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. 2011 Jan 25;19(5):969–978. doi: 10.1038/mt.2010.311

Functionalized Scaffold-mediated Interleukin 10 Gene Delivery Significantly Improves Survival Rates of Stem Cells In Vivo

Carolyn Holladay 1, Karen Power 2, Michael Sefton 3, Timothy O'Brien 4, William M Gallagher 2, Abhay Pandit 1
PMCID: PMC3086863  EMSID: UKMS33825  PMID: 21266957

Abstract

While stem cell transplantation could potentially treat a variety of disorders, clinical studies have not yet demonstrated conclusive benefits. This may be partly because transplanted stem cells have low survival rates, potentially due to host inflammation. The system described herein used two different gene therapy techniques to improve retention of rat mesenchymal stem cells. In the first, stem cells were transfected with interleukin-10 (IL-10) before being loaded into a collagen scaffold. In the second, unmodified stem cells were loaded into a collagen scaffold along with polymer-complexed IL-10 plasmids. The scaffolds were surgically implanted into the dorsum of syngeneic rats. At each endpoint, the scaffolds were explanted and cell retention, IL-10 level and inflammatory response were quantified. All treatment groups had statistically significant increases in cell retention after 7 days, but the group treated with 2 µg of IL-10 polyplexes had a significant improvement even at 21 days. This cell retention was associated with increased IL-10 and decreased levels of proinflammatory cytokines and apoptosis. The primary effect on the inflammatory response appeared to be on macrophage differentiation, encouraging the regulatory phenotype over the cytotoxic lineage. Improving cell survival may be an important step toward realization of the therapeutic potential of stem cells.

Introduction

Mesenchymal stem cell transplantation has been postulated as a treatment option for a number of ischemic conditions including hind-limb ischemia,1 stroke,2,3,4,5 and myocardial infarction (MI).6,7,8 In the case of MI, some early preclinical studies have reported significant therapeutic improvements that were associated with stem cell transplantation.9,10,11,12 However, clinical trials have failed to find major therapeutic benefits.13,14,15,16,17,18,19,20 One reason postulated for this failure is very low retention rates of stem cells in the ischemic myocardium after transplantation. For example, in a recent study by Pons et al., ~90% of the injected stem cells were lost within the first 24 hours, and 98% within the first week.21 The reason for this is unclear but may be related to the huge influx of inflammatory cells and proinflammatory factors associated with ischemia/reperfusion injury.22,23,24 As inflammatory cells (or cells belonging to the innate immune response) such as neutrophils and macrophages infiltrate a damaged area within a few hours of injury, they seem more likely to be responsible for the decrease in stem cell numbers than the adaptive immune system, which normally does not respond so quickly. Furthermore, significant cell death has been observed even in syngeneic systems where minimal immune-mediated rejection would be expected.

The inflammatory response is an integral component of the body's response to injury, but in many cases over-active inflammation over a prolonged period of time can be pathogenic. Upregulation of proinflammatory cytokines and increased activation of cytotoxic cells have been found to contribute to pathogenesis after MI,25,26 in diabetes,27,28,29 and many other diseases. Conversely, upregulation of cytokines such as interleukin-10 (IL-10) has been associated with improved survival in a variety of autoimmune,30,31 inflammatory32,33 and ischemic conditions.34,35,36 Thus, artificially modulating the inflammatory response is likely to be therapeutically beneficial in a variety of settings.

Reducing or controlling the inflammatory response is not a new concept; suppression of the immune and inflammatory reactions is a necessary aspect of whole organ transplantation. However, systemic suppression of the inflammatory and immune systems makes transplant patients extremely vulnerable to infection. In recent years, gene therapy localized to the site of transplantation has been proposed as a solution to this problem and applied in a number of studies.37,38,39

Nonviral gene therapy represents a promising therapeutic option for a variety of disorders, as the safety risks are relatively low and the production process is more suitable for scale-up.37,38,39,40 Scaffold-mediated gene therapy has been found effective in a variety of settings, as the scaffold can actually mediate higher levels of transfection as well as acting as a reservoir system for the gene therapy.41

In this study, IL-10 gene therapy was employed to modulate the inflammatory response after implantation of a collagen scaffold seeded with rat bone marrow-derived mesenchymal stem cells (rMSCs). IL-10 is considered a potent anti-inflammatory cytokine that is produced naturally42 and has been used in a number of studies to decrease or control inflammation.35,43,44,45 In fact, IL-10 secretion by MSCs has been postulated as one of their major therapeutic benefits in treating ischemic injury.34 Increasing the secretion rates of IL-10 by the implanted rMSCs by means of a functionalized scaffold it was expected to create a microenvironment in which the cells are protected from the host inflammatory reaction.

More specifically, it was hypothesized that IL-10 gene therapy can be used to increase the retention rate of rMSCs when loaded into a collagen scaffold. The primary objectives were to quantify the rMSC retention, compare the retention rate between treatment options and correlate with the effects of the therapy on the overall inflammatory response. This study also compares ex vivo genetic manipulation of the rMSCs with in situ transfection with plasmid-polymer complexes, or “polyplexes,” where the gene therapy is incorporated in the scaffold and transfection occurs in vivo.

Results

Higher stem cell retention within IL-10 modified samples

A significant increase in IL-10 levels was observed in samples containing IL-10 modified rMSCs implanted within collagen scaffolds, as shown in Figure 1a. The approximate increases in IL-10 levels from baseline (unmodified cells) were 1.7×, 1.9×, and 1.5× at 2, 7, and 21 days, respectively. While an increase in the retention rates of the IL-10 modified rMSCs was indicated by the trends, this effect was only statistically significant (6.5×, P < 0.05) at 7 days, as illustrated in Figure 1b. Quantification of inflammatory cell volume fractions, as shown in Figure 1c, indicated that fewer inflammatory cells were observed in the IL-10 modified group at both days 2 and 21 (statistically significant at day 21), but an increase in the volume fraction of inflammatory cells was observed after 7 days. No statistically significant effect was observed in IL-5 levels, as shown in Figure 1d, but a trend toward higher IL-5 in the IL-10 modified samples was observed.

