The Concurrent Effect of 5-Azacytidin and DMSO on In Vitro Differentiation Induction of Cardiomyocytes from Embryonic Carcinoma cells P19
Volume 5, Issue 3, Autumn 2014, Pages 317-324
https://doi.org/10.52547/JCT.5.3.317
H Gh, N R, R S, S A, M L, M M
Abstract Aim: In current study capability of differentiation of embryonic carcinoma cells P19 to cardiomyocyte through inducing effects of DMSO and 5-Azacytidin individually or in combinative form in laboratory condition were examined. Material and Methods: In order to differentiate induction of P19, Embryoid Bodies (EBs) formed through hanging drops method during two days. Then EBs have induced by 2 μM of 5-Azacytidin (5-Aza group), 0/5% DMSO (DMSO group) or both 5-Azacytidin and DMSO (Aza+DM Group) during ten more days. During differentiation beating number per minute counted every two days in all groups by invert Microscopy. Gene expression such as: α-Myosin Heavy Chain (α-MHC) and Myosin Light Chain (MLC) and Connexin-43 have examined by RT-PCR in last day. To more accuracy, F-actin protein expression also illustrated through immunocytochemistry method. Results: Morphological changes in differentiated cardiomyocytes derived from P19 illustrated in all groups. However the most changes such as significant increasing in size and number of process, their branches and attachment between branches happened in Aza+DM group. Daily beating counter per minute revealed increasing in beating number from 2+2 to 2+8 in all groups. RT-PCR analysis also revealed expression of both α-MHC and MLC in Aza+DM Group. However, F-action has expressed only in Aza+DM group. Conclusion: The Results have shown combination of two 5-Azacytidin and DMSO inducer caused effective cell culture differentiate of P19 to cardiomyocyte.