Mesenchymal stem cells (MSCs) from adult bone marrow have moved into a focus of attention for their immunomodulatory potential. Therefore, because MSCs can be easily obtained and propagated in culture, they could prove to be a valuable tool for cell-based tolerance-induction protocols (1–4). Mesenchymal stem cells reside in bone marrow as a nonhematopoietic cell population and are characterized by their ability to self-renew and differentiate into mesenchymal tissues such as bone, cartilage, or adipose tissue (5). Mesenchymal stem cells produce the stromal matrix, which constitutes the bone marrow microenvironment and supports the growth of hematopoietic progenitor cells, thereby preventing or mitigating graft-versus-host disease after bone marrow transplantation (5–8). Moreover, bone marrow–derived MSCs have been shown to exert immunoregulatory functions in vitro. For instance, baboon (9), human (6, 10–12), and rodent (13, 14) MSCs can effectively suppress T lymphocyte proliferation when added to a mixed-lymphocyte reaction (MLR).
Notwithstanding these in vitro data, the in vivo immunoregulatory potential of MSCs has yet to be described in detail to our knowledge. Thus far, it has been shown that intravenous administration of MSCs from donor-type adult baboon bone marrow to major histocompatibility complex (MHC)–mismatched recipients before skin grafting nonspecifically prolongs graft survival by a few days (9). In addition, MSCs favored the growth of subcutaneously injected melanoma cells in an allogenic mouse tumor model (13).
Indeed, the implementation of MSC therapy approaches for tolerance induction could be highly desirable to avoid the use of systemic immunosuppressive agents. Long-term systemic immunosuppression is increasingly recognized for its numerous serious side effects, providing intense motivation for seeking new tolerogenic strategies. Therefore, we tested the hypothesis that MSCs could have beneficial effects for allograft survival in vivo when applied to a primary vascularized model of solid organ transplantation in the rat. The present study outlines that, although MSCs have immunomodulative effects in vitro, tolerogenic effects in a solid organ transplant model were not found. Moreover, caution should be taken with regard to this concept because graft rejection was accelerated by MSCs in some cases.
MATERIALS AND METHODS
Male Lewis rats (MHC haplotype, RT1l; Charles River, Sulzfeld, Germany) and male ACI rats (RT1a) weighing approximately 250 g were maintained in our animal center. Regional authorities approved the animal procedures.
Isolation and Growth of Mesenchymal Stem Cells from Adult Rat Bone Marrow
Bone marrow cells were collected by flushing the long bones with HBSS medium (Biochrom, Berlin, Germany). Cells were cultivated in 175 cm2 cell culture flasks in α-MEM (Biochrom) supplemented with 20% fetal bovine serum (FBS), penicillin/streptomycin, insulin (5 μg/mL), transferrin (5 μg/mL), selenium (5 ng/mL), ascorbic acid (0.1 mM), linoleic acid (4.7 μg/mL), and dexamethasone (1×10–6 mM; Sigma-Aldrich, St. Louis). After 24, 48, and 72 hours, nonadherent cells were removed by changing the culture medium. Adherent cells were trypsinized (0.5% trypsin–EDTA), harvested, and plated into new flasks 2 weeks after the culture was started or each time when 80% confluency was achieved. Passage 5–8 MSCs were used for all experiments described. Hematopoietic cells were only identified rarely in these cell preparations.
Adipocyte Differentiation of Cultured Mesenchymal Stem Cells
Mesenchymal stem cells were cultured in 24-well plates with MSC culture medium until they reached confluency. For differentiation, cells were cultured for 3 days with α-MEM supplemented with 1% penicillin/streptomycin, insulin (5 μg/mL), transferrin (5 μg/mL), selenium (5 ng/mL), ascorbic acid (0.1 mM), linoleic acid (4.7 μg/mL), dexamethasone (1×10–6 mM), 5% goat serum (PromoCell, Heidelberg, Germany), and 3-isobutyl-1-methylxanthin (0.1 mg/mL; Sigma-Aldrich). Subsequently, cells were cultured for 5 days with MSC culture medium supplemented with 40 IU/mL of Rapid Insulin (Aventis, Soden, Germany) without FBS. Cell differentiation to adipocytes was confirmed by Oil Red O staining. For this, cells were washed with cold phosphate-buffered saline (PBS) solution and fixed with 10% formaldehyde at 4°C overnight. Cells were incubated with 5 mg/mL Oil Red O solution for 2 hours at room temperature.