Figure 1.

Figure 1

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Summary of interleukin-10 (IL-10) modified stem cell study. Rat bone marrow-derived mesenchymal stem cells (rMSCs) modified ex vivo to over-express IL-10 were seeded into scaffolds and implanted. In all figures, these IL-10 modified rMSCs (hatched bars) were compared to unmodified rMSCs (open bars). (a) A statistically significant increase in IL-10 concentration was observed at all timepoints. This effect was most dramatic after 7 days in vivo, where the samples containing IL-10 modified rMSCs produced almost double the amount of IL-10 observed in the unmodified samples. The volume fractions of (b) rMSCs and (c) inflammatory cells were then quantified to evaluate therapeutic efficacy. A statistically significant (6.5×) increase in rMSC volume fraction was observed after 7 days. The rMSC volume fraction in the unmodified group significantly decreased between 2 and 7 days whereas the IL-10 modified rMSC volume fraction did not decrease until after 7 days. The inflammatory cell infiltration appeared to be reduced after 2 and 21 days in the IL-10 modified group. However, the volume fraction of inflammatory cells was significantly increased in the IL-10 modified group after 7 days, as compared to the unmodified rMSCs. (d) The IL-5 concentrations had no statistically significant differences between groups, but the trend suggested more immune-related reaction at 2 days and at 7 days. (Data expressed as mean ± 95% confidence interval, n = 6, * represents significance for P < 0.05.)

IL-10 polyplex-mediated gene therapy

The second technique described herein used a collagen scaffold as a reservoir for both IL-10 polyplexes and seeded rMSCs. This system had previously been tested with reporter genes.41 In order to validate the system for a therapeutic gene, the IL-10 production from polyplex-loaded scaffolds in vitro was measured, as shown in Supplementary Figure S1. The maximum level of IL-10 measured in the media was just over 2,000 pg/ml. While the IL-10 level was increased in the treatment groups after only 24 hours in culture, this effect was not statistically significant.

When the 2 and 20 µg IL-10 polyplex-loaded scaffolds were tested in vivo, an increase in rMSC retention—both volume fraction and cell numbers/area—was observed at all timepoints. This effect was significant for both the 2 and 20 µg groups after 7 days, but only the 2 µg dose continued to be effective after 21 days. Figure 2a shows a representative set of fluorescent micrographs comparing scaffolds carrying only rMSCs to scaffolds treated with 2 or 20 µg IL-10. Figure 2b compares the volume fractions of the rMSCs over time, as quantified from the fluorescent images using stereology. A 9.2× increase in rMSC volume fraction was observed in the 2 µg group after 7 days as compared to untreated cells. After 21 days, the increase in retention rate in this group was ~2.7× that of untreated cells. Figure 2c shows a comparison of the cell numbers per area in each group over time. The approximate number rMSCs per area was highest in all groups after 2 days, but the only statistically significant decrease in rMSC numbers over time was observed in the untreated group between the 2 and 7 day timepoints. The statistically significant differences in rMSC numbers between the treatment and control groups within each timepoint mirrored the volume fraction results, although the differences were more dramatic (i.e., ~12× and 6× higher MSC numbers in the 2 µg group, after 7 and 21 days respectively, as compared to the untreated group).

Figure 2.

Figure 2

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Rat bone marrow-derived mesenchymal stem cells (rMSCs) retention within explanted scaffolds. (a) rMSCs retention is expressed as representative micrographs of rMSCs (red) counterstained with 4′,6-diamidino-2-phenylindole (blue) in patches treated with 0, 2, and 20 µg of interleukin-10 (IL-10) polyplexes, (b) the stereological quantification of these micrographs and (c) the number of rMSCs per mm2 as counted from the micrographs. rMSC volume fraction significantly decreased after 7 days in the untreated patches (open bars), but was unchanged until 21 days in the groups containing 2 (hatched bars) or 20 µg (stippled bars) of IL-10 polyplexes. Both the volume fraction and rMSC numbers/mm2 were significantly higher in the 2 µg group after 7 and 21 days and in the 20 µg group after 7 days, as compared to the untreated group. The average numbers of rMSCs in the 2 µg group did not significantly change over the course of the 21 days, but the mean value decreased by ~77% between the 2 and 7 day timepoints, whereas the number of rMSCs/mm2 in the untreated group significantly decreased after 2 days to <8% of the day 2 value. The cell numbers in the 20 µg group were relatively consistent over the 3 week period. (Scale bar represents 50 µm, graphical data are expressed as mean ± 95% confidence interval, n = 6, * represents significance for P < 0.05).

Increased IL-10 levels were also observed, which were statistically significant after 7 days, as shown in Figure 3c. At this timepoint, the mean IL-10 level in the 2 µg treated samples was 2.8× that of the untreated group. After 21 days, the IL-10 level in the 2 µg group was ~1.6× that of the untreated group. The 20 µg treatment group also had a significant increase in IL-10 levels after 7 days. Immunostaining for IL-10, as shown in Figure 3a,b, indicated that both the transplanted rMSCs and the host tissue were producing IL-10.

Figure 3.

