291 ENRICHMENT OF CARDIOMYOCYTES DERIVED FROM MURINE EMBRYONIC STEM CELLS TRANSFECTED OR NOT WITH BOVINE GROWTH HORMONE GENE (bGH)

Abstract

Embryonic stem cells (ESC) are a pluripotent cell type that may be differentiated into cell lineages derived from the three embryonic layers. In animal reproduction biotechnologies, one of the main applications of ESC is their use as a vehicle for the transference of mutant alleles into animal genoma. Transformed ESC can be used for production of chimeras or transgenic clones, searching for economically favorable genetic conditions. The objective of the present study was to evaluate the biological effects of transfection, based on cardiomyocyte differentiation of the H106 mES cell line transfected or not with bGH (mESC and mESCbGH). Transfection was performed using the Ecdysone-Inducible Mammalian System (InVitrogen Brasil, Ltda., Sao Paulo, Brazil) (Buck 2003 Acta Sci. Vet. 31, 270–271 abst), by means of electroporation of pIND plasmids containing the geneticin resistance gene and bGH linked to ecdysone inducible promoter, and lipofection of pVgRXR plasmids containing the zeocin resistance gene. Cells were purified by zeocin and geneticin selection, and bGH cDNA was demonstrated by PCR. bGH expression was not induced in the present study. Cells were cultivated in suspension using the hanging drop technique (Hopfl 2004 Germ Cell Protocols 2, 79–98), in 20-µL drops containing mESC suspension (100 000 cells mL^–1), divided into six groups: control mESC, dimethyl sulfoxide (DMSO) mESC, retinoic acid (RA) mESC, control mESCbGH, DMSO mESCbGH, and RA mESCbGH. Cells were resuspended in DMEM medium supplemented with 15% fetal calf serum, 0.1 mm 2-mercaptoethanol, 2 mm sodium pyruvate, 0.1 mm nonessential amino acids, 2 mm L-glutamine and 50 µg mL^–1 streptomycin; DMSO groups were supplemented with 1% DMSO on Day 0, and RA groups were supplemented with 1 × 10^–7 m all-trans-RA on Day 2, exchanging 10 µL medium containing 2 × 10^–7 m all-trans-RA. After 5 days, formed embryoid bodies (EBs) were transfered to 96-well gelatin-coated adherent plates, in medium without DMSO or RA. Medium exchange was carried out each 48 h. Beating cells were evaluated on Day 14. Immunocytochemistry was performed on Day 17. Statistical analyses to measure differences between treatments were performed using the chi-square test. Approximately 50 structures were formed in each group. Both cell lines (mESC and mESCbGH) exibited beating EBs on Day 14 (control mESC: 22.9%; control mESCbGH: 50.9%), and in both lineages DMSO increased beating structures (mESC DMSO: 48.9% (P < 0.01); mESCbGH DMSO: 71.2% (P < 0.05)). RA was toxic to both lineages, and the EBs dissociated into small clumps of cells, which did not form any beating cells. Beating EBs expressed cardiac Troponin I marker, characteristic of cardiomyocytes. Both cell lines, before and after transfection, differentiated into cardiomyocytes, increased the beating cell rate when exposed to DMSO, but did not form beating cells when exposed to RA. This demonstrates that transfection did not cause any detrimental biological effects during cardiomyocyte differentiation.