Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes

Abstract

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST-BODIPY-Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X-Rhod-1. Immunofluorescence experiments showed the presence of connexin-43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage-dependent calcium signaling in cocultured MSCs. MSCs showed a time-dependent increase of labeling of ST-BODIPY-Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non-fluorescent DHP, Nifedipine, completely abolished ST-BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization-induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage-dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte-like phenotype.

Bone Marrow-Derived Mesenchymal Stromal Cells Express Cardiac-Specific Markers, Retain the Stromal Phenotype, and Do Not Become Functional Cardiomyocytes In Vitro

“…This induction toward the cardiomyocyte lineage is presumably induced by local cell-to-cell contact and/or the presence of soluble factors in the coculture medium. For example, previous studies have shown that MSCs can establish connections with cardiomyocytes through gap junction channels composed of connexin-43 [21,39,[47][48][49][50], a gene that we found was induced in our GFP ϩ MSCs. MSCs with gap junction connections to cardiomyocytes do not generate APs (in agreement with our findings) but can affect electrical conduction by electrotonic interactions, thereby either inducing [48] or preventing [47,49,50] arrhythmias.…”

Mesenchymal stromal cell labeling by new uncoated superparamagnetic maghemite nanoparticles in comparison with commercial Resovist – an initial in vitro study

“…Rat bone marrow stromal cells were harvested and cultured as described by Gallo et al 19 Briefly, bone marrow cells were isolated from the bone marrow of femurs by flushing the medium into the bone shaft. The cell suspension was filtered through a 40 µm nylon filter (Falcon) and plated at a density 2.5×10 3 /cm 2 in chambers of 24-well plates.…”

Hepatocyte Growth Factor or Vascular Endothelial Growth Factor Gene Transfer Maximizes Mesenchymal Stem Cell–Based Myocardial Salvage After Acute Myocardial Infarction

“…28 It is unknown for how long the elevated cytokine concentrations are needed in the healing heart and for how long they remain beneficial. Too widespread and prolonged overexpression of growth and angiogenesis factors may increase the risk for hemangioma growth, and the observed fate of MSCs after 8 to 9 days may be advantageous.…”