Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells

Abstract

Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca(2+) indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25-40% of hMSCs (from 4 independent donors) acquired periodic Ca(2+) transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca(2+) current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca(2+) channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

Figure 1. hMSCs in co-culture with NRVMs express cardiac markers and activate human cardiac genes.

A,B) Representative confocal images of immunostaining for sarcomeric α-actinin (SAA) and cardiac troponin T (cTnT) from 7-day old hMSC-eGFP/NRVM co-cultures showing eGFP⁺/α-actinin⁺ (A) and eGFP⁺/cTnT⁺ (B) cells. (C) RT-PCR using rat- and human-specific primers shows activation of human cardiac genes (hTNNT2, hACTN2, hRYR2) in hMSC-eGFP/NRVM co-culture. Scale bar 50 μm.

Figure 2. hMSCs-gCaMP3 in co-culture with NRVMs exhibit both membrane voltage fluctuations and Ca²⁺ transients.

A) In co-cultures of hMSCs and NRVMs (phase contrast, top left), hMSCs transduced with Ca²⁺-sensitive indicator gCaMP3 exhibit flashes of gCaMP3 (examples shown by arrows) representing Ca²⁺ transients occurring in synchrony with NRVM contractions. Scale bar 100 μm. (B) Time snapshots during optically recorded gCaMP3 Ca²⁺ transient shown by pseudo-colored fluorescence intensity and corresponding time traces (below). C) Quantification of the percent of flashing hMSCs at 1 and 7 days of co-culture (n = 12 co-cultures, *p < 0.05, Student’s t-test). D) Representative sharp electrode recordings from 1 day-old hMSC-gCaMP3/NRVM co-cultures show that both flashing and non-flashing hMSCs have membrane voltage oscillations (bottom), but only flashing cells demonstrate synchronous gCaMP3 (Ca²⁺) transients (top). E) Unlike non-flashing hMSCs (left), flashing hMSCs from 1-day co-cultures demonstrate sensitivity to local superfusion with 10 mM caffeine (right).

Figure 3. Flashing hMSCs arise from fusion between hMSCs and NRVMs.

A) Representative images from 1-day co-cultures of NRVM-mCherry with hMSC-gCaMP3 with evidence of dually-labeled fused cells (arrows). (B) Nuclear labeling of 1-day co-culture of NRVM-H2B-mCherry and hMSC-gCaMP3/H2B-GFP shows both NRVM and hMSC nuclei in fused cells. (C) 1-day co-culture of NRVM-Cre with hMSC-LoxP.mCherry.STOP.LoxP.GFP shows Cre-mediated switching of hMSC labeling from mCherry to GFP, indicative of fusion with Cre-expressing NRVMs. All scale bars 50 μm. (D) Quantification of flashing hMSCs shows that nearly 100% of flashing hMSCs have markers of fused cells and virtually no non-fused hMSCs show flashing (n = 10 co-cultures). (E) These 3 independent methods to assess and quantify cell fusion all show that ~30% of hMSCs undergo fusion after 7 days of co-culture with NRVMs (nuclear: n = 2; cytoplasmic: n = 6; Cre-lox: n = 9 co-cultures). (F) Similar degree of fusion has been measured (by Cre-lox method) for hMSCs from 4 independent donors (n = 2–5 co-cultures).

Figure 4. Fused cells express various cardiac markers and couple to NRVMs.

A–D) Representative images of 7-day old hMSC-gCaMP3/NRVM-mCherry co-cultures showing that dually labeled fused cells express various cardiac markers including cardiac troponin I (cTnI, A), myosin heavy chain (MHC, B), sarcomeric α-actinin (SAA, C), and gap junctional protein Connexin-43 (Cx43, D). A non-fused hMSC is shown with arrow in B. All scale bars 50 μm.

Figure 5. Fused cells have electrophysiological properties in between hMSCs and NRVMs.

A) Representative traces of action potential depolarization in fused green cells (lines) and red non-fused hMSCs (circles and lines) in 1-day hMSC-LoxP.mCherry.STOP.LoxP.GFP/NRVM-Cre co-cultures. (B) Depolarization upstroke velocity (dV/dt_max) in fused cells and non-fused hMSCs; n = 5 cells each, *p < 0.02, Student’s t-test. (C) Representative example of whole-cell voltage clamp recording of GFP⁺ cell dissociated from 7-day co-culture of NRVM-Cre and hMSC-LoxP.mCherry.STOP.LoxP.GFP (i.e. fused cells) shows robust inward Ca²⁺ currents. (D) Average current-voltage relationships from freshly dissociated red non-fused hMSCs, green fused cells, and NRVMs from 7-day co-cultures show that total L-type Ca²⁺ currents in fused cells are in between those of non-fused hMSCs and NRVMs; n = 5–9 cells per group.

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.

Figure 7. Reversible inhibition of fusion via Myosin II and Ca²⁺ channel blockade.

A) Fraction of fused hMSCs in 1, 2, and 7-day co-cultures after application of various drugs. Drugs were added at the start of co-culture and removed after 1 day; n = 3–15 co-cultures; *p < 0.01, #p < 0.0001 relative to “Control, 1d in drug”, ANOVA with Tukey’s post-hoc test. (B) Mean squared displacement of hMSCs in co-culture with NRVMs during a 3.5 hr time-lapse imaging in the presence of various drugs. (C) Fraction of migrating cells across all co-cultures. (D) Parameter fitting from individual cells for speed, persistence time and mean path of migration; *p < 0.05. (B–D) n = 2–3 co-cultures, N = 25–54 cells per timelapse of co-culture.