Stem cells, due to their high proliferative and differentiation potential, have been transplanted into different animal models of neurodegenerative diseases with uneven results since an exhaustive comprehension of the interactions between transplanted cells and host tissues is still missing. Imaging is a new approach that allows in vivo, to investigated overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) thereby reducing interindividual variability and the number of experimental animals needed. The purpose of the study was to evaluate the feasibility of mcherry lentiviral infection for the direct visualization of bone marrow-derived human Mesenchymal Stem Cells (hMSC), by optical imaging, as a proof of principle for their long-term tracking in pre-clinical models. Commercial hMSC were infected with different concentrations of a lentiviral vector carrying the mcherry gene under the control of Phospho Glicerate Kinase promoter. Labeled hMSC were analyzed for viability, morphology, and differentiation capability along with maintenance of fluorescent labeling after extensive culture in vitro. Thereafter, we transplanted them in a rodent model of Parkinson’s disease based on the unilateral intrastriatal injection of 6-hydroxydopamine, a procedure that causes a progressive and retrograde degeneration of the nigrostriatal pathway. Our FACS analysis showed that hMSC can be efficiently transduced with the lentiviral vector bearing the mcherry protein since a high percentage of cells expressed the reporter gene (around 80-90%). The infected cells showed a high level of vector copy number inserted in their DNA able to stably express mcherry mRNA, as estimated by real-time PCR. Biological features of mcherry+ hMSC were not altered, even in long term cultures, since their doubling time and metabolic rates appeared comparable to control cells and no morphological alterations were retrieved by confocal analysis. Upon striatal transplantation, it was possible to visualize hMSC ex vivo in the whole brain by a sensitive CCD camera for fluorescent imaging. Finally, the presence of the mcherry+ cells at the injection site was also confirmed using human specific antibodies on frozen microscope slides. Our protocol efficiently labeled hMSC without altering their biological properties and allowed direct cell detection ex vivo by optical imaging. Insertion of mcherry fluorescent probe appears as a reliable technique to follow the fate hMSC upon transplantation and for studying their behaviour in vivo and ex vivo in order to establish efficient therapeutic strategies promptly applicable to patients.

Application to an experimental model of Parkinson’s disease of a reliable protocol to track human mesenchymal stem cells using a lentiviral vector expressing mCherry fluorescent protein / L. Cova, I.V. Libani, M.T. Armentero, V. Diana, L. Ottobrini, F. Blandini, V. Silani, G. Lucignani. ((Intervento presentato al convegno Neuroscience 2011 tenutosi a Washington nel 2011.

Application to an experimental model of Parkinson’s disease of a reliable protocol to track human mesenchymal stem cells using a lentiviral vector expressing mCherry fluorescent protein

I.V. Libani
Secondo
;
L. Ottobrini;V. Silani
Penultimo
;
G. Lucignani
2011

Abstract

Stem cells, due to their high proliferative and differentiation potential, have been transplanted into different animal models of neurodegenerative diseases with uneven results since an exhaustive comprehension of the interactions between transplanted cells and host tissues is still missing. Imaging is a new approach that allows in vivo, to investigated overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) thereby reducing interindividual variability and the number of experimental animals needed. The purpose of the study was to evaluate the feasibility of mcherry lentiviral infection for the direct visualization of bone marrow-derived human Mesenchymal Stem Cells (hMSC), by optical imaging, as a proof of principle for their long-term tracking in pre-clinical models. Commercial hMSC were infected with different concentrations of a lentiviral vector carrying the mcherry gene under the control of Phospho Glicerate Kinase promoter. Labeled hMSC were analyzed for viability, morphology, and differentiation capability along with maintenance of fluorescent labeling after extensive culture in vitro. Thereafter, we transplanted them in a rodent model of Parkinson’s disease based on the unilateral intrastriatal injection of 6-hydroxydopamine, a procedure that causes a progressive and retrograde degeneration of the nigrostriatal pathway. Our FACS analysis showed that hMSC can be efficiently transduced with the lentiviral vector bearing the mcherry protein since a high percentage of cells expressed the reporter gene (around 80-90%). The infected cells showed a high level of vector copy number inserted in their DNA able to stably express mcherry mRNA, as estimated by real-time PCR. Biological features of mcherry+ hMSC were not altered, even in long term cultures, since their doubling time and metabolic rates appeared comparable to control cells and no morphological alterations were retrieved by confocal analysis. Upon striatal transplantation, it was possible to visualize hMSC ex vivo in the whole brain by a sensitive CCD camera for fluorescent imaging. Finally, the presence of the mcherry+ cells at the injection site was also confirmed using human specific antibodies on frozen microscope slides. Our protocol efficiently labeled hMSC without altering their biological properties and allowed direct cell detection ex vivo by optical imaging. Insertion of mcherry fluorescent probe appears as a reliable technique to follow the fate hMSC upon transplantation and for studying their behaviour in vivo and ex vivo in order to establish efficient therapeutic strategies promptly applicable to patients.
Settore MED/26 - Neurologia
Settore MED/36 - Diagnostica per Immagini e Radioterapia
Application to an experimental model of Parkinson’s disease of a reliable protocol to track human mesenchymal stem cells using a lentiviral vector expressing mCherry fluorescent protein / L. Cova, I.V. Libani, M.T. Armentero, V. Diana, L. Ottobrini, F. Blandini, V. Silani, G. Lucignani. ((Intervento presentato al convegno Neuroscience 2011 tenutosi a Washington nel 2011.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/167275
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