Differentiation of human embryonic stem cells into cardiomyocytes in vitro
- VernacularTitle:人胚胎干细胞分化为心肌细胞的体外实验
- Author:
Xinlan FAN
;
Chunling MENG
;
Yougang MAI
;
Ling XU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;11(46):9413-9415
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro has been studied in great detail in the world. However, much of what is currently known about cardiomyocyte differentiation from ES cells has been learned from studies on mouse in China, few studies are on human ES cells.OB JECTIVE: To investigate the differentiation effcacy of human ES cells into functional cardiomycytes with the human H14 ES cell line.DESIGN: Suspending method was used to form pseudo-embryo proper of human ES cells so as to observe ratio of pseudo-embryo proper with rhythmic contraction and expression of specific gene of myocardium in various differentiated phases.SETTING: Molecular Biology Laboratory of Stanley Ho Center for Emerging Infectious Diseases, School of Public Health, the Chinese University of Hong Kong.MATERIALS: Human ES cell line H14 was obtained from WiCell Research Institute (Wisconsin, USA) with a license agreement.METHODS: The experiments were carried out in the Molecular Biology Laboratory of Stanley Ho Center for Emerging Infectious Diseases, School of Public Health, the Chinese University of Hong Kong from August to December of 2006.The H14 ES cell colony was used to form embryoid bodies (EBs) by using suspending method. Four days later,pseudo-embryo proper was cultured in gelatin-coating 6-well culture plate (5-10 embryo proper/well) and spontaneously differentiated into moving pseudo-embryo proper. Rhythmic contraction was observed under microscope and RT-PCR was used to detect expression of special genes of myocardium.MAIN OUTCOME MEASURES: Ratio of pseudo-embryo proper with rhythmic contraction and expression of specific gene of myocardium in various differentiated phasesRESULTS: Spontaneously contracting cells appeared as cluster and were identified in approximately 2% of EBs at differentiation day 8 and increased to as many as 10% of the EBs by day 16. The beating rate of contracting cells arranged at 70-100 beats per minute. RT-PCR analyses demonstrated that cells isolated from spontaneous beating areas within the EB expressed the cardiac transcription factors GATA-4 and Nkx2.5, cardiac progenitor gene Isl-1 and cardiomyocyte marker gene α-MHC.CONCLUSION: This is the first time to report human ES cells can effectively differentiate into functional cardiomyocytes in China.
