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- Materials and Methods
- Disclosure of Potential Conflicts of Interest
- Supporting Information
Author contributions: S.J.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; J.P.: financial support, data analysis and interpretation, manuscript writing, final approval of manuscript; M.M.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
Neural stem/progenitor cells (NSPCs) are potentially a promising treatment strategy for neurodegenerative disorders. There is a proof of concept from extensive animal studies, and a limited number of clinical trials, that intracerebral grafting of cells can have restorative effects [1, , –4]. NSPCs are self-renewing and have the potential to develop into neurons, astrocytes, and oligodendrocytes [5, 6]. Self-renewal facilitates the generation of sufficient cells for clinical use . Nevertheless, as with all transplantations, a major obstacle is the induction of a host-derived immune response followed by graft rejection.
Recognition of foreign major histocompatibility complex (MHC) antigens on grafted cells is thought to be a major determinant in the immunological rejection of neural grafts [8, , –11]. Notably, MHC-I is expressed by almost all cells, whereas MHC-II is generally expressed only by antigen-presenting cells . Recognition of foreign MHC-I antigens by cytotoxic T cells stimulates cell lysis, whereas the lack of MHC-I expression can induce cell lysis mediated by natural killer cells . MHC-II is detected by helper T cells that stimulate T-cell proliferation and antibody synthesis. Most immunosuppressive drugs that are used to ensure long-term graft survival are directed primarily against the MHC-mediated activation of T cells [14, –16].
Implantation into the brain exposes NSPCs to the presence of inflammatory cytokines that are involved both in immune response and the evolution of neurodegeneration. Inflammatory cytokines, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), are known to be potent immunomodulators . It has been reported that various types of stem cells express no or low levels of MHC, thus it appears that stem cells might be less immunogenic compared with other tissue/cells [18, , , –22]. Nevertheless, others have demonstrated that transplanted human stem cells can still induce an immune response resulting in graft rejection [23, 24].
In addition to an upregulation of MHC expression, cytokines can affect cell proliferation, differentiation, and migration and hence modulate the cell's repair potential [17, 25, –27]. High levels of cytokines are also detected in the fetal brain, suggesting a role of cytokines during development [28, , –31]. For instance, IFN-γ has been implicated in neural fate determination [32, –34]. Recent reports suggest that MHC expression may also play a role in synapse plasticity during development and after injury [35, , , –39]. Thus, it is possible that cytokines play a dual role as modulators of neural formation during development as well as inducing an immune response in a mature host environment following transplantation.
Animal models are commonly used to profile human NSPCs (hNSPCs) and their potential efficacy in treatment of neurodegenerative disorders. Immune recognition, as well as cellular functions, of xenotransplanted hNSPCs may be dependent on the local host cytokine environment. It is therefore necessary to characterize the immunogenicity and differentiation of hNSPC lines with clinical potential carefully. We here investigate the effect of IFN-γ, TNF-α, and IL-6 from human, monkey, and rat on MHC expression and differentiation in two conditionally immortalized hNSPC lines, one derived from striatum, the other from hippocampus.
- Top of page
- Materials and Methods
- Disclosure of Potential Conflicts of Interest
- Supporting Information
Transplantation of NSPCs is a potential new treatment for many neurodegenerative disorders. Inflammatory cytokines can upregulate MHC expression on transplanted cells, thereby rendering them more susceptible to graft rejection. Furthermore, cytokines also have a profound effect on cell differentiation, which can have a significant impact on the outcome of transplantation. Here, the effects of three inflammatory cytokines (IFN-γ, TNF-α, IL-6) from three different species (human, monkey, rat) on MHC expression and cell differentiation were investigated in two hNSPC lines generated from striatum and hippocampus. We find that h-IFN-γ and m-IFN-γ greatly upregulate MHC expression in both cell lines in a dose-dependent manner, whereas r-IFN-γ has an effect on MHC expression only in HPC cells. Both h-TNF-α and r-TNF-α also upregulate MHC expression in both lines, whereas IL-6 had no effect on MHC expression in either cell line. We further demonstrated that differentiation of the two hNSPC lines in the presence of cytokines affects cell fate determination. Differentiation in the presence of IFN-γ increased the neuronal yield in STR cultures. In contrast, addition of IFN-γ to HPC cultures increased oligodendrocyte numbers. Differentiation in the presence of TNF-α increased the number of astroglia, whereas IL-6 stimulated neurogenesis, in both cell lines. These findings have significant implications for the transplantation of hNSPCs to remedy neurodegenerative diseases.
