Figure 2.

Effects of hypoxic preconditioning on EDC proliferation rate, cardiosphere size, CDC proliferation rate. (A) Atrial heart tissue (n = 4) was separated into two equal portions and cultured under normoxia or hypoxia for up to 18 days. At day 0, the black clump shown at the corner (indicated by dashed arrows) was an edge of a freshly plated cardiac tissue explant. At day 6, fibroblast-like cells (indicated by dotted arrows) were found migrating away from the edge of the explants. At day 9, phase bright cells (indicated by solid arrows) were found to grow on top of this fibroblast-like monolayer. All these cells were collectively termed as explant-derived cells (EDCs). (B) EDCs generated under normoxia or hypoxia were harvested, and cell numbers were counted. At day 9, hypoxic explants generated approximately 1.4-fold more EDCs, compared with normoxic explants, and this rate was maintained up to 18 days. (C) Morphology of normoxic and hypoxic cardiospheres (Csp) were significantly different, with (D) hypoxic Csp 1.9-fold larger than the normoxic Csp. (E) Hypoxia also yielded higher cardiosphere-derived cell (CDC) numbers than normoxia at day 5 of each passage from P1 until P5 (*p < 0.05 vs. normoxia). (F) CDC proliferation assay was validated using Live/Dead^® Viability/Cytotoxicity kit (Molecular Probes). Hypoxic CDCs at P4 showed higher proliferation than normoxic P4 CDCs after 2 days in culture (*p < 0.05 vs. normoxia).