Maturation of Induced Cardiomyocyte Precursor Cells Created by Direct Reprogramming

Objective: Enriched population of proliferative induced cardiomyocyte precursors (iCMPs) generated from cardiac fibroblasts (CF) represent a potentially population for use in therapeutic application. However, if true regeneration is to take place, then these cells must be able to differentiate into fully functional cardiomyocytes. Therefore we sought to investigate the differentiation capability of iCMPs using defined media conditions and culture substrates.

Methods: Proliferative iCMPs were generated from CFs by forced expression of Gata4, Mef2c, Tbx5 and Myocd. Pure populations of iCMPs were obtained by molecular beacon (MB) based selection of MYH6/7 expression. The global transcriptome was compared between iCMP, CF, and mature cardiomyocytes (CMs) by RNA sequencing. To generate mature CMs, (1) iCMPs were treated with 5′-azacytidine and cultured in differentiation medium (DM) containing ascorbic acid and TGFß1; (2) iCMPs were maintained in serum free differentiation medium (SFDM) and cardiac differentiation medium (CDM) for 20 days with Wnt inhibitors IWP2 and IWR1 respectively; (3) iCMPs were cultured on cardiac extra cellular matrix (cECM).

Results: iCMPs expressed troponin T, α-actinin and myosin heavy chain (MHC) protein. RNAseq revealed that the iCMP gene expression profile is unique from those of the parental CFs and adult CMs. By Gene Ontology (GO) analysis, iCMPs show upregulation of genes associated with cardiac development, differentiation and morphogenesis, and downregulation of proliferation-associated genes. Individually, non-myocyte genes (COL3A1, VIM) were downregulated, while transcription factors (Gata4, Mef2a, Mef2c, Tbx5, MyocD, Hand1, Hand2) and functional and structural genes including ion channels (KCNH2, SCN5A) and contractile proteins (TNNT1, TNNT2) were upregulated. iCMPs gradually changed morphology after stimulation with 5-Aza, establishing morphologically heterogeneous cell populations. After 10 days in culture with SFDM and CDM, more cells expressed cardiac markers (cTnT, α-actinin, MHC) and lost CD31. cECM resulted in an increase in cardiac marker expression and cell cluster formation in SFDM, and a marked increase in α smooth muscle actin (αSMA).

Conclusion: Gene expression suggests that these iCMPs represent an intermediate state of cardiogenic population that can be expanded to yield therapeutic cell doses. However further studies evaluating the regeneration capacity are necessary.