Characterization of IPSCs-Derived Cardiomyocytes

Abstract

Purpose Functional cardiomyocytes are instrumental to in vitro studies of myocardial diseases, understanding toxicities, and studying new therapies. Because functional cardiomyocytes in tissue culture are scarce and difficult to obtain we sought to differentiate and characterize cardiomyocytes derived from induced pluripotent stem cells (iPSCs) in our lab. Methods Induced pluripotent stem cells (iPSCs) from dermal fibroblasts obtained from a healthy donor were utilized to derive cardiomyocytes. Cells were cultured until 70-85% confluent and differentiated to cardiomyocytes using differentiation kit that contain a high dose of GSK3B inhibitor to induce mesodermal lineage commitment for one day that will result in up-regulation of endogenous BMP4 and activin/NODAL followed by WNT signaling modulation for two days for Cardiac mesodermal induction. Immunocytochemistry for NKX2-5 and TNNT2 were used to characterize differentiated cardiomyocytes. NanoString micro RNA was used to profile commercial and lab differentiated cardiomyocytes. Results Cells start beating on day eight and stayed beating for 30 days in maintenance media. Furthermore, cells were positive for two key markers of the human cardiac lineage: NKX2-5 for early cardiac mesoderm and TNNT2/cTNT for cardiomyocytes. The percentage of differentiation to cardiomyocytes were about 70% of the total iPSCs. Both commercial and lab dedifferentiated cardiomyocytes have similar contractility function. We compared micro RNA profile of our differentiated cardiomyocytes to commercial iPSCs-derived cardiomyocytes and they cluster very close to each other. However, both were clustered differently from miRNA profile of normal adult tissue cardiomyocytes. Conclusion Unlimited iPSCs-derived cardiomyocytes can be obtained in a robust and cost-effective method from fibroblasts. The cells have similar miRNA profile, molecular markers and contractile function in comparison to commercially available cardiomyocytes but have unique patterns of gene expression in comparison to non-failing adult tissue.