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Adenoviral transduction of mesenchymal stem cells: in vitro responses and in vivo immune responses after cell transplantation.

Treacy O, Ryan AE, Heinzl T, O'Flynn L, Cregg M, Wilk M, Odoardi F, Lohan P, O'Brien T, Nosov M, Ritter T - PLoS ONE (2012)

Bottom Line: We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86.In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues.Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, Nursing and Health Sciences, School of Medicine, Regenerative Medicine Institute, National University of Ireland, Galway, Ireland.

ABSTRACT
Adult mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential which makes them attractive targets for regenerative medicine applications. However, to date, therapeutic success of MSC-therapy is limited and the genetic modification of MSCs using viral vectors is one option to improve their therapeutic potential. Ex-vivo genetic modification of MSCs using recombinant adenovirus (Ad) could be promising to reduce undesired immune responses as Ad will be removed before cell/tissue transplantation. In this regard, we investigated whether Ad-modification of MSCs alters their immunological properties in vitro and in vivo. We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86. Moreover, Ad-transduction caused no significant changes in terms of pro-inflammatory cytokine expression, chemokine and chemokine receptor and Toll-like receptor expression. In addition, Ad-modification of MSCs had no affect on their ability to suppress T cell proliferation in vitro. In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues. Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.

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Immunophenotyping and pro-inflammatory cytokine expression of untransduced and Ad-transduced MSCs.(A) Untransduced and Ad-transduced MSCs were stained for expression of the immunologically relevant markers MHC class I/II, CD80 and CD86 with specific antibodies and analysed by flow cytometry. (B) MSCs were transduced with Ad.GFP and after 24 h cells were collected and subjected to mRNA isolation and cDNA synthesis. RT-PCR analysis showing mRNA expression levels of IL-1β, IFN-γ and IL-6 from untransduced and Ad-transduced MSCs. Data shown are means ±SD of three independent experiments. 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copes of the internal control gene, β-actin.
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pone-0042662-g002: Immunophenotyping and pro-inflammatory cytokine expression of untransduced and Ad-transduced MSCs.(A) Untransduced and Ad-transduced MSCs were stained for expression of the immunologically relevant markers MHC class I/II, CD80 and CD86 with specific antibodies and analysed by flow cytometry. (B) MSCs were transduced with Ad.GFP and after 24 h cells were collected and subjected to mRNA isolation and cDNA synthesis. RT-PCR analysis showing mRNA expression levels of IL-1β, IFN-γ and IL-6 from untransduced and Ad-transduced MSCs. Data shown are means ±SD of three independent experiments. 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copes of the internal control gene, β-actin.

Mentions: MSCs can be readily transduced with adenoviral vectors at a MOI of 100 followed by spin centrifugation. The mean transduction efficiency in this study was 66% (n = 3, representative gating shown in figure S1A) as determined by fluorescence activated cell sorting (FACS) analysis. Due to limited transduction efficiencies, Ad.GFP MSCs were FACS sorted into GFP positive (+) and GFP negative (−) fractions (n = 3, figure S1A). From the two distinct populations, we proceeded to isolate RNA, synthesise cDNA and perform RT-PCR analysis (as described in Materials and Methods) to analyse the mRNA expression levels of the chemokines, chemokine receptors and TLRs listed in Table 1. Consistently, we observed non-significant differences in expression levels between the two cell populations (figure S1B) and, as a result, a transduction efficiency of 66% was deemed adequate for subsequent experiments. Moreover, it was decided that while an increased MOI could be used and thus improve transduction efficiency, we found that this leads to a substantial increase in cytotoxicity (unpublished observations) and therefore would compromise the viability of the Ad.GFP transduced MSCs in vivo. In order to understand if Ad-transduction of MSCs alters the immunologically relevant cell surface marker expression profile, transduction of MSCs using an adenovirus encoding for the reporter gene β-Galactosidase (Ad.β-Gal) at a MOI of 100 followed by spin centrifugation was carried out. After 24 h MSCs were collected and analysed for expression of specific cell surface markers by flow cytometry. As shown in Figure 2A, transduction of MSCs with Ad.β-Gal did not alter the cell surface expression profile of immunologically relevant cell surface markers. Neither an increase in MHC class I and II cell surface expression nor an increase in expression of co-stimulatory molecules CD80 or CD86 was observed upon transduction with Ad-vectors (n = 3).


Adenoviral transduction of mesenchymal stem cells: in vitro responses and in vivo immune responses after cell transplantation.

