The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes

Mesenchymal stem cells (MSCs) were recently found to form three-dimensional (3D) multicellular spheroids on chitosan membranes. The exact mechanism of spheroid formation, however, remains unclear. In this study, the regulation of spheroid formation for adipose derived adult stem cells (ADAS) grown on chitosan membranes was examined. By varying the membrane thickness, calcium concentration in culture medium, and acetylation extent of chitosan, the physico-chemical characteristics of chitosan that modulated spheroid formation was elucidated. The capacity of cardiomyogenic differentiation was further evaluated. Results suggested that the calcium binding capacity of chitosan may affect the cell–substrate and cell–cell interactions and critically influence the dynamics of spheroid formation. The intracellular calcium level was elevated for ADAS spheroids on chitosan. Chitosan-bound calcium was observed to enter the cells. The expression of N-cadherin was upregulated for ADAS spheroids on chitosan, evidenced by quantitative RT-PCR and Western blot. After the induction by 5-aza, the expression levels of cardiac marker genes (Gata4, Nkx2.5, Tnnt2, and Myh6) were remarkably enhanced (about four-fold) for ADAS on chitosan vs. tissue culture polystyrene or polyvinyl alcohol. Immunofluorescence staining confirmed the expression of cardiac-associated tight junction protein ZO-1 for ADAS grown on chitosan membranes. The gene expression of Wnt11 was significantly upregulated for ADAS spheroids on chitosan at 3 days and 12 days. We suggested that Wnt11 may be involved in the spheroid formation and cardiomyogenic differentiation of MSCs on chitosan membranes. Spheroids formed on the acetylated chitosan or polyvinyl alcohol membranes failed to show such behavior. The properties of MSC spheroids were therefore determined by the culture substrate.