PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium

Abstract

Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT–PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα+ cells. Clonal progeny of single Sca1+ SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα− cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα+/CD31− cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα−/CD31+ cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1+ stem/progenitor cell.

The Quest for the Adult Cardiac Stem Cell

“…62 Stratifying Clonal Growth Based on preparative cell sorting with various combinations of the discussed identifiers, single cells were deposited in 96-well plates to assess the subpopulations' clonal growth, self-renewal, and multilineage potential. 28 SP status was a potent predictor of clonal growth under these exacting conditions, as seen earlier by colony formation in methylcellulose. 41 However, the SP phenotype was dispensible: PDGFRα + Non-SP cells were clonogenic (2% efficiency in physiological hypoxia), yet the combined PDGFRα + SP phenotype was highly synergistic (cloning efficiency >25%).…”

“…41 However, the SP phenotype was dispensible: PDGFRα + Non-SP cells were clonogenic (2% efficiency in physiological hypoxia), yet the combined PDGFRα + SP phenotype was highly synergistic (cloning efficiency >25%). 28 Cells lacking PDGFRα but expressing CD31 were rarely if ever clonable under our conditions, even if positive for the SP phenotype. Cloned Sca1 + SP cells could be propagated for over 1 year, retained the cognate features (Sca1, SP status), maintained the cardiogenic gene signature, and were enriched for generating secondary clones, features together indicating self-renewal.…”

“…Cloned Sca1 + SP cells could be propagated for over 1 year, retained the cognate features (Sca1, SP status), maintained the cardiogenic gene signature, and were enriched for generating secondary clones, features together indicating self-renewal. 28 ATP-binding cassette transporters that mediate the SP dyeefflux phenotype include Abcg2 and Mdr1. Both were expressed at equal levels in SP and Non-SP cells, suggesting that differences in pump activity, not expression, might distinguish the 2 states.…”

“…Both were expressed at equal levels in SP and Non-SP cells, suggesting that differences in pump activity, not expression, might distinguish the 2 states. 28 …”

“…All the clones of Sca1 + SP cells tested expressed Pdgfra mRNA and PDGFRα. 28 Within 2 weeks of cardiac grafting, molecular markers of tri-lineage potential emerged (cardiac troponin I, myosin light chain 2v, sarcomeric α-actin, sarcomeric myosin heavy chain, CD31, von Willebrand factor, smooth muscle myosin heavy chain). At 12 weeks, donorderived differentiating cells were incorporated into the endothelial and smooth muscle layers of blood vessels, and, if cardiac, were typically rod-shaped, bi-nucleated, and striated ( Figure).…”