Study On Inducing Differentiation And Enrichment Of Mouse Embryonic Stem Cells Into Cardiomyocytes

The death of highly vulnerable cardiomyocytes leads to cardiac dysfunction, including heart failure, myocardium injury and infarction. Cardiomyocyte is end-stage differentiated cell and cann't regenerate. To find suitablely alternative donor cells is the focus of the reseachers. Embryonic stem cells (ESCs), accuring in the early stages of embryo development, can differentiate into a wide variety of cell types in defined medium and environment that make them attractive candidates for use in a series of innovative transplantation paradigms, especially transplantation of cardiomyocytes. Exploitation of ESCs-derived cardiomyocytes may facilitate the analysis of early cardiac development, and pave the way for novel therapeutic modalities for enhancing myocardial performance and treating cardiac disease. There are some difficulties in Inducing ESCs into specially cardiomyocytes, its essential to label and sort the pure differentiate cardiomyocytes at stages of differentiation. Cardiacα-actin is one of the earliest markers of cardiac cell development, which is expressed in the early developing heart and observed in all somites as they formed. Cardiacα-actin promoter has been shown to be sufficient to direct cardiac-specific gene expression in stable transformants and human cardiacα-actin promoter has been shown to be cardiac specific and switched on before the onset of spontaneous contractions in ESCs. Up to now many researches had used them to label and sort the pure differentiate cells which are derived from ESCs at stages of differentiation, so we selected human cardiacα-actin promoter for the gene targeting in this study.1. Construction and identification of pAd-actin and pA-CMV-actin which containingα-actin promoter.The cardiacα-actin promoter gene was amplified by PCR,which analysis of the sequencing result shows that it is a member of the human gene family and contains all regulatory factors of the standard promoter. and respectively cloned it into the adenoviral shuttle plasmid pAdTrack, which one was deleted CMV and the ather was not. After identification by restriction endonuclease analysis and sequence analysis, the recombinant shuttle was named pAdTrack-actin and pAdTrack-CMV-actin, then linearized them with Pme I, and transferred into E.coli BJ5183 cells pretransformed with the pAdEasy-1 plasmid. A recombinant adenoviral genomic named pAd-easy-actin and pAdEasy-CMV-actin were obtained by homologous recombination. By endonuclease Pac I digestion, pAd-easy-actin and pAdEasy-CMV-actin were transfected into HEK 293 cells to produce recombinant adenovirus pAd-actin and pA-CMV-actin before PCR identification, and the titer is respectively 6×108 pfu/ml and 5×108 pfu/ml. The recombinant adenovirus containingα-actin promoter has been constructed successfully, and provides a basis to monitor the differentiation of embryonic stem cells into cardiomyocytes.2. The research of mouse ESCs transfected with pAd-actinIn order to select cardiomyocytes from ESCs, we construct pAd-actin which consists ofα-actin promoter and GFP gene as positive selection markers. Then transfer it into mouse ESCs and compare the characteristic of GFP-ESCs with normal ESCs. Morphological observation demonstrated that GFP-ESCs expressed GFP stablely, but Proliferation and clony-forming were declined, which gradually got normal. Immunostaining assay and differentiation demonstrated that GFP-ESCs like normal mouse ESCs which express AP, SSEA-1, Nanog and Oct-4,and have pluripotency to differentiate into cells of cardiomyocytes, neural cells, vascular endothelial cells or insulin-producing cells. and GFP-ESCs have normal karyotype and express Oct-4 and Nanog by karyotype analysis and RT-PCR. It demonstrate that GFP-ESCs has typical characteristics of normal ESCs, which indicated that transfected with pAd-actin and gene expressing did not effect on the characteristics of ESCs.3. Differentiation and enrichment of mouse GFP-ESCs into cardiomyocytesUndifferentiated GFP-ESCs were incubated an d differentiate into EBs by hanging drop and suspension culture method, then EBs attached and continued to proliferate and differentiate with differentiation medium containing 0.75 % DMSO. GFP-ESCs-derived cardiomyocytes were characterized by RT-PCR, immunostaining and FACS. RT-PCR assays showed that differentiated cells specifically expressed cardiac transcription factors, including Nkx2.5 and GATA4. and cardiac specific myoglobin (myoglobin-positive cells were 34.3±2.8,25.6±1.7 ),α-actin (α-actin-positive cells were 44.5±1.4,38.3±2.1) andβ-tublin (β-tublin -positive cells were 35.6±2.2,25.6±3.4) were detected in differentiated cells by using immunostaining. The percent of cardiomyocytes(38.8 and 24.1) was evaluated by FACS, and GFP cells which selected by FACS expressedα-actin. The results suggest that hanging drop method was even benefit for mouse embryonic stem cells into cardiomyocytes, and human cardiacα-actin promoter may be useful in enrichment of mouse ESCs-cardiomyocytes.