Study On Inducing Differentiation And Enrichment Of Human Embryonic Stem Cells Into Atrial And Ventricular Myocytes

The adult heart has limited regenerative capacity. Therefore, any significant heart cell loss may lead to the development of progressive heart failure, one of the leading causes of worldwide morbidity and mortality. Myocardial cell replacement therapy is emerging as a novel therapeutic paradigm for myocardial repair.The recently described human embryonic stem cells (hESCs) lines may provide a possible solution for this cell sourcing problem. These unique pluripotent stem cell lines, derived from the inner cell mass of human blastocysts, can be propagated continuously in culture in the undifferentiated state and coaxed to differentiate into a variety of cell lineages. hESCs-derived cardiomyocytes resemble immature human fetal cardiomyocytes by multiple criteria, including electrophysiology, calcium handling, force generation, and contractile protein expression and myofibrillar structure. Despite these advances, cardiomyocyte differentiation strategies remain suboptimal. A high degree of heterogeneity is observed in the differentiated cultures, which is a potential cause of ventricular arrhythmias.alpha-myosin heavy chain (aMHC) is robust and specific expression in cardiomyocytes generated during the in vitro differentiation of the hESCs, which suggested that the aMHC promoter could be suitable for positive selection of hESCs-cardiomyocytes.Here, we constructed a plasmid with Blasticidin resistance/EGFP-labeled cassettes, aMHC-Blar-2A-EGFP-Rex-neo. Then we packaged the plasmid into lentivirus, infected hESCs with it and selected hESCs with G418. After selection, we established a stable hESCs line and differentiated into cardiomyocytes, enriched purified cardiomyocytes.1. Construction and identification of lentiviral vector containing aMHC promoter.The blasticidin resistant gene and the foot-and-mouth disease virus(FMDV) 2A segment ware amplified by PCR, and cloned the Blar-2A fragment into the lentiviral vector, aMHC-EGFP-Rex-neo, constructed a lentiviral vector with Blasticidin resistance/EGFP-labeled cassettes, aMHC-Blar-2A-EGFP-Rex-neo. After identifi-cation by restriction endonuclease analysis and sequence analysis, we expanded the aMHC-Blar-2A-EGFP-Rex-neo vector. Using three plasmids (transfer vector with expression construct, the packaging plasmid psPAX2, and the envelope protein expression plasmid pMD2.G) transfected into 293T. 2. hESCs infection and drug selection of stable hESCs line.Using lentiviral vectors to infect low passage hESCs. The infected hESCs have specific promoter Rex,which could promote the expression of neomycin resistant protein. After G418 selection, we obtained the infected hESCs, manual dissect the clones, and propagate them.We confirmed that infected hESCs have normal karyotype and express inserted fragment by karyotype analysis and PCR, and named neo-hESCs. It demonstrated that infected hESCs has typical characteristics of normal hESCs, which indicated that transfected with lentivirus and gene expressing did not effect on the characteristics of hESCs.3. Differentiation and enrichment of neo-hESCs into cardiomyocytes.Direct differentiate the neo-hESCs into atrial and ventricular myocytes by retinoid signals pathway. The cardiomyocytes were beating at day 10. The RNA of day 9, day 10, day 12 were extracted and synthesized cDNA, in order to detect the expression of aMHC, and the onset of endogenous expression of aMHC is as early as day 9.In the day 10 of differentiation, we selected the cardiomyocytes with Blasticidin. After Blasticidin selection, cardiomyocytes can be maintained as spheroidal clasters attached to the substratum. The immunostaining analysis showed the expression of typical cardiac marker cTnT. Therefore, the selected cardiomyocytes named as Blar-2A-EGFP-cardiomyocytes. EGFP were detected under fluorescence microscope, and the beating area is shwing EGFP. Our results demonstrated the appearance of cardiomyocytes after the selection that can be sorted using FACS, and the drug selection protocol yielded 95% ventricular myocytes, and 93% atrial myocytes. Furthermore, we examined the expression of ventricular specific gene—IRX4. Quantitative RT-PCR showed that IRX4 expression was 10-fold greater than the atrial myocytes.Our study indicates the feasibility of a specific selected hESCs line for cardiomyocytes purification, and provides an important platform to study the regulatory mechanism in cardiogenesis.