Figure 3

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Interleukin-10 (IL-10) content in explanted scaffolds. Representative micrographs showing (a) immunostained IL-10 (green) and rMSCs (red) counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) within explanted scaffolds and (b) a representative micrograph of IL-10 production from fibroblastic and inflammatory cells in the connective tissue surrounding a patch treated with 2 µg IL-10 polyplexes after 7 days. rMSCs mostly stained positive for IL-10, particularly in the 2 and 20 µg groups after 7 days. However, the majority of IL-10 production was from the infiltrating cells (macrophages and fibroblasts), not the rMSCs, and occurred even at a distance from the scaffold. The overall IL-10 levels in scaffolds treated with 0 (open bars), 2 (hatched bars), or 20 µg (stippled bars) of IL-10 polyplexes was also quantified with enzyme-linked immunosorbent assay (ELISA). (c) The IL-10 levels in the 2 and 20 µg groups were significantly increased after 7, and there was a trend toward higher IL-10 levels after 21 days. The IL-10 level in the 2 µg group after 7 days was ~2.8× that of the untreated group. (Scale bars in all cases represent 50 µm. Graphical data are expressed as mean ± 95% confidence interval, n = 6, * represents significance for P < 0.05).

Standard hematoxylin and eosin staining and stereological analysis was used to determine the volume fraction of inflammatory cells within the treatment area at each timepoint, as shown in Figure 4a. The only statistically significant effect was observed after 7 days where a significant reduction (43%, P < 0.05) in the inflammatory cell numbers was observed in the 2 µg group. However, no other changes in the numbers of inflammatory cells were observed. Thus, the effects of the therapy did not appear to be solely on the numbers of inflammatory cells. The volume fraction of CD68+ cells (all macrophages), as shown in Figure 4b, indicated no statistically significant differences between groups. Comparing Figure 4a,b it was observed that the majority of inflammatory cells at days 7 and 21 were macrophages, but after 2 days in vivo, there were significant numbers of other cell types (primarily neutrophils). The levels of a variety of proinflammatory cytokines were measured to obtain an alternate quantification of overall inflammation. Figure 4c,d,e show the levels of IL-1β, the rat analog of human IL-8 (aIL-8) (KC/GRO/CINC) and TNF-α respectively. While there is no statistically significant difference in the IL-1β levels, there was a trend toward a reduction in cytokine concentration in the 2 µg group compared to the untreated group after both 2 and 7 days. The reduction in aIL-8 levels was statistically significant in the 2 µg group after 7 and 21 days while the reduction in TNF-α levels was statistically significant after 2 and 7 days in the 2 µg group. There was no significant trend observed in IL-5 levels, as shown in Figure 4e, although there was a trend towards higher IL-5 levels after 2 and 7 days in the 20 µg group.

Figure 4.

Figure 4

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Overall quantification of inflammatory response. Inflammation was quantified in terms of (a) mean inflammatory cell volume fraction, (b) mean macrophage volume fraction, (c) interleukin-1β (IL-1β) concentration, (d) aIL-8 concentration, (e) tumor necrosis factor-α (TNF-α) concentration, and (f) IL-5 concentration in samples treated with 0 (open bars), 2 (hatched bars) or 20 µg (stippled bars) of IL-10 polyplexes. After 7 days, the inflammatory cell volume fraction in the 2 µg group was decreased by ~43% compared to the untreated control. There were no statistically significant differences in the macrophage volume fractions between groups at any timepoint, but there were statistically significant decreases in all groups between the day 2 and day 7 timepoint. The IL-1β levels were approximately half that of the untreated control after 2 and 7 days, but this difference was not statistically significant. The aIL-8 concentration was reduced by >50% in the 2 µg group at all timepoints but this effect was only statistically significant after 7 and 21 days. The TNF-α was similar to the IL-1β trend, but the decrease was statistically significant after both 2 and 7 days. No statistically significant effects were observed for the T helper cells (cytokine IL-5, but a trend toward higher IL-5 levels in the 20µg group was observed after both 2 and 7 days. (Data expressed as mean ± 95% confidence interval, n = 6, * represents significance for P < 0.05).

Staining for apoptosis indicated that transfection with IL-10 may have also improved rMSC retention by decreasing stem cell apoptosis, as illustrated in Figure 5. While apoptotic cells were observed in all groups, many apoptotic rMSCs were observed in the untreated group, whereas the majority of apoptotic cells observed in the 2 and 20 µg groups were endogenous. The largest numbers of apoptotic cells were observed in the samples from the 20 µg group after 2 days in vivo. The numbers of apoptotic cells decreased with time, and were comparatively lowest in the 2 µg group at each timepoint.

Figure 5.

Figure 5

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Qualitative investigation of the effects of interleukin-10 (IL-10) gene delivery on apoptosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining for apoptotic cells (green) counterstained with 4′,6-diamidino-2-phenylindole (blue) and rat bone marrow-derived mesenchymal stem cells (rMSCs) (red). Colocalization of red and green appears orange to yellow, and indicated apoptotic rMSCs. In all images, the scale bars represent 20 µm.

Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) for IL-10 indicated an increased level of IL-10 in treated samples (both 2 and 20 µg) as compared to unloaded scaffolds, as shown in Figure 3. Furthermore, significant IL-10 production was observed in many of the inflammatory cells (i.e., macrophages and fibroblasts) infiltrating the scaffolds. IHC for macrophages, as shown in Figure 6a indicated no significant effect on overall macrophage numbers, (CD68+ cells) but more CD80+ (M1) macrophages were observed in the untreated and 20 µg groups than the 2 µg group, as shown in Figure 6b. The fraction of M1/total macrophage numbers is shown in Figure 7a, where the fraction of M1 macrophages was significantly lower at all timepoints in the 2 µg group than in the untreated samples. Finally, IHC for CD163, a marker for the regulatory lineage of macrophages (M2), indicated that the numbers of M2 were significantly higher in the 2 µg group at all timepoints, as shown in Figure 6c and Figure 7b. Comparing the volume fractions of the two macrophage types in serial sections of representative samples, as shown in Figure 7c, indicated that the ratio of M2/M1 (CD163/CD80) was highest in the 2 µg samples at all timepoints, and M2 numbers outweighed M1 in these samples at both 7 and 21 days. In the samples from the 2 and 20 µg groups, overlaying images from serial sections suggested that in some cases the macrophages carried markers for both CD163 and CD80, as shown in Figure 6d.

Figure 6.

