Human myocardial extracellular matrix directs the differentiation of pluripotent stem cells toward a cardiomyocyte phenotype

Objectives: Cross-talk between organ-specific extracellular matrix (ECM) and stem cells has often been assumed but not been directly demonstrated. We have previously developed a protocol for preparation of cardiac ECM (cECM) that supports survival and proliferation of cardiomyocyte-like cells, and now studied whether cECM emit differentiation-relevant signals to pluripotent stem cells.

Methods: cECM was prepared from human myocardium by a 3-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS), and fetal bovine serum (FBS). Murine embryonic stem cells (ESC), induced pluripotent stem cells (iPS) and mesenchymal stromal cells (MSC) were seeded and cultured in standard culture, on cECM, or on commercially available non-specific ECM preparations or basement membrane extracts (Matrigel® or Geltrex®) for 20 days. Transcriptional activation of genes involved in early (Nkx2.5, Gata4, Mef2c) and late (Myh6, Tnnt2) myocardial development, pluripotency (Oct4, Sox2), endothelial (Vwf, VE cadherin), ectoderm (Ncam1) or endoderm (Sox17) commitment were monitored by quantitative rtPCR. Protein expression of selected markers was confirmed by immunohistology.

Results: In ESC and iPS cells, cardiac lineage commitment was favoured when grown on cECM, as evidenced by significantly increased gene expression of Myh6 (p = 0.03), Tnnt2 (p = 0.01), Nkx2.5 (p = 0.02) and Gata4 (p = 0.01) as well as positive immunohistology for sarcomeric α-actinin and troponin T. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSC showed no evidence of cardiomyocyte differentiation. Pluripotency markers were best preserved on Matrigel, while endothelial development was supported by both cECM and Matrigel. cECM suppressed endoderm and supported ectoderm development.

Conclusion: Human cardiac ECM conveys signals to pluripotent stem cells that seem to direct differentiation toward a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair.