Adult mesenchymal stem cells (MSC) isolated from adipose tissue represent a useful tool to regenerate damaged tissues in regenerative medicine protocols due to their high proliferative and differentiation potential and easily accessible, ethically-approved source. Nevertheless, interactions between transplanted cells and host tissues, is not completely understood. Imaging is a new in vivo approach that allows to investigate overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) thereby reducing inter-individual variability and the number of experimental animals needed. In this study the feasibility of luciferase lentiviral infection has been evaluated in order to achieve the direct visualization of mouse apidose tissue-derived adult mesenchymal stem cells by optical imaging, as a proof of principle for their long-term tracking in pre-clinical models. Adipose –derived MSCs from CD1 mice were infected with different concentrations of a lentiviral vector carrying the luciferase gene under the control of Phospho Glicerate Kinase promoter (PLW vector). Labeled MSCs were analyzed for viability, morphology, and osteogenic differentiation capability along with maintenance of luminescence labeling. Moreover, intracellular calcium dynamics following 1mM ATP stimulation was analysed by means of single cell calcium imaging recordings. Our analysis showed that MSCs can be efficiently transduced with PLW vector maintaining the proportion with the amount of virus used. The infected cells showed that biological features of luciferase-positive MSC were not altered. Moreover, cells maintained their physiological differentiation potential, quantitatively assayed by analyzing calcium deposits via Alizarin Red staining. Moreover, just like non-infected cells, they showed responsiveness to stimulation by extracellular ATP. Our protocol efficiently labeled Adipose–derived MSCs without altering their biological properties and could allow direct cell detection ex vivo and in vivo by optical imaging. Insertion of luciferase probe appears as a reliable technique to follow the fate Adipose–derived MSCs upon transplantation and for studying their behaviour in vivo and ex vivo in order to establish efficient therapeutic strategies.

Reliable protocol to track mesenchymal stem cells using a lentiviral vector expressing luciferase protein / I.V. Libani, C. Bossio, R. Mastrangelo, F. Bianco, G. Lucignani, L. Ottobrini. ((Intervento presentato al 24. convegno Convegno Annuale dell’Associazione Italiana di Colture Cellulari tenutosi a Roma nel 2011.

Reliable protocol to track mesenchymal stem cells using a lentiviral vector expressing luciferase protein

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

Abstract

Adult mesenchymal stem cells (MSC) isolated from adipose tissue represent a useful tool to regenerate damaged tissues in regenerative medicine protocols due to their high proliferative and differentiation potential and easily accessible, ethically-approved source. Nevertheless, interactions between transplanted cells and host tissues, is not completely understood. Imaging is a new in vivo approach that allows to investigate overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) thereby reducing inter-individual variability and the number of experimental animals needed. In this study the feasibility of luciferase lentiviral infection has been evaluated in order to achieve the direct visualization of mouse apidose tissue-derived adult mesenchymal stem cells by optical imaging, as a proof of principle for their long-term tracking in pre-clinical models. Adipose –derived MSCs from CD1 mice were infected with different concentrations of a lentiviral vector carrying the luciferase gene under the control of Phospho Glicerate Kinase promoter (PLW vector). Labeled MSCs were analyzed for viability, morphology, and osteogenic differentiation capability along with maintenance of luminescence labeling. Moreover, intracellular calcium dynamics following 1mM ATP stimulation was analysed by means of single cell calcium imaging recordings. Our analysis showed that MSCs can be efficiently transduced with PLW vector maintaining the proportion with the amount of virus used. The infected cells showed that biological features of luciferase-positive MSC were not altered. Moreover, cells maintained their physiological differentiation potential, quantitatively assayed by analyzing calcium deposits via Alizarin Red staining. Moreover, just like non-infected cells, they showed responsiveness to stimulation by extracellular ATP. Our protocol efficiently labeled Adipose–derived MSCs without altering their biological properties and could allow direct cell detection ex vivo and in vivo by optical imaging. Insertion of luciferase probe appears as a reliable technique to follow the fate Adipose–derived MSCs upon transplantation and for studying their behaviour in vivo and ex vivo in order to establish efficient therapeutic strategies.
23-nov-2011
Settore MED/36 - Diagnostica per Immagini e Radioterapia
Associazione Italiana di Colture Cellulari (ONLUS-AICC)
Reliable protocol to track mesenchymal stem cells using a lentiviral vector expressing luciferase protein / I.V. Libani, C. Bossio, R. Mastrangelo, F. Bianco, G. Lucignani, L. Ottobrini. ((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/2434/167274
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