Immunomodulatory Properties of Mesenchymal Stromal Cells Can Vary in Genetically Modified Rats

Abstract

Mesenchymal Stromal Cells (MSC) have been shown to exhibit immuno-modulatory and regenerative properties at sites of inflammation. In solid organ transplantation (SOT), administration of MSCs might lead to an alleviation of ischemia-reperfusion injury and a reduction of rejection episodes. Previous reports have suggested 'MSC-preconditioning' of macrophages to be partly responsible for the beneficial effects. Whether this results from direct cell-cell interactions (e.g., MSC trans-differentiation at sites of damage), or from paracrine mechanisms, remains unclear. Immunosuppressive capacities of MSCs from donors of different age and from genetically modified donor animals, often used for in-vivo experiments, have so far not been investigated. We conducted an in vitro study to compare paracrine effects of supernatants from MSCs extracted from young and old wild-type Wystar-Kyoto rats (WKY-wt), as well as young and old WKY donor rats positive for the expression of green fluorescent protein (WKY-GFP), on bone marrow derived macrophages (BMDM). Expression levels of Mannose receptor 1 (Mrc-1), Tumor necrosis factor α (TNFα), inducible NO synthase (iNos) and Interleukin-10 (IL-10) in BMDMs after treatment with different MSC supernatants were compared by performance of quantitative PCR. We observed different expression patterns of inflammatory markers within BMDMs, depending on age and genotype of origin for MSC supernatants. This must be taken into consideration for preclinical and clinical studies, for which MSCs will be used to treat transplant patients, aiming to mitigate inflammatory and allo-responses.

Keywords: cytokines; mesenchymal stem cells; transplantation.

Figure A1

Wystar Kyoto wildtype (WKY-wt) and transgenic WKY rats positive for green fluorescent protein (WKY-GFP) (a) were used as bone marrow donors for the extraction of wildtype MSCs (b) and GFP+ MSCs (c). Pictures of cells were taken using light microscopy at a 20× magnification for wildtype cells of all donors and for all passages. In (b) one representative picture of WKY-wt cells in culture, at a 20× magnification, from a young donor in passage 5 is shown. Using a confocal microscope to detect the green fluorescence in GFP+ cells, in (c), a representative picture of WKY-GFP cells in culture, at a 20× magnification, is shown. Cells were in passage 5 and from a young donor rat (6 weeks of age). Strong green fluorescence was present throughout all passages (c). Microscopic features were similar for both age groups and throughout passages.

Figure A2

Flow cytometry of extracted MSCs to confirm MSC phenotype. Results of Flow cytometry for MSCs after staining for the surface markers CD29, CD34, CD44, CD45 and CD90. GFP positivity was confirmed by gating for GFP and 92% of those MSCs were negative for CD34 and CD45 (a). 97% of all MSCs were positive for CD44 or CD29 whilst being negative for CD45 and CD34 (b). 60% of all MSCs were furthermore positive for CD90 and CD44 (c).

Figure A3

Confirmation of MSC differentiation into osteocytes and adipocytes. 10× magnification of MSCs after stimulation with osteogenic and adipogenic differentiation media. Alizarin-red-staining for cells after osteogenic differentiation is shown in (a). Oil-red-O staining for adipocytes after differentiation is shown in (b).

Figure A4

Scheme of in vitro supernatant transfer experiment. The experimental scheme of different in vitro treatments of BMDMs with different supernatants coming from MSCs is illustrated in the Figure. WT old = Wildtype Wistar Kyoto rat >6 months of age as a Mesenchymal Stromal Cell (MSC) donor. WT young = Wildtype Wistar Kyoto rat <6 months of age as MSC donor. LPS = lipopolysaccharide treatment. GFP old = transgenic Wistar Kyoto rat positive for ubiquitous expression of green fluorescent protein (GFP) >6 months of age as MSC donor. GFP young= transgenic Wistar Kyoto rat positive for ubiquitous expression of green fluorescent protein (GFP) <6 months of age as MSC donor. Mac ctrl= untreated macrophages in DMEM based medium as control. MSC ctrl = macrophage in DMEM based medium, treated with blank MSC medium. P1-P10 = passage 1–10; sup = supernatant.

