Maintenance and Expansion of Induced Cardiomyocyte Precursor Cells Created by Direct Reprogramming

Objective: We previously developed a protocol for direct reprogramming of cardiac fibroblasts into induced cardiomyocyte precursors (iCMPs) followed by transcriptional selection. Here, we evaluated different methods to support iCMP enrichment and maturation in vitro, prior to therapeutic intramyocardial transplantation.

Methods: For cellular reprogramming, murine cardiac fibroblasts were infected with a transduction cocktail of IRES-GFP VSV-G pseudotyped lentiviral vectors (LV) encoding for Gata4, Mef2c, Tbx5 and MyocD (GMTMy). For enrichment of viable iCMPs, molecular beacons (MB) were constructed that emit fluorescence upon binding to myosin heavy chain α/beta (MYH6/7). Two weeks post infection the cells were transfected with the MB using the non-viral XtremeGENE HP DNA nucleofection system. The sorted cells were maintained in culture to assess stability of phenotype (immunocytochemistry and FACS analysis) and proliferation over serial passages. Culture medium was supplemented with factors or cocktails known to support mesoderm induction (ascorbic acid, BMP4, FGF2, VEGF) or cardiac specification and maturation (RPMI/B27, insulin withdrawal, FBS, IWR-1-endo, CHIR) and cells were monitored for morphological changes, MYH6/7 expression levels and spontaneous contraction.

Results: Reprogramming resulted in heterogenic population of cells that expressed troponin T, α-actinin, and myosin heavy chain (MHC) protein by immunocytology. Molecular beacon-based FACsorting yielded a MYH6/7+ population with an initial purity of 80% that corresponded to 30% of the transduced cells. Over 28 days, cell number increased 57-fold without obvious signs of senescence, but purity decreased to 40–50%. By renewed FACsorting for MYH6/7 molecular beacon binding, purity could be raised to 70%. Neither incubation with RPMI/B27, VEGF, FGF2, BMP, IWR-1-endo, CHIR or combinations thereof reproducibly increased the MYH6/7+ iCMP fraction. Ascorbic acid, however, led to a robust and significant increase in iCMP purity to 99% at two weeks and 95% at 3 weeks, without signs of senescence, cell death or terminal differentiation and cell cycle exit.

Conclusion: Ascorbic acid alone is most effective in preserving iCMP phenotype throughout a post-reprogramming in vitro expansion period and facilitates the generation of adequate iCMP cell doses for therapeutic application.

 

Scroll to top

No conflict of interest has been declared by the author(s).