture. Culture medium with serum was added into the culture dishes to terminate the digestion. Cell fluid was centrifuged and supernatant was discarded. The sediments were prepared into suspension and inoculated at 2.5×104 with LIF-free culture medium. After 24-48 hours, embryonic bodies (EBs) were collected and replated in 1% Matrigel-coated dishes. When began to adhere to the dishes, EBs were cultured in 10% FBS/DMEM supplemented with 100μg/L activin A for 24 hours. Then EBs were switched to 10% FBS/DMEM for 6-8 hours as an interval. After this interval. EBs differentiated were cultured in 10% FBS/DMEM with 10<-6mol/L RA for another 24 hours followed by culture in 10% FBS/DMEM supplemented with 10μg/L bFGFs for 3-5 days. Finally, EBs differentiated were cultured in DMEM/F12 supplemented with N2 supplement, B27 supplement, 1μg/L laminin, 10μg/L bFGFs, and 10mmol/L nicotinamide for 3-5 days. Dithizone (DTZ) staining, inununofluorescent staining and reverse transcription-polymerase chain reaction (RT-PCR) were applied to detect insulin expression in the differentiated cells.MAIN OUTCOME MEASURES: Induction of ESCs, DTZ staining and immunofluorescent staining as wel as RT-PCR detection.RESULTS: Mouse ESCs growing on a feeder layer formed many colonies with clear boundary and dense structure. However, there was no obvious outer limit between these ESCs. EBs began to adhere to the dishes, which were coated with matrigel, on the 2nd day. After activin A and ATRA interval induction, EBs spread, and most of the living cells were epithelial cell-like when cultured in 10% FBS/DMEM supplemented with 10μg/L bFGFs. After culturing in DMEM/F12 supplemented with N2, B27, nicotinamide, bFGFs and laminin, the cells formed small clusters. The insulin-producing cells were stained dark red with DTZ, and the cells stained with primary antibody to insulin were insulin-positive. After 2 weeks of induction of activin A, ATRA, bFGFs and nicotinamide, the insulin-producing cells expressed insulin 2, Pdxl, Nkx6.1, Nkx2.2, PP, IAPP, Glut2, Somastatin, Hnf3β and Neuro D mRNA but did not express insulin 1 mRNA.CONCLUSION: Mouse ESCs call be induced to differentiate into insulin-producing cells by activin A, ATRA, bFGFs and nicotinamide in vitro. Induction time call be shortened to 2 weeks.