Implications for Immune Response After hNSPC Transplantation
Stem cells have been reported to express no or low levels of MHC and be less immunogenic compared with primary fetal material [18, 20, 43]. Here we show that already at baseline conditions, a low percentage of HPC cells expressed MHC-II. Furthermore, addition of a low dose of h-IFN-γ (0.05 ng/ml) was enough to induce a significant upregulation of MHC antigens on both lines. This “low” dose is comparable to levels of IFN-γ found in sera and cerebrospinal fluid of patients with neuroinflammatory conditions, such as multiple sclerosis or meningitis [44, 45], but is likely to be many folds higher at inflammatory sites (e.g., activated microglia in areas of damage). This is of concern as already a low MHC expression level can be enough to cause allorecognition by incompatible lymphocytes .
The dose-dependent increase in MHC expression in response to human IFN-γ and TNF-α in both cell lines is in accordance with other studies [18, 22, 47, 48]. Although IL-6 did not affect MHC antigens on undifferentiated cells, upon differentiation, 30% of NSPCs stimulated with IL-6 expressed MHC-I. This response mirrored the increase in IL-6-R expression. Interestingly, r-IFN-γ had no effect initially on STR cells, but strongly induced MHC expression in differentiated cells. This highlights the importance of characterizing the immunogenicity of cells under undifferentiated and differentiated conditions prior to undertaking clinical studies.
We have previously demonstrated that the mouse NSPC line MHP36 decreases MHC expression during differentiation and that this might underlie the lack of an immune response in immunocompetent rats [49, 50]. However, the IFN-γ-induced increase in MHC expression in undifferentiated MHP36 cells is needed for the conditional immortalization in these cells and can explain why there is a dramatic reduction in MHC expression in differentiated cells. Nevertheless, differentiated MHP36 cells also did not respond significantly to IFN-γ. Here we show that 100% of differentiating cells in two hNSPC lines express MHC-I in response to IFN-γ. Neural progenitors have been reported to either upregulate MHC-I expression [10, 11, 20, 51] or to be unaffected  by IFN-γ upon differentiation. Most likely these discrepancies reflect differences in the inherent properties of the investigated cells.
MHC-I is normally expressed by all nucleated differentiated cells, whereas MHC-II is usually expressed only by antigen-presenting cells , even after stimulation with IFN-γ . However, we here found that upon differentiation in the presence of IFN-γ, almost all cells in both lines expressed MHC-II. The most parsimonious explanation for the observed expression is that many of the cells are still in a progenitor/precursor state, as evidenced by the majority of the cells still expressing nestin. Indeed, if the cells are stimulated with IFN-γ for only the first 24 hours of differentiation, there is no significant difference in MHC-II expression compared with controls. Hence, the MHC-II expression observed is transient and is down-regulated in the absence of IFN-γ.
Differentiation of hNSPCs in an Inflammatory Environment
MHC expression and cytokine responsiveness are known to be cell type and region specific [53, 54]. For instance, differentiation of cortical NSPCs has been shown to generate more glutamatergic, but less DARPP-32+, neurons, compared with striatal NSPCs . Also the distribution of cytokine-specific receptors is region specific, with hippocampus known to express higher levels of MHC and cytokine receptors compared with the striatum [17, 53, 56]. Hippocampal hNSPCs here expressed 2- to 3-fold higher levels of IFN-γ-R subunits compared with striatal hNSPCs. However, although the STR cells expressed considerably lower levels of IFN-γ-R compared with HPC cells, these cells appear to be as responsive to human and monkey IFN-γ as HPC cells. Thus, it is unlikely that the difference in MHC expression in HPC and STR cells in response to rat IFN-γ is reflecting solely the difference in receptor levels.
