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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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Chemokine/chemokine receptor mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of a panel of chemokines and chemokine receptors from untransduced and Ad-transduced MSCs. Freshly isolated DCs 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): CX3CR1 protein expression analysis by Western blot, 48 h post-transduction or no transduction, untransduced vs. Ad-transduced MSCs. Tubulin expression served as control. 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-g003: Chemokine/chemokine receptor mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of a panel of chemokines and chemokine receptors from untransduced and Ad-transduced MSCs. Freshly isolated DCs 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): CX3CR1 protein expression analysis by Western blot, 48 h post-transduction or no transduction, untransduced vs. Ad-transduced MSCs. Tubulin expression served as control. 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: The enhanced secretion of chemokines upon genetic modification of MSCs may render the cells more susceptible for immune recognition by the host organism. Therefore we analysed if the chemokine and chemokine receptor mRNA expression profile of untransduced MSCs was altered after Ad-transduction. mRNA was isolated from either untransduced or Ad-transduced MSCs and subjected to real time RT-PCR analysis using specific primers and probes (sequences for all chemokines and chemokine receptors are shown in Table 1). Rat professional antigen presenting cells (dendritic cells, DCs) known to express multiple chemokine/chemokine receptors served as a positive control. As shown in Figure 3A, the majority of chemokines and chemokine receptors analysed in this study were expressed by both MSCs and rDCs (n = 4). Interestingly, CCL2 (MCP-1), CXCL12 (SDF-1α) and CX3CL1 (fractalkine) were the only chemokine mRNAs expressed at higher levels in MSCs compared to DCs which is in agreement with a number of other studies [17], [31], [32]. For all other studied molecules, however, mRNA expression levels were between factor 10 and 1×105 times lower in MSCs compared to DCs. Moreover, genetic modification of MSCs using Ad-vectors did not lead to a significant up- or down-regulation of chemokine or chemokine receptor mRNA expression (Figure 3A). It was, however, interesting to note that in Ad-transduced MSCs mRNA levels of CX3CR1 (the receptor for fractalkine) were profoundly reduced compared to untransduced MSCs. To investigate if this has any influence on the migratory capacity of Ad-transduced MSCs a cell migration experiment was set up by seeding either untransduced or Ad-transduced MSCs in transwells and allowing them to migrate in response to CX3CL1 (fractalkine) over a period of 18–20 hr. In general, we observed low levels of MSC migration in response to the chemoattractant and no difference in the migratory capacity of untransduced vs. Ad-transduced MSCs (data not shown). Moreover, Western blot analysis of CX3CR1 protein expression did not show any difference between untransduced and Ad-transduced MSCs (Fig. 3B). In summary these data indicate that Ad-transduction of MSCs does not induce major changes in their chemokine/chemokine receptor expression profile which also does not seem to affect their migration potential.


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)

Chemokine/chemokine receptor mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of a panel of chemokines and chemokine receptors from untransduced and Ad-transduced MSCs. Freshly isolated DCs 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): CX3CR1 protein expression analysis by Western blot, 48 h post-transduction or no transduction, untransduced vs. Ad-transduced MSCs. Tubulin expression served as control. 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-g003: Chemokine/chemokine receptor mRNA expression profile of untransduced and Ad-transduced MSCs.(A) RT-PCR analysis showing mRNA expression levels of a panel of chemokines and chemokine receptors from untransduced and Ad-transduced MSCs. Freshly isolated DCs 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): CX3CR1 protein expression analysis by Western blot, 48 h post-transduction or no transduction, untransduced vs. Ad-transduced MSCs. Tubulin expression served as control. 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: The enhanced secretion of chemokines upon genetic modification of MSCs may render the cells more susceptible for immune recognition by the host organism. Therefore we analysed if the chemokine and chemokine receptor mRNA expression profile of untransduced MSCs was altered after Ad-transduction. mRNA was isolated from either untransduced or Ad-transduced MSCs and subjected to real time RT-PCR analysis using specific primers and probes (sequences for all chemokines and chemokine receptors are shown in Table 1). Rat professional antigen presenting cells (dendritic cells, DCs) known to express multiple chemokine/chemokine receptors served as a positive control. As shown in Figure 3A, the majority of chemokines and chemokine receptors analysed in this study were expressed by both MSCs and rDCs (n = 4). Interestingly, CCL2 (MCP-1), CXCL12 (SDF-1α) and CX3CL1 (fractalkine) were the only chemokine mRNAs expressed at higher levels in MSCs compared to DCs which is in agreement with a number of other studies [17], [31], [32]. For all other studied molecules, however, mRNA expression levels were between factor 10 and 1×105 times lower in MSCs compared to DCs. Moreover, genetic modification of MSCs using Ad-vectors did not lead to a significant up- or down-regulation of chemokine or chemokine receptor mRNA expression (Figure 3A). It was, however, interesting to note that in Ad-transduced MSCs mRNA levels of CX3CR1 (the receptor for fractalkine) were profoundly reduced compared to untransduced MSCs. To investigate if this has any influence on the migratory capacity of Ad-transduced MSCs a cell migration experiment was set up by seeding either untransduced or Ad-transduced MSCs in transwells and allowing them to migrate in response to CX3CL1 (fractalkine) over a period of 18–20 hr. In general, we observed low levels of MSC migration in response to the chemoattractant and no difference in the migratory capacity of untransduced vs. Ad-transduced MSCs (data not shown). Moreover, Western blot analysis of CX3CR1 protein expression did not show any difference between untransduced and Ad-transduced MSCs (Fig. 3B). In summary these data indicate that Ad-transduction of MSCs does not induce major changes in their chemokine/chemokine receptor expression profile which also does not seem to affect their migration potential.

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