Labeling Embryonic Stem Cell With EGFP Differentiation Into Cardiomyocyte-Like Cells In Vitro And The Preliminary Study Of Embryoid Body Formation

Embryonic stem cells are pluripotent cells derived from the inner cellmass of pre-implantation mouse embryos. They have self-renewal ability andrepresent embryonic precursor cells that can differentiate into three embryonicgerm layers in vivo and in vitro. It is a tractable cellular system to investigatecellular and genetic programming of early development. In terms of clinicalbenefit, stem cells are generating many hopes for future regenerative medicine.Mouse embryonic stem cells are widely used for gene mutation analysis or forincorporation of marker genes. Molecular markers are useful in studying geneexpression profile during differentiation and in tracing and selecting particularsubsets of cells. The mES allows for studying genes of interest in vitro, but,more importantly, by creating chimeras it is an extremely useful tool inanalyzing genes during the embryo development in vivo. Green fluorescentprotein emits fluorescence independent any substrate and live cell directlydetect the green fluorescence by the 488 nm emission light under the inverted IIIfluorescent microscope. The GFP is introduced into ESC and screened thestable GFP-positive ESC. Finally the GFP-positive ESC can be tracked andmonitored by detecting the green fluorescent under the inverted fluorescentmicroscope. Some methodology of GFP transfection had been established byelectroporation or other way. In the present research, plasmid pEGFP N2 wasintroduced into the murine embryonic stem cell D3 by standard calciumphosphate precipitation. Transfected clones were screened out under thefluorescence microscope at the 488 nm emission light in the presence of G418.Strong fluorescent EGFP clones were picked out and further proliferated.Stable EGFP ES cells were subcultured and maintained on a feeder layer ofmitomycin-C treated mouse embryonic fibroblasts (MEF) and underwenttwenty passages. PCR analysis and sequencing for GFP DNA showed thatundifferentiated EGFP ES cells at passage 8 and 18 carried the GFP gene.Alkaline phosphatase staining, embryoid body (EB) and teratoma formationwere carried to analyze the differentiation status and potential of the EGFP ESD3 cells. The cells derived from EB were able to differentiate into beatingcardiomyocytes with green fluorescence clearly observable under the confocallaser scanning microscopy. 30-40% of cells from embryoid bodies werecapable to differentiate cardiomyocyte-like cells, which appeared to be lowerthan the non-transfected ES cells, which could be 60-70% under the samecondition. Immunocytochemistry staining indicated that the contracting cellswere cadiomyocytes based on the presence of cardiac specific molecularmarker cTnT. Results showed that the stable EGFP ES cell line retained thetypical characteristics of ES cells. The EGFP transfected cells stably yieldingbright green fluorescence in real time and in situ rendered it a powerful tool incell transplantation and tissue engineering the researches. ESC maintained proliferation and undifferentiation in vitro on the mouseembryonic fibroblast or present of LIF. When remove the inhibitor ESCundergo differentiation. ESC forms the three-dimensional aggregates of cellscalled embryoid bodies (EBs). EBs increase in cell number and complexity as IVcells from the three embryonic germ layers are formed. However EBs play animportant role in the ESC differentiation, there are few report about themethodology and morphology of EBs. In this study, we established threemethods to form the EBs, such as STO, suspension culture, collagenase,compared with traditional method: hanging drop, which evaluate normal EBsformation by detecting the cardiomyocytes beating in different culture system.The results show STO, suspension culture, collagenase, compared withtraditional hanging drop, obtained different percentage of beatingcardiomyocytes EBs. The statistic indicated th...