In the last years, mesenchymal stem cells (MSC) have been extracted from adult tissues other than bone marrow (e.g. muscle, skin, adipose tissue, dental pulp). In vitro characterization revealed that MSC from dental pulp (DP-MSC) are multipotent and show excellent differentiation potentiality in response to specific stimuli. In spite of their multipotency DP-MSC are characterized by an early compartmentalization which protects them against differentiating stimuli from the environment. Since in cocolture with neonatal cardiomyocytes DP-MSC have been seen to differentiate into cardiomyocytes, the present investigation aims at characterizing them and studying their early homing in normal and infarcted isolated rat hearts. Dental pulp-mesenchimal stem cells were obtained from the teeth of anaesthetized (ketamine (90 mg/kg) and xylazine (10 mg/kg)) and decapitated adult Wistar rats. The cells were characterized with RT-PCR and immunofluorescence. 10-6 DP-MSC marked with carboxyfluorescin were implanted in the ventricular apex of Langendorff isolated and perfused hearts rapidly excised from syngenic rats killed as above. The hearts were randomly assigned to two groups: in the Control group the cells were implanted immediately after stabilization and no other maneuver was performed. In another group the cells were implanted after 30 min of ligature of the left anterior descending coronary artery and 5 min of reperfusion (Ischaemia/Reperfusion = I/R group). After 4 hours from the implantation, the location of the cells was assessed in both groups and the injured area in I/R group was highlighted with trypan blue staining. Characterization indicated that DP-MSC express some myocardial precursor markers, like NKX2.5, GATA-4, and MEF2C. In addition, RT-PCR showed the transcription of β2-adrenergic receptors. Carboxyfluorescin allowed to recognize DP-MSC in the host tissue by their green color. In Control group DP-MSCs remained in the site of injection as round-shaped cell clusters, while in I/R group they migrated towards the injured areas, as evidenced by trypan blue staining. Yet, in I/R group some DP-MSCs were elongated in parallel with cardiomyocytes and showed the presence of connexin-43 on the membrane. This observation is in contrast with that of a previous investigation where bone marrow MSCs maintained their round shape when injected near the infarcted area after 30 min reperfusion. The results indicate that in infarcted hearts DP-MSCs can migrate and integrate within the peri-infarcted area in already 4 hours. In addition the characterization suggests that they can differentiate into either cardiomyocytes or vascular smooth muscle fibers. Further studies should identify factors that influence the apparently different responses of these cells.
Early cross-talk between dental pulp stem cell and ischaemic cardiomyocytes in an ex vivo model
RASTALDO, Raffaella;SPRIO, ANDREA ELIO;DI SCIPIO, FEDERICA;FOLINO, Anna;SALAMONE, PAOLINA;LOSANO, Giovanni;PAGLIARO, Pasquale;GEUNA, Stefano;BERTA, Giovanni Nicolao
2010-01-01
Abstract
In the last years, mesenchymal stem cells (MSC) have been extracted from adult tissues other than bone marrow (e.g. muscle, skin, adipose tissue, dental pulp). In vitro characterization revealed that MSC from dental pulp (DP-MSC) are multipotent and show excellent differentiation potentiality in response to specific stimuli. In spite of their multipotency DP-MSC are characterized by an early compartmentalization which protects them against differentiating stimuli from the environment. Since in cocolture with neonatal cardiomyocytes DP-MSC have been seen to differentiate into cardiomyocytes, the present investigation aims at characterizing them and studying their early homing in normal and infarcted isolated rat hearts. Dental pulp-mesenchimal stem cells were obtained from the teeth of anaesthetized (ketamine (90 mg/kg) and xylazine (10 mg/kg)) and decapitated adult Wistar rats. The cells were characterized with RT-PCR and immunofluorescence. 10-6 DP-MSC marked with carboxyfluorescin were implanted in the ventricular apex of Langendorff isolated and perfused hearts rapidly excised from syngenic rats killed as above. The hearts were randomly assigned to two groups: in the Control group the cells were implanted immediately after stabilization and no other maneuver was performed. In another group the cells were implanted after 30 min of ligature of the left anterior descending coronary artery and 5 min of reperfusion (Ischaemia/Reperfusion = I/R group). After 4 hours from the implantation, the location of the cells was assessed in both groups and the injured area in I/R group was highlighted with trypan blue staining. Characterization indicated that DP-MSC express some myocardial precursor markers, like NKX2.5, GATA-4, and MEF2C. In addition, RT-PCR showed the transcription of β2-adrenergic receptors. Carboxyfluorescin allowed to recognize DP-MSC in the host tissue by their green color. In Control group DP-MSCs remained in the site of injection as round-shaped cell clusters, while in I/R group they migrated towards the injured areas, as evidenced by trypan blue staining. Yet, in I/R group some DP-MSCs were elongated in parallel with cardiomyocytes and showed the presence of connexin-43 on the membrane. This observation is in contrast with that of a previous investigation where bone marrow MSCs maintained their round shape when injected near the infarcted area after 30 min reperfusion. The results indicate that in infarcted hearts DP-MSCs can migrate and integrate within the peri-infarcted area in already 4 hours. In addition the characterization suggests that they can differentiate into either cardiomyocytes or vascular smooth muscle fibers. Further studies should identify factors that influence the apparently different responses of these cells.
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