Phenotypic Analysis of Mesenchymal Stem Cells
For flow cytometry, MSCs from the eighth passage were washed with PBS, trypsinized, and resuspended in PBS solution containing 10% FBS. The cell suspensions were incubated with monoclonal antibodies for 30 minutes at 4°C with combinations of saturating amounts of purified monoclonal antibodies conjugated with fluorescein isothiocyanate or phycoerythrin. Fluorescein isothiocyanate–conjugated antibodies against CD4 and phycoerythrin-conjugated antibodies against CD25 were purchased from Caltag Laboratories (Burlingame, CA). Purified anti–rat CD45, CD49b, CD73, CD80, CD86, Thy1 (i.e. CD90) and pan anti–rat MHC class II (i.e. RT1B) were purchased from Becton Dickinson (Franklin Lakes, NJ). Anti–rat RT1.Al was originally purified and provided by K. Wonigeit (Hannover Medical School, Hannover, Germany). Each fluorescence analysis included the appropriate fluorescein isothiocyanate– or phycoerythrin-conjugated isotype antibody controls (Caltag Laboratories). Cells were analyzed with an FACS Vantage laser flow cytometry system (Becton Dickinson), and data analysis was performed with Win MDI software (Becton Dickinson).
Analysis of Immunomodulative Effects of Mesenchymal Stem Cells In Vitro
For the in vitro analysis of MSC effects, 1.2×104 MSCs from Lewis rats were cultured in 12-well plates overnight in MSC expansion medium, which formed a confluent monolayer. Spleens obtained from ACI rats were minced by passage through a stainless steel mesh into HBSS medium; erythrocytes were lysed by NH4Cl buffer solution. Splenocytes were stained by carboxyfluorescein diacetate and succinimidyl ester (i.e. CFDA SE) with use of the Vybrant CFDA SE cell tracer kit (Molecular Probes Europe, Leiden, The Netherlands). Then, 7.5×105 carboxyfluorescein succinimidyl ester–labeled ACI splenocytes were added to wells containing MSCs and incubated at 37°C and 5% CO2.
Mixed Lymphocyte Reactions with Mesenchymal Stem Cells
ACI T cells were enriched from spleens making use of Degalan bead columns (Degussa, Teterboro, NJ) as described in detail previously (15, 16). Briefly, a single-cell suspension of ACI spleen cells was passed over Degalan beads coated with rabbit anti–rat immunoglobulin G heavy- and light-chain–specific antibody (MP Biomedicals, Irvine, CA). Cells not binding to the column were stained with CFDA SE according to the manufacturer’s instructions (as described earlier). CFDA SE–labeled ACI responder cells (2.5×105) were cocultured in 24-well plates with 7.5×105 irradiated (20 Gy) Lewis spleen cells as stimulators. Cocultures also contained MSCs from Lewis rats at ratios of 1:1 and 1:100 in RPMI medium (Gibco Invitrogen, Karlsruhe, Germany) supplemented with 10% FBS with or without 800 U/mL rat interleukin-2 (IL-2; BD Bioscience, Bedford, MA). Transwell inserts were used in some wells (0.4-μm pore size; BD Falcon, San Jose, CA). After incubation CFSE-labeled T cells were resuspended in PBS and T cell proliferation was assessed by counting labeled T cells in a Neubauer chamber. Thereby, the CSFE labeling was only used as a marker dye for responder lymphocytes to assess their number (29, 30). All experiments were carried out in duplicate wells.
Application of Mesenchymal Stem Cells in a Heterotopic Rat Heart Transplantation Model
Lewis rats were used as heart transplant donors and ACI rats were used as recipients. Heterotopic heart transplantation was performed as previously described (17). Mesenchymal stem cell injections were performed immediately after heart transplantation, with an additional injection on day 3 in some experiments. In one group, 5×06 MSCs were diluted in 1 mL of saline solution and injected one time into the inferior vena cava or portal vein. In another group of animals, 2×106 MSCs were injected via the liver portal venous system on day 0 and a subsequent injection of 2×106 MSCs was performed via the tail vein on day 3. In specified experiments, 200×106 Lewis splenocytes were injected intravenously as a control cell population known to promote allograft survival (18). Cyclosporin A (CsA; Sandimmune; Novartis, Nuernberg, Germany) was diluted in PBS solution and injected intraperitoneally at a dosage of 0.5 mg/kg/d from day 5 to day 9 as indicated. All experimental groups are summarized in Table 1. Heart grafts were checked daily for signs of rejection. Graft rejection time was defined as the day at which no cardiac contractions were palpable, with verification of rejection by direct inspection of the allograft via laparotomy.