Figure 6

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Qualitative investigation of the effects of interleukin-10 (IL-10) gene delivery on macrophage phenotype. Immunohistochemistry for all macrophages (a) was carried out using anti-CD68 antibody while cytotoxic macrophages (b) were identified using anti-CD80 antibody and regulatory macrophages (c) were identified using anti-CD163 antibodies. The marker is shown as green counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and arrows are used to highlight the CD80+ (M1) and CD163+ (M2) cells. While there was no apparent trend in overall numbers of macrophages, the numbers of CD80+ macrophages decreased over time and were consistently lowest in the group treated with 2 µg of IL-10 polyplexes. The numbers of CD163+ macrophages increased over time and the highest numbers were observed in the 2 µg group after 21 days. The final micrograph (d) shows an overlaid image of serial sections showing CD80 (red) and CD163 (green) counterstained with DAPI. The arrows highlight the cells that appear to be positive for both markers. In all images, the scale bars represent 20 µm.

Figure 7.

Figure 7

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Quantitative investigation of the effects of interleukin-10 (IL-10) gene delivery on macrophage phenotype. Stereological analysis of immunohistochemistry micrographs was used to estimate the volume fractions of CD68+, CD80+, and CD163+ cells. (a) The ratio of the CD80+ macrophages compared to the overall macrophage volume fraction (CD68+) indicated that the untreated samples (open bars) were primarily CD80+ at day 2 and day 7, whereas the 2 (hatched bars) and 20 µg (stippled bars) samples had a significantly lower fraction of these cells. (b) The ratio of the CD163+ macrophages compared to the overall macrophage volume fraction indicated that the group treated with 2 µg of IL-10 polyplexes consistently had the highest ratio of CD163+ cells, while the 20 µg group did not differ significantly from the control until after 21 days. (c) The untreated samples had a far lower volume fraction of CD163+ than CD80+ cells, with a maximum CD163/CD80 ratio of <25% after 7 days. The 2 µg group had relatively high ratios of CD163/CD80, especially after 7 and 21 days. The 20 µg group had significantly increased CD163/CD80 ratios after 7 and 21 days, but these ratios remained significantly lower than the 2 µg group. The estimated error of these measurements was ~25% as the error associated with each individual measurement was compounded by dividing two volume fraction estimations. Considering this error, * represents a significant change compared to the control and † represents a significant change as compared to the 20 µg group. Data show quantification of images from two representative animals per group and per timepoint.

Discussion

IL-10 modification of rMSCs yielded a significant increase in IL-10 levels, as compared to unmodified cells, at all timepoints. rMSC retention was also increased at all timepoints, but this effect was only statistically significant (6.5×, P < 0.05) at 7 days. In fact, the IL-10 modified rMSC retention rate reduced to almost the same level as unmodified cells by day 21, with no statistical difference between the two groups. In order to explain this observation, inflammatory cell volume fractions were compared between the groups. While fewer inflammatory cells were observed in the IL-10 modified group at both days 2 and 21 (statistically significant at day 21), an increase in the volume fraction of inflammatory cells was observed after 7 days. This increase was also associated with a trend toward increased levels of IL-1β (1.7×) and IL-8 (1.2×). Thus, the altered trend in the profile of inflammation appeared to be related to the eventual decrease of the IL-10 modified rMSC numbers.

Another possible explanation for the reduction in rMSC numbers at day 21 could be associated with the immune response, as the IL-10 modified cells were cultured and manipulated ex vivo for a significant length of time, and might therefore have altered their phenotype to become more immunogenic. The level of IL-5, a Th2 cytokine, was used to estimate the overall immune-mediated response. While no significant differences were observed, there was a trend toward higher IL-5 levels at days 2 and 7, as shown in Figure 1d, suggesting that the immune-mediated reaction may have contributed towards the eventual decrease in rMSC retention. While stem cells are generally described as hypoimmunogenic,34 there is evidence to suggest that, in some cases, they do suffer from immune-associate rejection.46 The most likely explanation is that the two factors were combined, as the immune reaction is a component of the inflammatory reaction, and many of the inflammatory mediators have crossover effects.

Thus, while IL-10 modification appeared to significantly increase rMSC retention 7 days after implantation, unexpected side-effects limited the duration of this benefit and an alternate technique was thus investigated. A nonviral scaffold-based transfection system had been previously found to induce significant upregulation of transgenes in a variety of cell types for up to 3 weeks in vitro. This system was considered ideal as it required minimal ex vivo manipulation of cells, was effective for up to 3 weeks in vitro, and was highly effective at transfecting progenitor cell types like the rMSCs.41 In vitro testing of this system, as shown in Supplementary Figure S1, validated its efficacy with a therapeutic gene. The most efficacious group yielded more than fivefold higher IL-10 levels than unmodified rMSCs after two days, and approximately double the normal IL-10 production for the remainder of 2 weeks. It should be noted that the unmodified rMSCs naturally produced a low level of IL-10, a property which has been previously demonstrated to be therapeutically relevant in treating ischemic injury.34 In a parallel in vivo reporter gene transfection study, statistically significant transfection of host cells was observed after 7 and 14 days in vivo. This suggests that the system prolonged the transfection activity of the polyplexes (M. Monaghan, C. Holladay, W. Wang, and A. Pandit, unpublished results).

