Abstract 1142: Effects of Culture Conditions on Cardiomyogenic Differentiation and Expansion of Bone Marrow-Derived Myocardial Tissue-Committed Stem Cells
The bone marrow harbors a population of Sca-1+/Lin-/CD45- tissue-committed stem cells (TCSCs) that express early cardiac markers. Given the rarity (approx. 0.01% of all cells) of these cells, optimization of culture conditions for cardiac differentiation and expansion is crucial for clinical translation. Thus, TCSCs were isolated from bone marrow of EGFP transgenic mice using FACS. Cardiac differentiation and expansion were induced on a feeder layer of bone marrow stromal cells in basic medium (BM, containing FBS, and IGF-1), BM supplemented with dynorphin B (DynB), oxytocin (OT), FGF-2 or insulin (for differentiation), or BM with different doses of FBS supplemented with combinations of LIF, SCF, EGF, PDGF-B and TPO (for expansion). After 30 days of culture, differentiation was quantitated by the expression of cardiac-specific transcription factors and intracellular antigens in EGFP+ cells by confocal microscopy (Fig. 1A⇓). Expansion was assessed by counting the number of EGFP+ TCSCs after 30 days of culture. BM alone, BM+DynB, BM+insulin, BM+FGF-2, and BM+OT induced cardiac differentiation in 12.0±2.5%, 8.2±0.6%, 8.2±0.3%, 17.1±0.3% and 20.6±9.0% of TCSCs, respectively (Fig. 1B⇓). Under expansion conditions, TCSCs survived only in media with low concentrations of FBS (2 and 5%) and in the presence of LIF alone or combinations of LIF, EGF and PDGF-B. Addition of SCF failed to enhance expansion (Fig.1C⇓). These results indicate that TCSCs differentiate into a cardiac phenotype in vitro and that this process is profoundly affected by culture conditions. However, additional strategies need to be formulated to effectively expand these primitive cells.