Expansion and Characterization of Mesenchymal Stem Cells from Adult Rat Bone Marrow
Rat MSCs from adult bone marrow were purified by adherence to plastic culture flasks as previously described (19–21) (Fig. 1A,1B). Adherent MSCs had a spindle-shaped fibroblastic morphology after expansion. At least 17 passages could be obtained from each primary population. Culturing in adipocyte-differentiation media induced differentiation of MSCs into adipocytes, as verified by Oil Red O staining of intracellular fat deposits (Fig. 1C). In addition, phenotypic analysis of Lewis MSCs was performed by flow cytometry (Fig. 2). Mesenchymal stem cells expressed MHC class I molecules (i.e. RT1Al), but did not express MHC class II molecules (i.e. RT1B, nonpolymorphic determinant). The expression of costimulatory molecules was very weak or absent (CD80/dim and CD86/negative). Mesenchymal stem cells were positive for Thy1 (i.e. CD90) and CD73, but lacked expression of CD4, CD25, CD45, and CD49b.
The isolation of MSCs usually results in a heterogeneous cell population with hematopoietic contaminations (14, 19, 20). To exclude that residual hematopoietic cells in the culture would influence the in vivo experiments, we performed FACS analysis for CD45+ hematopoietic cells at different time points. In passage 1 and passage 2 cultures, the frequencies of CD45+ cells were 34% and 10%, respectively. However, when passaged further, the number of contaminating cells was reduced to very low levels (passage 4, 0.3%; passages 6–17, <0.2%).
Immunomodulatory Effects of Mesenchymal Stem Cells In Vitro
To examine the influence of MSCs on the recipient’s antidonor response in vitro, two distinct coculture experiments were set up. First, Lewis MSCs were cultured in MSC expansion medium overnight to achieve adherence. CFSE-labeled ACI spleen cells were then added to the culture. This resulted in the killing of ACI spleen cells by MSCs; cell death was evidenced by MSC phagocytosis of the target cells (Figs. 3,4). However, it cannot be ruled out that some of these events were caused by strong attachment of MSCs to lymphocytes.
In a second set of experiments, purified T cells harvested from ACI spleens were cocultured with different relative numbers of Lewis MSCs (ratios of MSCs to spleen cells of 1:1 vs. 1:100), together with irradiated Lewis spleen cells as stimulators. Proliferation of CFSE-labeled T cells was suppressed when cocultured with an equal number of MSCs (Fig. 5A), whereas the lower relative number of MSCs (1:100) did not suppress T cell proliferation. This suppressive effect was also observed when activation of T cells in the coculture was enhanced by the addition of IL-2 (Fig. 5B). Cell–cell contact was not required, at least in the latter experiment, as the suppressive effect was observed even when MSCs and spleen cells were cultured on opposite sides of a transwell membrane.
Application of Mesenchymal Stem Cells to a Model of Allogeneic Heart Transplantation
To elucidate whether the immunomodulative effects of rat MSCs could also modify the antidonor response in vivo, we applied MSCs to a model of solid organ transplantation. In our model, we injected MSCs alone after transplantation or in combination with delayed low-dose CsA treatment. The combination experiments were set up in analogy to the window of opportunity concept, whereby immunologic interactions are promoted for a short period after organ transplantation, before initiation of general immunosuppression (18, 22). Results from these experiments are summarized in Table 1.
Our results show that, when 5×106 donor-type MSCs were injected one time after heart transplantation, intravenously (group 2; Table 1) or via the liver portal system (group 3), graft survival was not prolonged compared with the no-treatment group (group 1). Because the intravenous injection of MSCs resulted in a high frequency of emboli in the lung (six of 11 cases), we omitted the intravenous injection of high numbers of MSCs. In another set of experiments, 2×106 MSCs were injected into the liver portal venous system on day 0 and into the tail vein on day 3, without any animal mortality after injection. However, again, graft survival was not significantly prolonged (groups 4 and 5). To compare the MSC effect with the injection of cells previously reported to promote transplant survival in the same model, 200×106 donor-type spleen cells were injected one time after transplantation. Indeed, treatment with donor splenocytes prolonged graft survival significantly compared with the no-treatment group (group 6), suggesting the model system is appropriate for testing the potential of MSCs to promote allograft survival.
In the second set of experiments, we examined MSC effects on transplant survival in the presence of immunosuppression. Mesenchymal stem cells were injected on days 0 and 3 before the start of low-dose CsA therapy (0.5 mg/kg) from days 5 through 9. Interestingly, the application of MSCs combined with CsA therapy only served to accelerate graft rejection compared with the control group treated with CsA only (group 7). In comparison, if CsA was given in the same way together with spleen cells, graft rejection was prolonged (group 10). Therefore, MSCs do not appear to promote allograft survival with or without concurrent CsA immunosuppression in a rat heart transplant model.
