The lack of effective treatments for neurodegenerative diseases prompted scientists to study stem cells (SC) as a feasible therapeutic alternative. In particular, human mesenchymal SC (hMSC) derived from bone marrow, show relevant neuroprotective and neuroregenerative outcomes when grafted into pathological animal models. However, an important, still unsolved, challenge is to track grafted SC to follow overtime their location and fate. In this work we tested a specific labeling protocol for direct visualization of hMSC ex vivo by Optical imaging as a proof of principle for their long-term tracking in pre-clinical models. Commercial hMSC were infected with a lentiviral vector carrying the m-cherry gene under the control of Phospho Glicerate Kinase promoter, afterwards we checked transfection efficiency by FACS analysis. Infected hMSC showed a high reporter gene inclusion, as also confirmed by elevated m-cherry mRNA expression, estimated by real time PCR. Moreover, we observed that proliferation, survival and morphology of the infected cells were similar to non infected controls, demonstrating that the labeling protocol did not alter the main biological features of SC. m-cherry positive hMSC were then grafted into the striatum of an experimental rat model of Parkinson’s Disease, based on the unilateral injection of 6-hydroxydopamine. By a CCD camera it was possible to visualize transplanted hMSC ex vivo in the whole brain. Furthermore, imaging of frozen brain sections, observed both by fluorescent microscopy and CCD camera, confirmed the striatal cell localization. In conclusion we set up an efficient protocol to visualize hMSC grafted in animals ex vivo, also translable to in vivo tracking. This method will also allow to establish future efficient therapeutic strategies applicable to patient.

Ex vivo visualization of transfected human mesenchymal stem cells after transplantation: a reliable cell-labeling protocol for optical imaging / V. Diana, L. Cova, I.V. Libani, L. Ottobrini, M.T. Armentero, F. Blandini, G. Lucignani, V. Silani. ((Intervento presentato al 5. convegno Meeting On Molecular Mechanisms Of Neurodegeneration tenutosi a Milano nel 2011.

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

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

Abstract

The lack of effective treatments for neurodegenerative diseases prompted scientists to study stem cells (SC) as a feasible therapeutic alternative. In particular, human mesenchymal SC (hMSC) derived from bone marrow, show relevant neuroprotective and neuroregenerative outcomes when grafted into pathological animal models. However, an important, still unsolved, challenge is to track grafted SC to follow overtime their location and fate. In this work we tested a specific labeling protocol for direct visualization of hMSC ex vivo by Optical imaging as a proof of principle for their long-term tracking in pre-clinical models. Commercial hMSC were infected with a lentiviral vector carrying the m-cherry gene under the control of Phospho Glicerate Kinase promoter, afterwards we checked transfection efficiency by FACS analysis. Infected hMSC showed a high reporter gene inclusion, as also confirmed by elevated m-cherry mRNA expression, estimated by real time PCR. Moreover, we observed that proliferation, survival and morphology of the infected cells were similar to non infected controls, demonstrating that the labeling protocol did not alter the main biological features of SC. m-cherry positive hMSC were then grafted into the striatum of an experimental rat model of Parkinson’s Disease, based on the unilateral injection of 6-hydroxydopamine. By a CCD camera it was possible to visualize transplanted hMSC ex vivo in the whole brain. Furthermore, imaging of frozen brain sections, observed both by fluorescent microscopy and CCD camera, confirmed the striatal cell localization. In conclusion we set up an efficient protocol to visualize hMSC grafted in animals ex vivo, also translable to in vivo tracking. This method will also allow to establish future efficient therapeutic strategies applicable to patient.
Settore MED/36 - Diagnostica per Immagini e Radioterapia
Settore MED/26 - Neurologia
Ex vivo visualization of transfected human mesenchymal stem cells after transplantation: a reliable cell-labeling protocol for optical imaging / V. Diana, L. Cova, I.V. Libani, L. Ottobrini, M.T. Armentero, F. Blandini, G. Lucignani, V. Silani. ((Intervento presentato al 5. convegno Meeting On Molecular Mechanisms Of Neurodegeneration tenutosi a Milano nel 2011.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/168263
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