Figure 6. Fusion of hMSCs with NRVMs occurs rapidly and leads to loss of sarcomeric structures.

A) Percent of fused cells increases with time of hMSC/NRVM co-culture, reaching a steady-state after only 12 hrs (4day: n = 8 co-cultures; all others: n = 2–3 co-cultures). (B1–4) Representative snapshots from a live-cell time-lapse video of hMSCs-gCaMP3/NRVM-mCherry co-culture. Note rapid spread of gCaMP3 label from hMSC to NRVM (arrow in B2 and B3) and occurrence of mCherry in hMSC (arrowhead in B2 and B3). Within 1 hr, membrane contour of the fused cell (yellow dashed line) significantly changes (B4). Elapsed time in minutes shown at bottom left. (C) Representative immunostaining of sarcomeric α-actinin (SAA, red) after 6 hrs of hMSC-gCaMP3/NRVM co-culture showing breakdown of sarcomeres in fused cell (white dashes – border of hybrid cell, roughly 25% of which has intact striations). (D) Percent area of a fused cell covered by cross-striations decreases over time, indicating loss of sarcomeres; n = 4–10 cells per time point. All scale bars 50 μm.