A Novel Method to Characterize Functional Human Embryonic Stem Cells Derived Cardiomyocytes

Background: As key factors in innate immunity and activators of signals that are critically involved in the commencement of adaptive immune responses, Toll-Like receptors (TLRs) leave a trace in heart diseases, cardiomyocyte (CM) protection, contractility, and maturation. Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, being invaluable resources for cell therapy to restore heart function after damage. Objectives: The main goal of this novel study, TLRs expression during cardiomyocyte differentiation, is to determine whether this assessment can be considered a potential procedure to characterize functional cardiomyocytes. Methods: Royan H5 and Royan H6 hESC lines were cultured in suspension and differentiated into CMs according to the Laflamme protocol, and hESC-CMs were characterized by immunostaining for cytoskeletal proteins. The gene expression for TLR2, TLR3, TLR4, TLR5, and TLR9 was evaluated by RT-PCR and Q-PCR during differentiation. Moreover, the secretion of interleukin (IL)-6 and IL-8 was measured by enzyme-linked immunosorbent assay (ELISA). Results: The cell membrane receptors, TLR2 and TLR4, both represented the downward trend after day 14. However, the expression of endosomal representative receptors (TLR3 and TLR9) surged during differentiation and hit the highest point in fully differentiated CMs. Conclusion: It can be concluded that the generated CMs in this study are functional due to the expression pattern of TLRs being in favor of obtaining desired CMs. Meanwhile, manipulation and optimization of such receptors? expression is suggested in subsequent studies to obtain highly mature CMs as a gold candidate in cardiac cell-based therapy.