In vivo, this system was found to effectively increase IL-10 levels and improve rMSC retention. Both the volume fraction of rMSCs and the number of rMSCs per unit area were significantly increased by loading the scaffolds with 2 and 20 µg of IL-10. This effect was not found to be statistically significant at 2 days, but was significant in the 2 µg group after 7 and 21 days. A significant decrease in rMSC retention in the untreated group was expected after 2 days as very low retention rates of normal stem cells (i.e., <10% after 24 hours) have been observed in previous studies.21 In this study, the volume fraction of the untreated rMSCs after 2 days would correspond to ~26% of the volume fraction of cells in the scaffolds prior to implantation and the rMSC numbers per area correspond to ~13% (preimplantation volume fraction = 0.235 ± 0.05, cell numbers/mm2 = 8,970 ± 420). This suggested that the scaffold itself might have provided some level of protection to the cells, as observed by Zhang et al.47 However, this effect was short-lived as by day 7 the number of untreated cells per mm2 had decreased to ~1% of the original number of implanted cells. Scaffolds loaded with IL-10 polyplexes showed no significant decrease in rMSC numbers per area. After 7 days, the point at which the greatest improvement was observed, the retention rate of the rMSCs in the 2 µg treatment group was ~12.4%, compared to ~1% rMSC retention in the control.

Analysis of the rMSC volume fraction and rMSC numbers per area suggested that that there was a change in cell volume between days 2 and 7. While there was no statistically significant change in either parameter between days 2 and 7 in the treatment groups, there appeared to be a large decrease in cell number per area while the volume fraction appeared to increase. The overall correlation between cell number and volume fraction was quite low (R = 0.426). This was likely due to variation in cell size and distribution, as the micrographs from the day 2 timepoint had more clumps of tightly packed, compact cells than the later timepoints.

It should be noted that the cell numbers per area figure likely underestimated the actual cell numbers at the later timepoints as the preimplantation values were used as the positive control for comparison, but those values would be relatively high for two reasons; first, the dye is freshly added to the cells and thus at its brightest, and second, the cells are still highly compact and thus all nuclei colocalize with the dye. After a week or 3 weeks in vivo, it is likely that some dye would have leached out of the cells, and the cells are so spread out that the nuclei would not necessarily appear in the brightest part of a cell. Thus, both the cell numbers estimation and the volume fraction estimation are valuable indicators of total rMSC retention.

The optimal dose of polyplexes used in this study was 2 µg, while the higher dose (20 µg) appeared to have limited efficacy. This may be due to the significantly higher dose of the polymer component of the IL-10 polyplex. The polymer used in this study, a commercially available transfection reagent, is known to have cytotoxic effects at high doses in vitro (Supplementary Figure S2). This hypothesis is furthered by the observation of large numbers of apoptotic cells in the 20 µg treatment group at the 2 and 7 day timepoints. However, there was a statistically significant benefit of this dose after 7 days which suggests that the IL-10 production may have modulated the cytotoxicity or at least minimized the cell death due to the inflammatory response. While IL-10 levels in the 20 µg group were significantly increased after 7 days compared to the untreated control, IL1β, aIL-8, and TNF-α levels were not decreased. In fact, the levels of these cytokines were significantly higher than observed in the 2 µg samples at this timepoint. Furthermore, while the numbers of cytotoxic (CD80+) macrophages were reduced after 2 and 7 days in the 20 µg group as compared to the control, the only significant increase in regulatory (CD163+) macrophages occurred after 21 days. Furthermore, at this timepoint, the ratios of CD80+ and CD163+ cells indicated that a portion of the macrophages expressed both markers, as the sum of the volume fraction ratios was greater than unity. This was visualized by overlaying images of serial sections immunostained for the different markers (Figure 6d).

Thus, while IL-10 production was increased by treatment with 20 µg of IL-10 polyplexes, the higher dose was also associated with higher levels of proinflammatory cytokines and the benefit of the IL-10 was thus diminished. While the adaptive immune system may have also contributed to the decrease in efficacy, as suggested by the trend toward higher levels of IL-5 in the 20 µg group after 2 and 7 days, this effect was not significant and thus unlikely to have had a significant contribution.

The increase in rMSC retention rates was associated with a significant increase in IL-10 levels and a decrease in the levels of a variety of proinflammatory cytokines. In the 2 µg group (as compared to the control) after 7 days, the IL-10 level was more than doubled, the levels of aIL-8 and TNF-α were decreased by >50%, and the inflammatory cell volume fraction was also significantly decreased. Thus, the increase in the rMSC retention appeared to be related to a significant downregulation of the inflammatory response.

Analysis of indicated that IL-10 transfection, as compared to the control, may have also had a protective effect on rMSC retention, as very few rMSCs in the 2 µg group were apoptotic unlike many rMSCs in the control, as highlighted in Figure 5. The most apoptotic cells were observed in the 20 µg group, especially at the earliest timepoint, suggesting that the higher dose of complex may have induced greater levels of cell death via apoptosis. Further analysis would be required to conclusively demonstrate the relationship between apoptosis and stem cell survival but preliminary results further the hypothesis that IL-10 has protective effects via prevention of apoptosis in addition to its effects on the inflammatory response.

Correlating the levels of IL-10 to the levels of a variety of proinflammatory cytokines indicated that the strongest negative relationships existed between IL-10 and IL-1β, aIL-8, and TNF-α. These three proinflammatory cytokines are associated with the cytotoxic macrophage (M1) phenotype. IL-10, conversely, is associated with the regulatory macrophage (M2) phenotype. Thus, it seemed plausible that the main effect of the therapy was not actually on the number of inflammatory cells but rather on the phenotype of the macrophages. This seemed credible, as one of the main roles of endogenous IL-10 is to direct the maturation of immature macrophages toward the M2 phenotype.42

IHC for IL-10, as shown in Figure 3a, indicated IL-10 in rMSCs as well as cells with macrophage and fibroblastic morphology, especially in the 2 µg group after 7 days. As reporter gene studies have indicated that transfection is possible from these scaffolds in vivo, some host cells were probably expressing the exogenous IL-10. However, it is unlikely that all of the IL-10 production was due to transfection by the encapsulated polyplexes, especially considering the limited efficacy of nonviral techniques. Nevertheless, highly efficient transfection was not necessary because even low levels of IL-10 appeared sufficient to direct immature macrophages toward a regulatory phenotype. This regulatory phenotype secretes, among other things, IL-10. Thus, a positive feedback system may have come into effect. It seems likely that only a small portion of the observed IL-10 level was directly induced by delivering the IL-10 polyplexes, but this small level was sufficient to stimulate endogenous IL-10 production which was responsible for the rest of the secretion.