Figure 1

Mrc-1 expression levels in BMDM after treatment with MSC-wildtype versus MSC-GFP+ supernatants of different age groups and passages. The graph shows the influence of different supernatants from Mesenchymal Stromal Cells on the expression levels of Mannose receptor-1 (Mrc-1) in BMDM extracted from a Wistar Kyoto wildtype (WKY-wt) rat. Supernatants from MSC-wt led to a significantly stronger upregulation of Mrc-1 than supernatants from MSC-GFP+ cells, both in native BMDM (a), as well as in macrophages after an inflammatory stimulus with Lipopolysaccharide (LPS) (b). Furthermore, for supernatants from MSC-wt cells, the difference between the effects from young versus old cell supernatants were significant (c), which was not the case within MSC-GFP+ supernatant treatment groups (d). Relative mRNA expression levels of Mrc-1 were calculated using real time (RT)-PCR and the ΔΔCt-method was used for calculations, with untreated BMDM as control and HPRT-1 as housekeeping gene. Statistical analysis was performed using Mann Whitney U test. Statistical significance: no statistical significance (ns; no asterisk); p < 0.05 (*); BMDM = Bone marrow derived macrophage, Mrc-1= Mannose receptor-1, WT= supernatants coming from MSC-wild-type cells, GFP = supernatants from MSC-GFP+ cells. P1-8= passages 1-10. o = supernatants from cells from donor rats > 6 months of age, y = supernatants from cells from donor rats < 6 months of age.

Figure 2

iNOS expression levels in BMDM after treatment with MSC-wildtype versus MSC-GFP+ supernatants of different age groups and passages. The graph shows the influence of different MSC supernatants on the expression levels of Inducible Nitric Oxide Synthase (iNOS) on BMDM coming from a WKY-wt rat. Supernatants from MSCs extracted from MSC-wt donors led to lower iNOs expression levels than supernatants from MSC-GFP+ cells. Furthermore, within supernatants from older donors, this difference was significant (a). After LPS stimulation of BMDM, supernatants from GFP+ cells and supernatants from WT cells had similar effects on BMDMs in culture (b). Within supernatants from both, MSC-wt and MSC-GFP+ supernatants, young groups inhibited expression of iNOS more efficiently in native BMDM than supernatants from MSCs extracted from old donors, however without statistical significance (c,d). Statistical evaluation: Mann-Whitney U test. mRNA levels were calculated using RT-PCR and the ΔΔCt-method. Untreated macrophages served as control and HPRT-1 was the housekeeping gene. Statistical significance: no statistical significance (ns; no asterisk); p < 0.05 (*). BMDM = bone marrow derived macrophages, iNOS = Inducible Nitric Oxide Synthase, WT= supernatants coming from wild-type cells, GFP = supernatants from GFP+ cells. Old = supernatants from cells from donor rats >6 months of age, young = supernatants from cells from donor rats < 6 months of age.

Figure 3

TNFα expression levels in BMDMs after treatment with MSC-wildtype versus MSC-GFP+ supernatants of different age groups and passages. The graph shows the influence of different supernatants from Mesenchymal Stromal Cells on the expression levels of Tumor necrosis factor α (TNFα) on BMDMs extracted from a WKY-WT rat. Supernatants from MSCs extracted from GFP+ donors led to a significantly better reduction of TNFα expression levels after stimulation with lipopolysaccharide (LPS) than supernatants from WT-MSCs (p = 0.04, calculated using Mann-Whitney U test, (a)). Particularly supernatants from older GFP+ rats led to a decrease of TNFα expression levels (b). Within the MSC-GFP+ treated group, TNFα expressions were particularly downregulated after treatment with supernatants coming from the older donors, when compared to supernatants from younger donors (c). mRNA levels were calculated using RT-PCR and the ΔΔCt-method. Untreated BMDM served as control and HPRT-1 was the housekeeping gene. Statistical significance: no statistical significance (ns; no asterisk); p < 0.05 (*); p < 0.005 (**). BMDM= bone marrow derived macrophages, TNFα = Tumor necrosis factor α, WT = supernatants coming from wild-type cells, GFP = supernatants from GFP + cells. Old = supernatants from cells from donor rats > 6 months of age, young= supernatants from cells from donor rats < 6 months of age.

Figure 4

IL-10 expression levels in BMDMs after treatment with MSC-wildtype versus MSC-GFP+ supernatants of different age groups and passages. Figure 4. the graph shows the influence of different MSC supernatants on the expression levels of Interleukin-10 (IL-10) on BMDMs extracted from a WKY-WT rat. Supernatants from MSCs from GFP+ donor rats led to a higher upregulation of IL-10 expression levels in native BMDMs than supernatants from WT-MSCs (a). Particularly supernatants from older GFP+ rats led to an increase of IL-10 expression levels (b). In untreated BMDMs, results indicated a more potent up-regulation of IL-10 after treatment with supernatants from old MSC donors in comparison to their young equivalents (c). mRNA levels were calculated using RT-PCR and the ΔΔCt-method. Untreated macrophages served as control and HPRT-1 was the housekeeping gene. Statistical significance: no statistical significance (ns; no asterisk); BMDM = bone marrow derived macrophages, IL-10 = Interleukin-10, WT = supernatants coming from wild-type cells, GFP = supernatants from GFP+ cells. Old = supernatants from cells from donor rats > 6 months of age, young = supernatants from cells from donor rats < 6 months of age.