Introduction: The use of adult stem cells in cell-mediated therapies is an area of considerable interest within tissue regeneration research. However, important parameters such as the distribution of the injected cells, cell survival, target organ localisation, cell proliferation and differentiation cannot be evaluated in vivo. Here we propose multiple labelling protocols for in vivo visualisation by MRI and Optical fluorescence imaging of implanted murine neural stem cells originating from the subventricular zone of the adult murine brain. Methods: Murine neural stem cells were isolated and cultured as described elsewhere[1]. Cells were labelled for 24 or 48 hours with different amounts of SPIOs (0 - 100 - 200 - 400 μg Fe/ml Endorem®) in presence or absence of transfection agents (Poly-L-Lysine or Polybrene), and with PKH26 and qDOTS as indicated by the manufacturer. Cells were washed with PBS and immediately analysed for viability, iron content (Perl’s Staining), morphology, and fluorescence, or plated for further analyses on multipotency capability. Results: Murine neural stem cells incubated for 24 or 48h with different amounts of Endorem® did not show significant differences in terms of viability and proliferation rate between labelled/non-labelled cells in presence or not of Poly-L-Lysine, Polybrene or Protamine Sulfate[2]. The percentage of viable cells after labelling with 200 ug/mL and PLL was 77% in comparison to the 95% of the control. On the contrary, the percentage of iron-positive cells increased in proportion to the iron content in the medium and in the presence of PLL. In particular we obtained 62% of iron-positive cells in the samples incubated with 200 μg Fe/ml for 24h in the presence of PLL. Moreover stem cells incubated with qDOTs and PKH26 showed a very high labelling efficiency (98%), as evaluated by Fluorescence microscopy. In both cases, labelled cells were able to give rise to floating neurospheres as observed by optical microscopy after further 5 days of culture, demonstrating their maintenance of the self-renewal capability. Conclusions: These results show that adult neural stem cells can be efficiently labelled with different molecules without significantly perturbing physiological stem cell features and self-renewal capability. In conclusion we hypothesise future application of these labelling protocols for the in vivo visualisation by MRI or Fluorescence imaging of the distribution of stem cells after their transplantation into a recipient murine model of disease

Magnetic and fluorescence labelling for in vivo neural stem cell transplantation monitoring / D. Merli, R. Lui, G. Marfia, M. Clerici, L. Ottobrini, G. Lucignani, S. Carelli, F. Marra, A. M. Di Giulio, A. Gorio. ((Intervento presentato al 34. convegno Congresso SIF tenutosi a Rimini nel 2009.

Magnetic and fluorescence labelling for in vivo neural stem cell transplantation monitoring

D. Merli;R. Lui;G. Marfia;M. Clerici;L. Ottobrini;G. Lucignani;S. Carelli;F. Marra;A. M. Di Giulio;A. Gorio
2009-10-14

Abstract

Introduction: The use of adult stem cells in cell-mediated therapies is an area of considerable interest within tissue regeneration research. However, important parameters such as the distribution of the injected cells, cell survival, target organ localisation, cell proliferation and differentiation cannot be evaluated in vivo. Here we propose multiple labelling protocols for in vivo visualisation by MRI and Optical fluorescence imaging of implanted murine neural stem cells originating from the subventricular zone of the adult murine brain. Methods: Murine neural stem cells were isolated and cultured as described elsewhere[1]. Cells were labelled for 24 or 48 hours with different amounts of SPIOs (0 - 100 - 200 - 400 μg Fe/ml Endorem®) in presence or absence of transfection agents (Poly-L-Lysine or Polybrene), and with PKH26 and qDOTS as indicated by the manufacturer. Cells were washed with PBS and immediately analysed for viability, iron content (Perl’s Staining), morphology, and fluorescence, or plated for further analyses on multipotency capability. Results: Murine neural stem cells incubated for 24 or 48h with different amounts of Endorem® did not show significant differences in terms of viability and proliferation rate between labelled/non-labelled cells in presence or not of Poly-L-Lysine, Polybrene or Protamine Sulfate[2]. The percentage of viable cells after labelling with 200 ug/mL and PLL was 77% in comparison to the 95% of the control. On the contrary, the percentage of iron-positive cells increased in proportion to the iron content in the medium and in the presence of PLL. In particular we obtained 62% of iron-positive cells in the samples incubated with 200 μg Fe/ml for 24h in the presence of PLL. Moreover stem cells incubated with qDOTs and PKH26 showed a very high labelling efficiency (98%), as evaluated by Fluorescence microscopy. In both cases, labelled cells were able to give rise to floating neurospheres as observed by optical microscopy after further 5 days of culture, demonstrating their maintenance of the self-renewal capability. Conclusions: These results show that adult neural stem cells can be efficiently labelled with different molecules without significantly perturbing physiological stem cell features and self-renewal capability. In conclusion we hypothesise future application of these labelling protocols for the in vivo visualisation by MRI or Fluorescence imaging of the distribution of stem cells after their transplantation into a recipient murine model of disease
Settore MED/04 - Patologia Generale
Settore MED/36 - Diagnostica per Immagini e Radioterapia
Settore BIO/14 - Farmacologia
Società Italiana di Farmacologia
Fondazione Cariplo
Magnetic and fluorescence labelling for in vivo neural stem cell transplantation monitoring / D. Merli, R. Lui, G. Marfia, M. Clerici, L. Ottobrini, G. Lucignani, S. Carelli, F. Marra, A. M. Di Giulio, A. Gorio. ((Intervento presentato al 34. convegno Congresso SIF tenutosi a Rimini nel 2009.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/69401
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