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 to investigate overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) in vivo, thereby reducing interindividual variability and the number of experimental animals needed. Here we evaluate the feasibility of m-cherry lentiviral infection (transduction) as specific labelling protocol for the real time visualization of bone marrow-derived human Mesenchymal Stem Cells (hMSC) in vivo 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 m-cherry gene under the control of Phospho Glycerate Kinase promoter (PGK). 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 a high percentage of hMSCs expressed the reporter gene (87,2% and 92.4% with a MOI equal respectively to 2,5 and 5; non infected control cells: less than 1%) indicating that the cells can be efficiently transduced with the lentiviral vector bearing the m-cherry. Infected cells showed a high level of vector copy number inserted in their DNA able to stably express the corresponding mRNA, as estimated by real-time PCR. Biological features of m-cherry-positive hMSC were not altered, even in long term cultures, since their doubling time and metabolic rates were comparable to control cells and no morphological alterations were detected 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 m-cherry-positive 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 m-cherry 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.

Ex vivo visualization of transfected human mesenchymal stem cells after transplantation: a reliable cell-labeling protocol for optical imaging / I.V. Libani, L. Cova, V. Diana, M.T. Armentero, F. Blandini, G. Lucignani, L. Ottobrini, V. Silani. ((Intervento presentato al 24. convegno Convegno Annuale dell’Associazione Italiana di Colture Cellulari tenutosi a Roma nel 2011.

Ex vivo visualization of transfected human mesenchymal stem cells after transplantation: a reliable cell-labeling protocol for optical imaging

I.V. Libani
Primo
;
G. Lucignani;L. Ottobrini
Penultimo
;
V. Silani
Ultimo
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 to investigate overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) in vivo, thereby reducing interindividual variability and the number of experimental animals needed. Here we evaluate the feasibility of m-cherry lentiviral infection (transduction) as specific labelling protocol for the real time visualization of bone marrow-derived human Mesenchymal Stem Cells (hMSC) in vivo 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 m-cherry gene under the control of Phospho Glycerate Kinase promoter (PGK). 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 a high percentage of hMSCs expressed the reporter gene (87,2% and 92.4% with a MOI equal respectively to 2,5 and 5; non infected control cells: less than 1%) indicating that the cells can be efficiently transduced with the lentiviral vector bearing the m-cherry. Infected cells showed a high level of vector copy number inserted in their DNA able to stably express the corresponding mRNA, as estimated by real-time PCR. Biological features of m-cherry-positive hMSC were not altered, even in long term cultures, since their doubling time and metabolic rates were comparable to control cells and no morphological alterations were detected 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 m-cherry-positive 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 m-cherry 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/36 - Diagnostica per Immagini e Radioterapia
Settore MED/26 - Neurologia
Associazione Italiana di Colture Cellulari (ONLUS-AICC)
Ex vivo visualization of transfected human mesenchymal stem cells after transplantation: a reliable cell-labeling protocol for optical imaging / I.V. Libani, L. Cova, V. Diana, M.T. Armentero, F. Blandini, G. Lucignani, L. Ottobrini, V. Silani. ((Intervento presentato al 24. convegno Convegno Annuale dell’Associazione Italiana di Colture Cellulari tenutosi a Roma nel 2011.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/167302
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