The IFN-γ-R is species specific with limited cross-reaction. Monkey and human IFN-γ has been shown to cross-react , whereas chick and mouse IFN-γ has been shown to have a low or no cross-reactivity with the human IFN-γ-R [58, –60]. Several studies have demonstrated that the extracellular binding sequence on IFN-γ-R-α is species specific, whereas the intracellular region is not [61, 62]. However, it is evident from our results that treatment with r-IFN-γ induces MHC expression on our NSPC lines.
IFN-γ binds to the membrane-bound receptor, and can subsequently activate several different signaling pathways, where the Jak/Stat pathway is the most characterized [63, 64]. IFN-γ, together with IFN-γ-R-α, can also be internalized and translocated to the nucleus [63, 65]. Thus, it is possible that the difference in MHC expression between the cell lines after stimulation with r-IFN-γ reflects differences in activation of signaling pathways and downstream targets.
Differentiation of hNSPCs in the presence of cytokines also influenced neurogenesis or gliogenesis. For instance, IFN-γ increased the neuronal yield threefold in STR cultures and increased the number of oligodendrocytes twofold in HPC cultures. That the same cytokine can be either neurogenic or gliogenic can again partly be explained by region-specific differences. IFN-γ has been reported either to stimulate neurogenesis in neural precursor cells generated from spinal cord  and the subventricular zone , or to induce gliogenesis in hippocampal precursor cells [66, 67]. IL-6 increased neuronal differentiation in both cell lines and is known to stimulate neurogenesis and to promote neurite outgrowth in neural progenitor cells . Despite reports that IL-6 can stimulate astrocytic differentiation, no effect on gliogenesis was observed [69, 70]. Our finding that TNF-α increased the number of astroglia and reduced neurogenesis irrespective of cell line is in accordance with other studies [71, 72]. Interestingly, even a brief exposure of cytokines for merely 24 hours was sufficient to affect cell fate determination.
Intriguingly, treatment with rat cytokines had no or little effect on neural differentiation in both NSPC lines despite having an effect on MHC expression. It is possible that this reflects the low cytokine cross-reactivity between species reported by others [57, –59]. Another possibility is that although the level of receptor expression appears to not affect MHC expression, it could still influence differentiation. For example, NSPCs with high expression of FGF receptors differentiate into higher number of neurons when stimulated with FGF-2 compared with NSPCs with lower levels of FGF receptor expression .
A Cautionary Note on Animal Testing
Although STR and HPC cells responded in a comparable fashion to human and monkey cytokines, only HPC cells strongly upregulated MHC expression in response to r-IFN-γ. Moreover, rat cytokines had little effect on differentiation patterns in both hNSPC lines. If cytokines are required to upregulate MHC expression, the lack of responsiveness to rat cytokines would not allow a thorough assessment of the immunological properties of STR cells in rat models of neurodegeneration. However, once transplanted into humans, these cells would respond to the inflammatory cytokines in areas of damage and elicit an immune response.
Not only do these findings have implications for the evaluation of the immunological response, but the lack of effect of rat cytokines on cell differentiation also has implications for their potential mechanism of action. If differentiation of NSPCs is increased in the presence of cytokines, transplanted cells will differentiate more in areas of damage compared with “intact” areas. Previous in vivo studies have already provided evidence that transplantation into the intact brain results in a poor survival, differentiation, and migration [50, 74, 75]. Extrapolation of findings using hNSPCs in animal models to the human situation therefore requires caution. The use of monkey models prior to implementation into humans is likely to be an indispensable validation of stem cell therapy.
Today the most common strategy to avoid graft rejection is treatment with immunosuppressive drugs. However, long-term immunosuppressive treatment has severe side effects. Immunosuppressive treatment in animal models with stem cell transplants has a limited effect on cell survival and could potentially even be counterproductive with respect to functional recovery [24, 50, 74, , –77]. Whether immunosuppressive treatment, or other strategies such as knocking down MHC expression on stem cells prior to transplantation, is necessary, or even desirable, needs to be studied further prior to using stem cell transplantation as a clinical approach.