Directed differentiation of embryonic stem cells towards thyroid cells in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND: Embryonic stem cells fESCs) develop from early blastular inner cell mass.Under proper condition,ESCs can maintain undifferentiated state in vitro,normal diploid nuclear type,and proliferative potential.In addition,ESCs possess multi-directional differentiation capacity and can differentiate into all sorts of cells of three germ layers. OBJECTIVE: To investigate the feasibility of directed differentiation of ESCs towards thyroid cells in vitro as well as related molecular expression. DESIGN,TIME AND SETTING: A cell-based observational experiment was performed at the Bank of Cord Blood,Second Hospital Affiliated to Sun Yat-sen University between January 2004 and December 2006.MATERIALS Balb/c pregnant mice at pregnancy 12.5-14.5 days were used for preparation of embryonic fibroblast feeder layer.E14 mouse embryonic stem cell (ESC) strains were gifted by professor Xu from Harvard University.METHODS: Murine El4 ESCs were cultured in methylcellulose semisolid medium to form embryoid bodies (Ebs).These Ebs were transferred for further inductive culture with the stepwise addition of growth factors- thyrotropin (TSH),insulin and kalium iodidum (KI).The cultured thyroid cells of adult Kunming mice were taken as positive control.MAIN OUTCOME MEASURES: ①Cellular morphological change in the process of differentiation.② Detection of expression levels of TSHR,PAX8,TTF-2,and TIF-1 by immunofluorescence assay.③ Detection of expression levels of TSHR,PAX8,NIS,TPO,and Tg by reverse transcription-polymerase chain reaction (RT-PCR).RESULTS: The differentiated cells had clear boundary,exhibiting round,oval,shuttle-shaped,or polygon adhesive growth.On day 6 of inductive ifferentiation,the differentiated cells showed the expression of PAX8,NIS,TPO,Tg,and TSHR,the specific gene of thyroid ceils.On day 8 of inductive differentiation,the expression of TSHR,TIF-1,PAX8,and TIF-2 was detectable in the differentiated cells with morphous similar to thyroid cells.CONCLUSION: ESCs can differentiate towards thyroid cells under given inductive conditions.