Recombinant mouse SPARC promotes parietal endoderm differentiation and cardiomyogenesis in embryoid bodies

Abstract

In the absence of leukemia inhibitory factor, murine embryonic stem cells cultured in vitro spontaneously aggregate to from three-dimensional embryoid bodies that differentiate to produce hematopoietic, endothelial, muscle, and neuronal cell lineages in a manner recapitulating the events of early embryogenesis. Cardiomyogenesis in embryoid bodies was recently demonstrated to be promoted by PYS-2-derived native SPARC (secreted protein, acidic and rich in cysteine), whose expression is upregulated in parietal endoderm at the onset of the epithelial to mesenchymal transition. Here, we confirm the stimulatory effects of mouse SPARC on cardiomyogenesis using a recombinant baculovirus-produced protein (rmSPARC). Embryoid bodies cultured in the presence of glycosylated rmSPARC, or an unglycosylated peptide spanning the C-terminal EF-hand domain, developed greater numbers of beating cardiomyocytes than did time-matched controls, with enhanced expression of cardiac marker genes including Nkx2.5, Troponin, BMP-2, and MHCalpha. Histochemical analysis revealed an expansion of the peripheral endoderm, with thicker layers of extracellular matrix (ECM) material observed atop underlying cells. Embryoid bodies treated with SPARC also displayed increased adherence to polystyrene culture dishes, with enhanced expression of ECM mRNAs including collagen IValpha3, collagen IValpha5, and laminin alpha1. These results indicate that, in addition to the promotion of cardiomyogenesis, SPARC may also help regulate the molecular composition and organization of ECM secreted by the mesenchymal parietal endoderm.