Dental pulp mesenchymal stem cells (DP-MSC) show excellent differentiation potential in response to specific stimuli. In apparent contrast with such a potential, when implanted they are protected against environmental differentiating stimuli by an early compartmentalization. Since in co-culture with neonatal cardiomyocytes DP-MSC have been seen to differentiate into cardiomyocytes, aims of the present investigation were to identify the main characterizing markers of DP-MSC and to study their early homing in isolated rat hearts either in normal conditions or after regional ischemia. Adult Wistar male rats were anesthetized with intra-peritoneal injection of ketamine (90 mg/kg) and Xylazine (10 mg/kg) and killed by decapitation. DP-MSC were then obtained from avulsed teeth and characterized with RT-PCR and immunofluorescence. Characterization revealed that DP-MSC express some precursor markers of cardiomyocytes such as GATA-4, Nkx2.5 and MEF2C. RT-PCR also indicated the transcription of β2-adrenergic receptors. 106 cells were marked with carboxyfluorescin and implanted in the ventricular apex of Langendorff isolated syngenic rat hearts perfused with oxygenated Krebs-Henseleit buffer. These hearts were obtained from rats killed as described above. Two groups of hearts were considered: in control group, DP-MSC were implanted after 20 min of stabilization without ischemia, while in ischemic/reperfused group (I/R group) they were implanted after 20 min of stabilization, 30 min of regional ischemia and 5 min of reperfusion. Four hours after the implantation, the location of DP-MSC was determined in both groups. In the host tissue DP-MSC were identified by their green color due to carboxyfluorescin. In the I/R group the injured area was evidenced by trypan blue staining. In the control group DP-MSC remained in the site of implantation as round-shaped clusters, while in the I/R group they migrated towards the injured area. Particularly, in the I/R group some DP-MSC were elongated in parallel with cardiomyocytes and showed the presence of connexin-43 on the membrane. It may be concluded that in the infarcted heart DP-MSC can migrate close to the infarcted area within about 4 hours and possibly integrate with the surviving cardiomyocytes. Moreover the transcription of β2-adrenergic receptors suggests that DP-MSC can differentiate not only into myocardial cells but also into coronary smooth muscle fibers.
IMPLANTATION OF DENTAL PULP STEM CELLS IN ISOLATED RAT HEART IN THE ABSENCE AND IN THE PRESENCE OF REGIONAL ISCHEMIA
RASTALDO, Raffaella;FOLINO, Anna;SPRIO, ANDREA ELIO;DI SCIPIO, FEDERICA;SALAMONE, PAOLINA;GEUNA, Stefano;PAGLIARO, Pasquale;BERTA, Giovanni Nicolao;LOSANO, Giovanni
2010-01-01
Abstract
Dental pulp mesenchymal stem cells (DP-MSC) show excellent differentiation potential in response to specific stimuli. In apparent contrast with such a potential, when implanted they are protected against environmental differentiating stimuli by an early compartmentalization. Since in co-culture with neonatal cardiomyocytes DP-MSC have been seen to differentiate into cardiomyocytes, aims of the present investigation were to identify the main characterizing markers of DP-MSC and to study their early homing in isolated rat hearts either in normal conditions or after regional ischemia. Adult Wistar male rats were anesthetized with intra-peritoneal injection of ketamine (90 mg/kg) and Xylazine (10 mg/kg) and killed by decapitation. DP-MSC were then obtained from avulsed teeth and characterized with RT-PCR and immunofluorescence. Characterization revealed that DP-MSC express some precursor markers of cardiomyocytes such as GATA-4, Nkx2.5 and MEF2C. RT-PCR also indicated the transcription of β2-adrenergic receptors. 106 cells were marked with carboxyfluorescin and implanted in the ventricular apex of Langendorff isolated syngenic rat hearts perfused with oxygenated Krebs-Henseleit buffer. These hearts were obtained from rats killed as described above. Two groups of hearts were considered: in control group, DP-MSC were implanted after 20 min of stabilization without ischemia, while in ischemic/reperfused group (I/R group) they were implanted after 20 min of stabilization, 30 min of regional ischemia and 5 min of reperfusion. Four hours after the implantation, the location of DP-MSC was determined in both groups. In the host tissue DP-MSC were identified by their green color due to carboxyfluorescin. In the I/R group the injured area was evidenced by trypan blue staining. In the control group DP-MSC remained in the site of implantation as round-shaped clusters, while in the I/R group they migrated towards the injured area. Particularly, in the I/R group some DP-MSC were elongated in parallel with cardiomyocytes and showed the presence of connexin-43 on the membrane. It may be concluded that in the infarcted heart DP-MSC can migrate close to the infarcted area within about 4 hours and possibly integrate with the surviving cardiomyocytes. Moreover the transcription of β2-adrenergic receptors suggests that DP-MSC can differentiate not only into myocardial cells but also into coronary smooth muscle fibers.
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