Mesenchymal stem cells were first described as a nonhematopoietic stem cell population within adult bone marrow that supports the constant generation of hematopoietic cells. Moreover, MSCs can give rise to a typical set of stromal elements such as bone, cartilage, and adipose tissue (1). Recently, it has been demonstrated that MSCs can follow atypical pathways of differentiation, thereby becoming nonmesenchymal cells such as hematopoietic cells or liver, lung, or intestinal epithelium (2). The phenotypic character of MSCs is conserved among different species including baboons (9), humans (6, 10–12), and mice (13, 14). Our data are consistent with this observation, in that our rat MSCs have the same phenotype since they express MHC class I molecules but lack expression of MHC class II, mature leukocyte antigens, and costimulatory molecules.
Mesenchymal stem cells are especially interesting for transplantation medicine because they have been reported to escape immunity (23) and to downregulate the immune response. The phenotypic pattern of our MSCs supported the working hypothesis that they would be of low immunogenicity. In classic mixed-lymphocyte reactions, MSCs suppress T-cell proliferation independent of MHC matching between T cells and MSCs (10). A recent report by Glennie et al. (24) introduce a detailed mechanism for the MSC-induced suppression of T cells involving division arrest anergy. Interestingly, IL-2 did not restore the ability of T cells to proliferate after the removal of MSCs in their study, demonstrating a persistent block of the ability of T cells to respond to antigen. Additional mechanisms for MSC-induced modulation of the T cell alloresponse have been considered, including that MSCs influence distinct sets of antigen-presenting cells, such as monocytes, dendritic cells, or natural killer cells (25–27). The effects on antigen-presenting cells are thought to cause deviation in the immune response via pathways of indirect antigen presentation in favor of immune tolerance. Data from our present study confirm the ability of MSCs to suppress T cell proliferation in vitro for the rat system. This was particularly clear with the enhancement of T cell proliferation by the addition of IL-2. Although it cannot be ruled out that MSCs may have suppressed T cell proliferation nonspecifically via competition for soluble factors, this must be considered unlikely because significant suppression was not observed in the transwell system without IL-2.
Although it is controversial whether T cell suppression by MSCs needs cell-to-cell contact (10, 11), our data suggest that MSC suppression of T cell proliferation is contact-independent. Therefore, the suppression of T cell proliferation by MSCs in vitro is likely based on the secretion of a soluble factor, rather than on cell-to-cell contact. This hypothesis is consistent with the observation that T cell downregulation by MSCs does not require MHC matching. In summary, we were able to confirm that MSCs have a tolerogenic profile in vitro in the rat allogeneic system used in the present study.
However, in clear contrast to the promising in vitro capabilities of MSCs, injection of MSCs resulted in accelerated graft rejection in our transplant model in vivo. Neither the injection of 5×106 MSCs cells one time nor the injection of 2×106 MSCs at two peritransplantation time points prolonged graft survival. Moreover, when MSCs were applied together with low-dose delayed CsA, graft rejection was accelerated. Le Blanc et al. (28) reported that the immunosuppressive effects of MSCs and CsA did not interfere in vitro, suggesting that they could function together. However, in our study, the immunosuppressive effect of CsA was at least partially abrogated by MSCs, indicating a potential interaction between MSC and CsA activities. One possible explanation for the immunogenicity of MSCs may relate to the observation that they can upregulate MHC class I and possibly induce MHC class II molecule expression via an interferon-γ–dependent mechanism (12), leading to increased T cell alloreactivity. However, alloreactivity from induced MHC class II cannot provide a complete explanation, as graft rejection was also accelerated in those animals injected with recipient MSCs, making a nonspecific stimulation by MSCs more likely. Therefore, the mechanism for the allograft sensitization will require further investigation.
In conclusion, we have demonstrated that MSCs from adult rat bone marrow have clear immunomodulative effects in vitro, but graft survival was not prolonged when donor or syngenic MSCs were applied in a rat allogeneic heart transplantation model in vivo. Moreover, when MSCs were administered with delayed low-dose CsA, protective effects of CsA therapy on the allograft were reversed. Therefore, our results suggest that previously considered tolerogenic properties of MSCs need to be carefully investigated before these cells are used therapeutically in human solid organ transplantation.
The authors thank Irina Kucuk, Natascha Engelhardt, Anna Hoehn, and Erika Frank for their expert technical assistance.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
Cell-based immunosuppressive therapy; Cyclosporin A; Donor leukocytes; Mesenchymal stem cells; Rat heart transplantation