To conclusively test the macrophage polarization hypothesis, immunostaining for M1 and M2 macrophages was carried out. Briefly, it was found that the scaffolds with increased IL-10 levels were also found to have lower numbers of CD80+ M1 cells and higher numbers of CD163+ M2 cells. No clear trends were observed in the overall macrophage numbers, but the fraction of the total number of macrophages that were positive for CD80, the M1 marker, was significantly higher in the untreated control than in the 2 µg treatment group. The 20 µg treatment group showed an initial decrease in the fraction of M1, but after 21 days there actually appeared to be a higher fraction of M1 in that group than in the control, suggesting a delay in the inflammatory response. IHC for CD163, the M2 marker, showed a major increase in the number of M2 macrophages in the 2 µg treatment group, especially at the later timepoints. Comparing the ratio of M2 to M1 cells, the 2 µg group had a higher volume fraction of M2 than M1 cells at both 7 and 21 days, while the untreated control had far more M1 than M2 cells. While it may be more correct to consider three macrophages phenotypes, as described by Mosser et al.,48 the primary effect in this study appears to be sufficiently explained by the classical consideration of cytotoxic M1 and regulatory M2 macrophages.49 Thus, treatment with the optimal dose of IL-10 polyplexes appeared to reduce the volume fraction of proinflammatory macrophages and increase the fraction of regulatory macrophages, thereby reducing the overall production of proinflammatory cytokines and increasing the anti-inflammatory response.

As the system described herein is biodegradable with no harmful by-products, it may serve as an ideal cell and gene delivery system in vivo. A single procedure would allow a relatively long period of cell and gene mediated therapy with no need for a retrieval operation. However, after 21 days in vivo, the scaffold was largely degraded, which would preclude its use as a gene delivery system for longer than a few weeks. Furthermore, the scaffold is readily infiltrated by inflammatory cells and thus it offers no physical protection to the cells seeded within. While a small benefit was observed in stem cell retention in the untreated scaffolds after 2 days as compared to the literature (12.3% in the control samples versus ~10% after 24 hours observed by Pons et al.21) this benefit was relatively modest and unlikely to have therapeutic relevance. Only with the addition of IL-10 gene therapy did the improvement in rMSC retention become large and extended enough to potentially have therapeutic significance.

In conclusion, the retention rate of rat mesenchymal stem cells was found to be significantly increased by scaffold-mediated gene therapy with IL-10. Genetic modification of rMSCs to produce IL-10 significantly increased the IL-10 secretion at all timepoints, but had unexpected effects on the inflammatory and immune reaction which were ultimately associated with a limited therapeutic benefits on the rMSC retention rate. Thus, an alternative technique was employed where the rMSCs were implanted in scaffolds loaded with IL-10 polyplexes. This method significantly increased the retention rates of the rMSCs, even after 21 days in vivo. The increased rMSC retention rate was associated with increased levels of IL-10, decreased levels of proinflammatory cytokines and a decrease in the overall volume fraction of inflammatory cell. The primary effect of the IL-10 gene therapy appeared to be on the phenotype of the infiltrating macrophages, decreasing the fraction of cytotoxic macrophages and increasing the number of regulatory macrophages. The number of apoptotic rMSCs was also lowest in the optimal treatment group, implying an additional protective effect. Thus, treatment with the anti-inflammatory cytokine IL-10 significantly improved the retention rate of implanted rMSCs by modulating the inflammatory response and promoting stem cell survival. Application of this technique to the treatment of ischemic or degenerative conditions might yield significant improvement of stem cell survival, which may be an important step toward the realization of the therapeutic potential of stem cells.

Materials and Methods

Animal handling. Female Lewis rats were obtained from Harlan Laboratories (Bicester, UK) and allowed to acclimatize to housing conditions for at least 7 days prior to use. All animals received humane care in compliance with federal and institutional guidelines, and all procedures were approved by the institutional animal ethics committee and the federal board under the Cruelty to Animals Act.

Preparation of scaffolds. 1 mg collagen sponges were prepared by freeze-drying 0.3 wt% bovine atelocollagen (isolated in-house), then crosslinking with 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide/N-hydroxysuccinimide as described elsewhere.41 Crosslinked sponges were washed extensively with phosphate-buffered saline, distilled water and 70% ethanol to sterilize them. The scaffolds were then freeze-dried again.

IL-10 modification of stem cells. Male Lewis rMSCs were isolated as described elsewhere.50 Briefly, bone marrow was flushed from the tibial and femoral compartments of male Lewis rats, resuspended, centrifuged, and plated on tissue-culture plastic. The mesenchymal cell population was isolated based on adherence to plastic and hematopoietic cells were removed by regular medium changes. Adherent, spindle-shaped cells were characterized by uniform expression of MSC surface markers CD29, CD73, and CD90. No contamination with CD3 or CD11c positive cells was observed. All stem cell characterization antibodies were purchased from BD Pharmingen, Oxford, UK. Osteogenic, chondrogenic, and adipogenic assays were used to confirm cell multipotency.

These mesenchymal stem cells were transfected in T25 flasks with IL-10 polyplexes (pCMV-driven mouse IL-10 with neomycin resistance). After 2 days, G418 (Sigma, Dublin, Ireland) was added to the media in the flask at a concentration of 0.5 µg/ml based on previously conducted dose–response curves. After 14 days, stable clones were selected and seeded into new flasks. The cells were allowed to proliferate until confluence was reached. The IL-10 modified cells were used between passages 4 and 8.

