In vitro differentiation and fusion of hBMSCs to CMCs, ECs, and SMCs. (A–L) Coculture of hBMSCs with NRCMs. On day 3, DiI-labeled hBMSCs were added to the cultured NRCMs at a 1:4 ratio. IF images show cocultured DiI-labeled hBMSCs (B, F, and J) as red and NRCMs stained with CMC-specific proteins cTnI (C), ANP (G), and α-MHC (K) as green. Double-fluorescent cells in the merged images (D, H, and L) indicate that a subpopulation of hBMSCs exhibit CMC phenotypic markers (arrows). Arrowheads in B, D, J, and L indicate nontransdifferentiated hBMSCs. DAPI nuclear counterstaining (A, E, and I) shows no overlap of nuclei. mRNA expression of cardiac transcription factors was evaluated by RT-PCR (M). Before coculture, GATA-4 and Nkx2.5 were not expressed in hBMSC (lane 1) and NRCM (lane 2) cultures. Coculture (lane 3) induced de novo expression of GATA-4 and Nkx2.5. (N–Q) Coculture of prelabeled NRCMs and hBMSCs for investigation of cell fusion. NRCMs were labeled with the green fluorescence dye CFDA-SE (N) and DiI-labeled hBMSCs (O). Seven days after coculture, cells were stained for cTnI expression (blue fluorescence) (P). In the IF images (N–Q), triple-fluorescent cells (arrows) are fusion cells expressing cTnI protein. An hBMSC that has differentiated into CMC lineage is illustrated by red and blue fluorescence without green fluorescence (arrowheads). (R–Y) To determine the contribution of fusion and differentiation to phenotypic changes of hBMSCs into ECs (R–U) or SMCs (V–Y), CFDA-SE–labeled RAECs or RVSMCs were cocultured with DiI-labeled hBMSCs. Cells were stained with VE-cadherin or α-SMA (blue fluorescence). Arrows in R–U indicate fused RAECs (green) and hBMSCs (red) that express VE-cadherin. Arrowheads in R–U illustrate ECs differentiated from hBMSCs. Arrows in V–Y indicate fused RVSMCs (green) and hBMSCs (red) that express α-SMA. Scale bar in A–L: 50 μm; scale bar in N–Y: 100 μm.