The Role Of Wnt3a In Cardiomyocyte Differentiation Of Mouse Embryonic Stem Cells

Embryonic stem (ES) cell lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into derivatives of all three primary germ layers, including cardiomyocytes, depending on the cultivation conditions. During differentiation, cardiac-specific proteins, receptors, and ion channels are expressed in adevelop -mental continuum, which closely recapitulates the developmental pattern of early cardiogene- sis.Wnt signaling is involved in the development of many organs of various species, including cardiogenesis. Wnt signaling includes canonical Wnt pathway and noncano-nical Wnt pathway. There are two kinds of point about the role of canonical Wnt pathway in cardiogenesis, one is canonical Wnt pathway plays a negative role in cardim yocyte differentiation, the other is canonical Wnt pathway plays a positive negative role.AimBy using the mouse ES cell line from, we demonstrate the expression and role of the Wnt3a in order to illustrate the role of canonical Wnt pathway in cardiomyocyte differentiation of mouse ES cells. MethodsThe ES cells were obtained form the the Stem Cell Center of Zhengzhou University and cultured in MEF. The ES cells were identified by SSEA-1, OCT-4, AKP.To induce differentiatin, the cells were cultured in hanging dorps to form embryoid bodies for 4 days in different medium: GroupA (essential medium without inducer), GroupB (essential medium+ 1%DMSO) and GroupC (essential medium+ 1%DMSO+Frizzled-8/fc). Then ES cells were grown on gelatin-coated tissue culture plate.To elucidate Wnt3a mRNA transcripts, the total celluar RNA was extracted from the cells through day1 to day6 and detected using RT-PCR.Identification of induced cardiac cells:1. Examinations were done under an inverted microscope.2. Alpha-actin and cardiac-specific troponin T were detected through immunocytochemistry.3. MLC - 2v mRNA and alpha-MHC mRNA transcripts were detected by the method of reverse transcription polymerase chain reaction (RT-PCR).ResultsThe ES cells were grown in MEF and were poseitive for SSEA-1, OCT-4, AKP.In groupB and GroupC, Wnt3a gene expression was induced at 2-4 day after treatment with 1% DMSO. After 4 day its expression was down regulation. In group A Wnt3a was not detected.In group B, ES cell aggregates treated with dimethyl sulfoxide (DMSO) was induced into cardiomyocytes. Immunocytochemistry revealed that DMSO-treated cells expressed alpha-actin and cardiac-specific troponin T; cardiac MLC - 2v and alpha-MHC were detected by RT-PCR.In groupA and GroupC, spontaneous beating was not observed. Alpha-actin and cardiac-specific troponin T were not deceted by Immunocytochemistry in groupA and GroupC. Cardiac MLC - 2v and alpha-MHC were not detected by RT-PCR in groupA and GroupC. ConclusionExpression of Wnt3a was induced by DMSO in the ES cells.Wnt3a plays a positive role in cardiomyocyte differentiation of mouse ES cells.