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

Shadrin IY, Yoon W, Li L, Shepherd N, Bursac N - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC.

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.

No MeSH data available.


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.
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f7: 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.

Mentions: To investigate the mechanism of hMSC/NRVM fusion, we assessed percentage of fused cells following a 24 hr drug application at the time of hMSC seeding onto the NRVMs. We found that treatment with myosin II inhibitors (10 μM blebbistatin, 100 μM BTS, or 15 mM BDM) or L-type Ca2+ channel blockers (10 μM nifedipine or 10 μM verapamil) significantly reduced percent of fused hMSCs (in average 1.4 ± 0.43% in treated vs. 23 ± 2.76% in control, Fig. 7A, Supplementary Fig. S10). Following removal of these drugs after 24 hrs, fusion events were gradually restored by 7 days of co-culture to control levels (in average 25 ± 1.51% vs. 28.43 ± 1.52% in control, Fig. 7A), indicating reversibility of the fusion blockade. Interestingly, no effect on fusion was observed with application of numerous other drugs, including DMSO (vehicle for blebbistatin, BTS, nifedipine and verapamil), methyl-beta cyclodextrin, gadolinium, streptomycin, phenylephrine, and isoproterenol, while cytochalasin-D prevented spreading of hMSCs and caused rounding of NRVMs (Supplementary Fig. S10). Additionally, hMSCs treated with either blebbistatin or nifedipine for 24 hrs prior to addition to NRVMs still fused similar to untreated cells (not shown), suggesting a role of these drugs during the actual process of fusion.


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

Shadrin IY, Yoon W, Li L, Shepherd N, Bursac N - Sci Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498233&req=5

f7: 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.
Mentions: To investigate the mechanism of hMSC/NRVM fusion, we assessed percentage of fused cells following a 24 hr drug application at the time of hMSC seeding onto the NRVMs. We found that treatment with myosin II inhibitors (10 μM blebbistatin, 100 μM BTS, or 15 mM BDM) or L-type Ca2+ channel blockers (10 μM nifedipine or 10 μM verapamil) significantly reduced percent of fused hMSCs (in average 1.4 ± 0.43% in treated vs. 23 ± 2.76% in control, Fig. 7A, Supplementary Fig. S10). Following removal of these drugs after 24 hrs, fusion events were gradually restored by 7 days of co-culture to control levels (in average 25 ± 1.51% vs. 28.43 ± 1.52% in control, Fig. 7A), indicating reversibility of the fusion blockade. Interestingly, no effect on fusion was observed with application of numerous other drugs, including DMSO (vehicle for blebbistatin, BTS, nifedipine and verapamil), methyl-beta cyclodextrin, gadolinium, streptomycin, phenylephrine, and isoproterenol, while cytochalasin-D prevented spreading of hMSCs and caused rounding of NRVMs (Supplementary Fig. S10). Additionally, hMSCs treated with either blebbistatin or nifedipine for 24 hrs prior to addition to NRVMs still fused similar to untreated cells (not shown), suggesting a role of these drugs during the actual process of fusion.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC.

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.

No MeSH data available.