Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes.
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.
Gallo
Maria Pia
MP
Department of Animal and Human Biology, University of Torino, Torino, Italy. mariapia.gallo@unito.it
Ramella
Roberta
R
Alloatti
Giuseppe
G
Penna
Claudia
C
Pagliaro
Pasquale
P
Marcantoni
Andrea
A
Bonafé
Francesca
F
Losano
Gianni
G
Levi
Renzo
R
D000185:Actinin / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001896:Boron Compounds; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D018031:Connexin 43 / Q000378:metabolism; D004095:Dihydropyridines; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D009543:Nifedipine / Q000494:pharmacology; D051381:Rats; D012518:Sarcomeres / Q000378:metabolism
D001896:Boron Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018031:Connexin 43; D004095:Dihydropyridines; D005456:Fluorescent Dyes; C094861:STBODIPY dihydropyridine; D000185:Actinin; D049452:Green Fluorescent Proteins; D009218:Myosins; D009543:Nifedipine; D002118:Calcium