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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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TLR mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of TLRs 1–10 from untransduced and Ad-transduced MSCs. rDCs served as a positive control. Expression levels were normalized to the mRNA expression level of the constitutively expressed housekeeping gene β-actin. Data shown are means ±SD of four independent experiments. (B) Functional activation of TLR 2 and 9 following ligand-specific stimulation. RT-PCR analysis showing mRNA expression levels of TLR 2 and 9 by MSCs following stimulation with Pam3CSK4 and ODN2395. *: p<0.05 compared to control. (C) FACS analysis of GFP-expressing MSCs following transfection with pNFκB-d2EGFP, and subsequent transduction with Ad.β-Gal (black line) or stimulation with Pam3CSK4 (grey line), respectively, compared to untreated MSCs (filled histogram). 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copies of the internal control gene, β-actin.
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pone-0042662-g004: TLR mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of TLRs 1–10 from untransduced and Ad-transduced MSCs. rDCs served as a positive control. Expression levels were normalized to the mRNA expression level of the constitutively expressed housekeeping gene β-actin. Data shown are means ±SD of four independent experiments. (B) Functional activation of TLR 2 and 9 following ligand-specific stimulation. RT-PCR analysis showing mRNA expression levels of TLR 2 and 9 by MSCs following stimulation with Pam3CSK4 and ODN2395. *: p<0.05 compared to control. (C) FACS analysis of GFP-expressing MSCs following transfection with pNFκB-d2EGFP, and subsequent transduction with Ad.β-Gal (black line) or stimulation with Pam3CSK4 (grey line), respectively, compared to untreated MSCs (filled histogram). 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copies of the internal control gene, β-actin.

Mentions: Next we analysed the TLR mRNA expression profile of MSCs 24 h after transduction with Ad-vectors. mRNA was isolated from either untransduced or Ad-transduced MSCs and subjected to real time RT-PCR analysis using specific primers and probes (sequences shown in Table 1). Rat DCs known to express high levels of TLRs served as a positive control [33]. As shown in Figure 4A, both MSCs and DCs express TLRs 1–10 at the mRNA level (n = 4). However, our results indicate that MSCs have much lower TLR mRNA expression levels compared to DCs. Importantly, we found that genetic modification of MSCs with Ad-vectors only resulted in negligible differences between the Ad-transduced and untransduced MSCs. Of particular interest was the finding that TLR2 and TLR9, both of which play a key role in Ad-induced inflammation [34], were expressed approximately 100 and 570 times, respectively, less in Ad-transduced MSCs compared to DCs.


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)

TLR mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of TLRs 1–10 from untransduced and Ad-transduced MSCs. rDCs served as a positive control. Expression levels were normalized to the mRNA expression level of the constitutively expressed housekeeping gene β-actin. Data shown are means ±SD of four independent experiments. (B) Functional activation of TLR 2 and 9 following ligand-specific stimulation. RT-PCR analysis showing mRNA expression levels of TLR 2 and 9 by MSCs following stimulation with Pam3CSK4 and ODN2395. *: p<0.05 compared to control. (C) FACS analysis of GFP-expressing MSCs following transfection with pNFκB-d2EGFP, and subsequent transduction with Ad.β-Gal (black line) or stimulation with Pam3CSK4 (grey line), respectively, compared to untreated MSCs (filled histogram). 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copies of the internal control gene, β-actin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412834&req=5

pone-0042662-g004: TLR mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of TLRs 1–10 from untransduced and Ad-transduced MSCs. rDCs served as a positive control. Expression levels were normalized to the mRNA expression level of the constitutively expressed housekeeping gene β-actin. Data shown are means ±SD of four independent experiments. (B) Functional activation of TLR 2 and 9 following ligand-specific stimulation. RT-PCR analysis showing mRNA expression levels of TLR 2 and 9 by MSCs following stimulation with Pam3CSK4 and ODN2395. *: p<0.05 compared to control. (C) FACS analysis of GFP-expressing MSCs following transfection with pNFκB-d2EGFP, and subsequent transduction with Ad.β-Gal (black line) or stimulation with Pam3CSK4 (grey line), respectively, compared to untreated MSCs (filled histogram). 2E-ΔCT = 2−ΔCT→ number of copies of gene of interest relative to the number of copies of the internal control gene, β-actin.
Mentions: Next we analysed the TLR mRNA expression profile of MSCs 24 h after transduction with Ad-vectors. mRNA was isolated from either untransduced or Ad-transduced MSCs and subjected to real time RT-PCR analysis using specific primers and probes (sequences shown in Table 1). Rat DCs known to express high levels of TLRs served as a positive control [33]. As shown in Figure 4A, both MSCs and DCs express TLRs 1–10 at the mRNA level (n = 4). However, our results indicate that MSCs have much lower TLR mRNA expression levels compared to DCs. Importantly, we found that genetic modification of MSCs with Ad-vectors only resulted in negligible differences between the Ad-transduced and untransduced MSCs. Of particular interest was the finding that TLR2 and TLR9, both of which play a key role in Ad-induced inflammation [34], were expressed approximately 100 and 570 times, respectively, less in Ad-transduced MSCs compared to DCs.

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