Treacy O, Ryan AE, Heinzl T, O'Flynn L, Cregg M, Wilk M, Odoardi F, Lohan P, O'Brien T, Nosov M, Ritter T - PLoS ONE (2012)

Immunophenotyping and pro-inflammatory cytokine expression of untransduced and Ad-transduced MSCs.(A) Untransduced and Ad-transduced MSCs were stained for expression of the immunologically relevant markers MHC class I/II, CD80 and CD86 with specific antibodies and analysed by flow cytometry. (B) MSCs were transduced with Ad.GFP and after 24 h cells were collected and subjected to mRNA isolation and cDNA synthesis. RT-PCR analysis showing mRNA expression levels of IL-1β, IFN-γ and IL-6 from untransduced and Ad-transduced MSCs. Data shown are means ±SD of three independent experiments. 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copes of the internal control gene, β-actin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412834&req=5

pone-0042662-g002: Immunophenotyping and pro-inflammatory cytokine expression of untransduced and Ad-transduced MSCs.(A) Untransduced and Ad-transduced MSCs were stained for expression of the immunologically relevant markers MHC class I/II, CD80 and CD86 with specific antibodies and analysed by flow cytometry. (B) MSCs were transduced with Ad.GFP and after 24 h cells were collected and subjected to mRNA isolation and cDNA synthesis. RT-PCR analysis showing mRNA expression levels of IL-1β, IFN-γ and IL-6 from untransduced and Ad-transduced MSCs. Data shown are means ±SD of three independent experiments. 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copes of the internal control gene, β-actin.
Mentions: MSCs can be readily transduced with adenoviral vectors at a MOI of 100 followed by spin centrifugation. The mean transduction efficiency in this study was 66% (n = 3, representative gating shown in figure S1A) as determined by fluorescence activated cell sorting (FACS) analysis. Due to limited transduction efficiencies, Ad.GFP MSCs were FACS sorted into GFP positive (+) and GFP negative (−) fractions (n = 3, figure S1A). From the two distinct populations, we proceeded to isolate RNA, synthesise cDNA and perform RT-PCR analysis (as described in Materials and Methods) to analyse the mRNA expression levels of the chemokines, chemokine receptors and TLRs listed in Table 1. Consistently, we observed non-significant differences in expression levels between the two cell populations (figure S1B) and, as a result, a transduction efficiency of 66% was deemed adequate for subsequent experiments. Moreover, it was decided that while an increased MOI could be used and thus improve transduction efficiency, we found that this leads to a substantial increase in cytotoxicity (unpublished observations) and therefore would compromise the viability of the Ad.GFP transduced MSCs in vivo. In order to understand if Ad-transduction of MSCs alters the immunologically relevant cell surface marker expression profile, transduction of MSCs using an adenovirus encoding for the reporter gene β-Galactosidase (Ad.β-Gal) at a MOI of 100 followed by spin centrifugation was carried out. After 24 h MSCs were collected and analysed for expression of specific cell surface markers by flow cytometry. As shown in Figure 2A, transduction of MSCs with Ad.β-Gal did not alter the cell surface expression profile of immunologically relevant cell surface markers. Neither an increase in MHC class I and II cell surface expression nor an increase in expression of co-stimulatory molecules CD80 or CD86 was observed upon transduction with Ad-vectors (n = 3).

Bottom Line: We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86.In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues.Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, Nursing and Health Sciences, School of Medicine, Regenerative Medicine Institute, National University of Ireland, Galway, Ireland.

ABSTRACT
Adult mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential which makes them attractive targets for regenerative medicine applications. However, to date, therapeutic success of MSC-therapy is limited and the genetic modification of MSCs using viral vectors is one option to improve their therapeutic potential. Ex-vivo genetic modification of MSCs using recombinant adenovirus (Ad) could be promising to reduce undesired immune responses as Ad will be removed before cell/tissue transplantation. In this regard, we investigated whether Ad-modification of MSCs alters their immunological properties in vitro and in vivo. We found that Ad-transduction of MSCs does not lead to up-regulation of major histocompatibility complex class I and II and co-stimulatory molecules CD80 and CD86. Moreover, Ad-transduction caused no significant changes in terms of pro-inflammatory cytokine expression, chemokine and chemokine receptor and Toll-like receptor expression. In addition, Ad-modification of MSCs had no affect on their ability to suppress T cell proliferation in vitro. In vivo injection of Ad-transduced MSCs did not change the frequency of various immune cell populations (antigen presenting cells, T helper and cytotoxic T cells, natural killer and natural killer T cells) neither in the blood nor in tissues. Our results indicate that Ad-modification has no major influence on the immunological properties of MSCs and therefore can be considered as a suitable gene vector for therapeutic applications of MSCs.

Show MeSH
Related in: MedlinePlus