Preparation and loading of polyplexes into scaffolds. Polyplexes were prepared by incubating mouse IL-10 plasmids with a partially degraded dendrimer (Superfect, Qiagen, Crawley, UK). Polyplexes were added to the crosslinked collagen scaffolds and the polyplexes were allowed to adsorb to the scaffold for 3 hours. The mass of plasmid is treated as the dose, i.e., 2 µg IL-10 polyplexes refers to 2 µg of IL-10 plasmid which was then polyplexed at the optimized weight ratio of dendrimer:DNA (15:1).

Seeding cells onto scaffolds. Prior to seeding, male rMSCs between passage 2 and 6 were incubated with Celltracker CM-DiI (Invitrogen, Dun Laoghaire, Ireland) at a concentration of 4 µmol/l for 30 minutes at 37 °C. These fluorescently labeled rMSCs were seeded into the scaffolds. The seeded scaffolds were incubated overnight to allow the cells to attach to the collagen scaffold.

In vitro analysis of scaffolds. Dose and seeding optimization studies were carried out in vitro to select the parameters for the in vivo study. Transfection optimization studies were carried out to select the optimal weight ratio between the dendrimer and DNA, optimal polyplex dose, and optimal cell number. Briefly, scaffolds were loaded and seeded as described. IL-10 levels were quantified using ELISA for mouse IL-10. Cellular metabolic activity was measured with the AlamarBlue assay (Invitrogen). To measure IL-10 secretion over time, rMSCs were seeded into polyplex-loaded scaffolds and allowed to proliferate for 14 days. The media was changed at the specified timepoints. Antibodies used for sandwich ELISA were purchased from BD Pharmingen.

Implantation of scaffolds. The cell and polyplex-loaded scaffolds were implanted into the dorsum of female Lewis rats. Briefly, rats were anaesthetized, shaved, and a 4 cm long incision made along the centre of the dorsum. Blunt dissection was used to separate the fascia from the skeletal muscle, and patches were sutured directly onto the muscle. Four scaffolds were implanted, two on each side of the midline. Spacing between the patches was at least 1 cm. At the relevant timepoints (2, 7, and 21 days), the animals were euthanized and the patches explanted.

Tissue preservation and cryosectioning. Half of each patch was fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, then embedded in optimal cutting temperature media and flash-frozen. The optimal cutting temperature embedded samples were cryosectioned at a thickness of 5 µm. A total of nine sections at three levels were analyzed, each level 200 µm apart. The remaining half of each explant was immediately flash-frozen for protein analysis.

Fluorescent imaging and immunohistochemistry. Fluorescent micrographs of cryosectioned samples, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen), were analyzed stereologically to quantify the volume fraction of rMSCs in each tissue sample. Briefly, samples were brought to room temperature in phosphate-buffered saline, stained with DAPI, and mounted using aqueous mounting media (Vectashield, Vector labs, Burlingame, CA). IL-10 immunohistochemically stained sections were blocked in goat serum, incubated with goat antimouse IL-10 antibody (R&D Systems, Minneapolis, MN), washed, and incubated with fluorescein isothiocyanate-anti-goat antibody (Vectashield, Vector labs). ED-1 (anti-CD68) antibody (Abcam, Cambridge, UK) was used to stain for all macrophage types, anti-CD80 antibody (AdB Serotec, Kidlington, UK) was used to assess for presence of the M1 phenotype, and anti-CD163 antibody (AdB Serotec) was used to assess for presence of the M2 phenotype. Fluorescein isothiocyanate-antimouse antibodies (Vector labs) were used as a secondary antibody for all macrophage markers. These sections were also counterstained with DAPI. To control for nonspecific binding of the secondary antibody, negative control sections were incubated with phosphate-buffered saline instead of primary antibody before treatment with the secondary antibody. All negative control sections were found to have minimal fluorescent signal.

IL-10 level quantification. Protein analysis was conducted on tissue homogenate with sandwich ELISA for IL-10 using the protocol optimized in vitro. Briefly, tissue samples were suspended in Tissue Extraction Reagent (Sigma) at 20 mg/ml. The samples were incubated for 5 minutes then homogenized using a TissueRuptor (Qiagen). Homogenate was centrifuged at 10,000g for 10 minutes to remove particulates. The IL-10 content in the supernatant was then analyzed and normalized to the total protein content, as analyzed using the bicinchoninic acid assay (Pierce, Rockford, IL).

Multiplex cytokine measurements. A rat inflammatory cytokine multiplex array (Meso Scale Discovery, Gaithersburg, MD) was used to analyze the relative levels of a variety of different inflammatory cytokines. Briefly, tissue lysates from the ELISA analysis were added to plates carrying antibodies for IFN-γ, IL-1β, IL-4, IL-5, IL-13, KC/GRO/CINC (which is the rat analog of human IL-8), TNF-α, and IL-10. The plate was then imaged with a SECTOR Imager 2400 (Meso Scale Discovery).

Hematoxylin and eosin staining. Standard hematoxylin and eosin staining followed by stereological analysis was used to quantify overall numbers of infiltrating inflammatory cells at every timepoint, as described elsewhere.50

Terminal deoxynucleotidyl transferase dUTP nick end labeling staining. An in situ cell death detection kit (Roche, Clarecastle, Ireland) was used to detect apoptotic cells within cryosections as per the manufacturer's instructions. The negative control was stained with the label solution without any enzyme solution to control for background fluorescence.

Stereology. Stereological analysis was used for quantification of stem cell and inflammatory cell volume fractions, as described elsewhere.50 Briefly, a 192 point grid was superimposed over the image and all points that fell within the scaffold or reaction area were counted. The number of points intersecting with cells of interest was counted and the volume fraction calculated as a function of the ratio of cells to total scaffold. Three sections from three different levels were analyzed (a total of 9 sections), each separated by 200 µm, to ensure accurate representation of the entire scaffold volume. A minimum of six fields of view were analyzed per section. Cell counting was also conducted on the same sections to quantify the number of rMSCs per mm2. The number of nuclei that colocalized with the CM-DiI rMSC stain was considered the number of rMSCs. The stereological analysis used to quantify the volume fractions of macrophages expressing CD68, CD80, or CD163 was necessarily less exhaustive. Two representative patches were selected per treatment group and timepoint, and six fields of view per patch were analyzed. While the difference in mean volume fraction ratios between the patches was <15% in all cases, a 25% margin of error was applied to correct for the limited number of samples and the error inherent in combining two volume fractions (5% each). To ensure variations in cell size were not responsible for the variation in macrophage volume fractions, analysis of macrophage area was conducted, as shown in Supplementary Figure S3 and Supplementary Figure S4.

