title: Identification, selection, and enrichment of cardiomyocyte precursors.
abstract: The large-scale production of cardiomyocytes is a key step in the development of cell therapy and tissue engineering to treat cardiovascular diseases, particularly those caused by ischemia. The main objective of this study was to establish a procedure for the efficient production of cardiomyocytes by reprogramming mesenchymal stem cells from adipose tissue. First, lentiviral vectors expressing neoR and GFP under the control of promoters expressed specifically during cardiomyogenesis were constructed to monitor cell reprogramming into precardiomyocytes and to select cells for amplification and characterization. Cellular reprogramming was performed using 5'-azacytidine followed by electroporation with plasmid pOKS2a, which expressed Oct4, Sox2, and Klf4. Under these conditions, GFP expression began only after transfection with pOKS2a, and less than 0.015% of cells were GFP(+). These GFP(+) cells were selected for G418 resistance to find molecular markers of cardiomyocytes by RT-PCR and immunocytochemistry. Both genetic and protein markers of cardiomyocytes were present in the selected cells, with some variations among them. Cell doubling time did not change after selection. Together, these results indicate that enrichment with vectors expressing GFP and neoR under cardiomyocyte-specific promoters can produce large numbers of cardiomyocyte precursors (CMPs), which can then be differentiated terminally for cell therapy and tissue engineering.
authors.affiliation: Department of Biophysics, Gene Therapy Investigation Center, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Bianca Ferrarini
authors.initials: BF
authors.lastname: Zanetti
authors.forename: Walter José
authors.initials: WJ
authors.lastname: Gomes
authors.forename: Sang Won
authors.initials: SW
authors.lastname: Han
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D051381:Rats; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D049452:Green Fluorescent Proteins