Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation?

OBJECTIVE: The potential use of bone marrow stromal cells (MSCs) as a cellular therapy for chronic cardiac diseases relies on the ability of the cell to replicate extensively in vitro and to give rise to myogenic cells that can replace the damaged cardiomyocytes. For this reason the present study investigated the replication lifespan and chemical-induced cardiomyogenic differentiation of rat MSCs in vitro.

METHODS: The primary and the successively passaged Wistar rat MSCs were exposed to different concentrations (3, 5 and 10 microM) of 5-azacytidine using different methods (single- or repeat-treatment). The growth properties and the fate of the cells were compared to their untreated counterparts by cell counting, immunocytochemistry and Western analysis.

RESULTS: When seeded at a density of 2845 cells/cm(2) and cultured under common conditions, rat MSCs could be expanded up to 21.94 cell doublings in 30 days of successive subcultures. This was accompanied by a gradual loss of their replication ability with passages. When treated with 5-azacytidine for 24 h at day 3 of primary culture and the first subculture, the growth properties of the MSCs were not obviously affected. Neither the spontaneously beating cells nor the formation of myotubes were found in the primary and first passaged MSCs after a single treatment with 5-azacytidine and in cultures which underwent repeated 5-azacytidine-treatments during continuous subculturing to passage 2. The expressions of cardiac troponin I, cardiac myosin heavy chain and connexin 43 by the 5-azacytidine-treated MSCs were also undetectable at both immunocytochemistry and Western blot levels. The specificity and reliability of the detection methods were technically confirmed with cultured rat cardiomyocytes.

CONCLUSIONS: Rat MSCs cannot be extensively expanded in vitro or be induced to differentiate in an expected cardiomyogenic way by 5-azacytidine-treatment, if the cells are not immortalized.