Statistics. Data are expressed as mean ± 95% confidence interval. Cumulative averages were calculated from the stereological tests. All statistical comparisons were carried out using SPSS Statistics 17.0. ANOVA was used to determine statistical significance followed by Bonferoni's multiple test correction to determine which groups were statistically different. Bivariate correlations were used to investigate the relationships between different inflammatory cytokines, the rMSC volume fraction, and the inflammatory cell volume fraction over time. Statistical significance was set at P < 0.05 and post-hoc corrections were used. Outliers were considered as any point outside of three interquartile ranges.

SUPPLEMENTARY MATERIAL Figure S1. IL-10 secretion levels over time in vitro. The highest level of IL-10 in the media was just over 2000 pg/mL which was secreted 2 days after the rMSCs were seeded into the polyplex loaded scaffolds. The highest response was seen in scaffolds loaded with 2μg of IL-10 polyplexes. On average, the IL-10 secretion from the patches treated with 2μg IL-10 was approximately double the level secreted by unmodified cells. (Data expressed as mean ± standard deviation, n=3). Figure S2. Metabolic activity index of rMSCs treated with varying doses of IL-10 polyplexes in vitro. As the polyplex dose increases, the metabolic activity of the cells decreases, suggesting increasing toxicity of the polyplexes in vitro. (Data expressed as mean ± standard deviation, n=3). Figure S3. Mean macrophage area from IHC using CD68, CD80 and CD163. The mean macrophage area in micrographs immunostained for each marker was quantified to ensure that the relative macrophage sizes were the same and thus variability in area fraction was due to changes in macrophage numbers. (Data expressed as mean ± 95% CI). Figure S4. CE ratios for stereological measurements. The mean intra-sample and the inter-sample coefficient of error (CE) ratios are reported in this table for the stereological measurements of stem cell volume fraction.

Acknowledgments

This material is based upon works supported by the Science Foundation Ireland under grant no. 07/SRC/B1163. The authors would like to thank Prof Jeffrey Medin (University of Toronto) for providing the IL-10 plasmid, Dr Thomas Ritter and Prof Peter Dockery (National University of Ireland, Galway) for scientific advice and critique and Dr Mary Murphy (Regenerative Medicine Institute, National University of Ireland, Galway) for providing the rMSCs. Special thanks to Louise Unwin (University College Dublin) and Estelle Collin (NFB, National University of Ireland) for assistance in laboratory techniques. The main fraction of the work contained in this manuscript was carried out at the National University of Ireland, Galway, Ireland.

Supplementary Material

Figure S1.

IL-10 secretion levels over time in vitro. The highest level of IL-10 in the media was just over 2000 pg/mL which was secreted 2 days after the rMSCs were seeded into the polyplex loaded scaffolds. The highest response was seen in scaffolds loaded with 2μg of IL-10 polyplexes. On average, the IL-10 secretion from the patches treated with 2μg IL-10 was approximately double the level secreted by unmodified cells. (Data expressed as mean ± standard deviation, n=3).

Click here for additional data file. (29KB, pdf)
Figure S2.

Metabolic activity index of rMSCs treated with varying doses of IL-10 polyplexes in vitro. As the polyplex dose increases, the metabolic activity of the cells decreases, suggesting increasing toxicity of the polyplexes in vitro. (Data expressed as mean ± standard deviation, n=3).

Click here for additional data file. (18.9KB, pdf)
Figure S3.

Mean macrophage area from IHC using CD68, CD80 and CD163. The mean macrophage area in micrographs immunostained for each marker was quantified to ensure that the relative macrophage sizes were the same and thus variability in area fraction was due to changes in macrophage numbers. (Data expressed as mean ± 95% CI).

Click here for additional data file. (38.4KB, pdf)
Figure S4.

CE ratios for stereological measurements. The mean intra-sample and the inter-sample coefficient of error (CE) ratios are reported in this table for the stereological measurements of stem cell volume fraction.

Click here for additional data file. (25KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

IL-10 secretion levels over time in vitro. The highest level of IL-10 in the media was just over 2000 pg/mL which was secreted 2 days after the rMSCs were seeded into the polyplex loaded scaffolds. The highest response was seen in scaffolds loaded with 2μg of IL-10 polyplexes. On average, the IL-10 secretion from the patches treated with 2μg IL-10 was approximately double the level secreted by unmodified cells. (Data expressed as mean ± standard deviation, n=3).

Click here for additional data file. (29KB, pdf)
Figure S2.

Metabolic activity index of rMSCs treated with varying doses of IL-10 polyplexes in vitro. As the polyplex dose increases, the metabolic activity of the cells decreases, suggesting increasing toxicity of the polyplexes in vitro. (Data expressed as mean ± standard deviation, n=3).

Click here for additional data file. (18.9KB, pdf)
Figure S3.

Mean macrophage area from IHC using CD68, CD80 and CD163. The mean macrophage area in micrographs immunostained for each marker was quantified to ensure that the relative macrophage sizes were the same and thus variability in area fraction was due to changes in macrophage numbers. (Data expressed as mean ± 95% CI).

Click here for additional data file. (38.4KB, pdf)
Figure S4.

CE ratios for stereological measurements. The mean intra-sample and the inter-sample coefficient of error (CE) ratios are reported in this table for the stereological measurements of stem cell volume fraction.

Click here for additional data file. (25KB, pdf)

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