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Background: Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. Results: First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSCNT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. Conclusion: In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo. [ABSTRACT FROM AUTHOR]

Transplantation of neural stem cells (NSC) in diseased or injured brain tissue is widely studied as a potential treatment for various neurological pathologies. However, effective cell replacement therapy relies on the intrinsic capacity of cellular grafts to overcome hypoxic and/or immunological barriers after transplantation. In this context, it is hypothesized that structural support for grafted NSC will be of utmost importance. With this study, we present a novel decellularization protocol for 1.5 mm thick mouse brain sections, resulting in the generation of acellular three-dimensional (3D) brain sections. Next, the obtained 3D brain sections were seeded with murine NSC expressing both the eGFP and luciferase reporter proteins (NSC-eGFP/Luc). Using real-time bioluminescence imaging, the survival and growth of seeded NSC-eGFP/Luc cells was longitudinally monitored for 1–7 weeks in culture, indicating the ability of the acellular brain sections to support sustained ex vivo growth of NSC. Next, the organization of a 3D maze-like cellular structure was examined using confocal microscopy. Moreover, under mitogenic stimuli (EGF and hFGF-2), most cells in this 3D culture retained their NSC phenotype. Concluding, we here present a novel protocol for decellularization of mouse brain sections, which subsequently support long-term 3D culture of undifferentiated NSC. [ABSTRACT FROM AUTHOR]

Although implantation of cellular material in the central nervous system (CNS) is a key direction in CNS regenerative medicine, this approach is currently limited by the occurrence of strong endogenous immune cell responses. In a model of mesenchymal stem cell (MSC) grafting in the CNS of immune-competent mice, we previously described that MSC grafts become highly surrounded and invaded by Iba1+ myeloid cells (microglia and/or macrophages). Here, following grafting of blue fluorescent protein (BFP)-expressing MSC in the CNS of CX3CR1+/− and CX3CR1−/− mice, our results indicate: (1) that the observed inflammatory response is independent of the fractalkine signalling axis, and (2) that a significant spatial distribution of Iba1+ inflammatory cells occurs, in which Iba1+ CX3CR1+ myeloid cells mainly surround the MSC graft and Iba1+ CX3CR1− myeloid cells mainly invade the graft at 10 days post transplantation. Although Iba1+ CX3CR1+ myeloid cells are considered to be of resident microglial origin, Iba1+ CX3CR1− myeloid cells are most likely of peripheral monocyte/macrophage origin. In order to confirm the latter, we performed MSC-BFP grafting experiments in the CNS of eGFP+ bone marrow chimeric C57BL/6 mice. Analysis of MSC-BFP grafts in the CNS of these mice confirmed our observation that peripheral monocytes/macrophages invade the MSC graft and that resident microglia surround the MSC graft site. Furthermore, analysis of major histocompatibility complex class II (MHCII) expression revealed that mainly macrophages, but not microglia, express this M1 pro-inflammatory marker in the context of MSC grafting in the CNS. These results again highlight the complexity of cell implantation immunology in the CNS. [ABSTRACT FROM AUTHOR]

Transplantation of neural stem cells (NSC) is hoped to become a promising primary or secondary therapy for the treatment of various neurodegenerative disorders of the central nervous system (CNS), as demonstrated by multiple pre-clinical animal studies in which functional recovery has already been demonstrated. However, for NSC therapy to be successful, the first challenge will be to define a transplantable cell population. In the first part of this review, we will briefly discuss the main features of ex vivo culture and characterisation of NSC. Next, NSC grafting itself may not only result in the regeneration of lost tissue, but more importantly has the potential to improve functional outcome through many bystander mechanisms. In the second part of this review, we will briefly discuss several pre-clinical studies that contributed to a better understanding of the therapeutic potential of NSC grafts in vivo. However, while many pre-clinical animal studies mainly report on the clinical benefit of NSC grafting, little is known about the actual in vivo fate of grafted NSC. Therefore, the third part of this review will focus on non-invasive imaging techniques for monitoring cellular grafts in the brain under in vivo conditions. Finally, as NSC transplantation research has evolved during the past decade, it has become clear that the host micro-environment itself, either in healthy or injured condition, is an important player in defining success of NSC grafting. The final part of this review will focus on the host environmental influence on survival, migration and differentiation of grafted NSC. [ABSTRACT FROM AUTHOR]

• Ovariectomy causes metabolic alterations in the hypothalamus in Tg2576 mice. • Ovariectomy alters cortical and sub-cortical resting state functional connectivity in Tg2576 and WT mice. • Ovariectomy alters white matter integrity in TG2576 mice. • The level of GFAP immunoreactivity and amyloid beta was not affected by ovariectomy. Premenopausal bilateral ovariectomy is considered to be one of the risk factors of Alzheimer's disease (AD). However, the underlying mechanisms remain unclear. Here, we aimed to investigate long-term neurological consequences of ovariectomy in a rodent AD model, TG2576 (TG), and wild-type mice (WT) that underwent an ovariectomy or sham-operation, using in vivo MRI biomarkers. An increase in osmoregulation and energy metabolism biomarkers in the hypothalamus, a decrease in white matter integrity, and a decrease in the resting-state functional connectivity was observed in ovariectomized TG mice compared to sham-operated TG mice. In addition, we observed an increase in functional connectivity in ovariectomized WT mice compared to sham-operated WT mice. Furthermore, genotype (TG vs. WT) effects on imaging markers and GFAP immunoreactivity levels were observed, but there was no effect of interaction (Genotype × Surgery) on amyloid-beta-and GFAP immunoreactivity levels. Taken together, our results indicated that both genotype and ovariectomy alters imaging biomarkers associated with AD. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common form of dementia in the elderly. According to the amyloid hypothesis, the accumulation and deposition of amyloid-beta (Aβ) peptides play a key role in AD. Soluble Aβ (sAβ) oligomers were shown to be involved in pathological hypersynchronisation of brain resting-state networks in different transgenic developmental-onset mouse models of amyloidosis. However, the impact of protein overexpression during brain postnatal development may cause additional phenotypes unrelated to AD. To address this concern, we investigated sAβ effects on functional resting-state networks in transgenic mature-onset amyloidosis Tet-Off APP (TG) mice. TG mice and control littermates were raised on doxycycline (DOX) diet from 3d up to 3 m of age to suppress transgenic Aβ production. Thereafter, longitudinal resting-state functional MRI was performed on a 9.4 T MR-system starting from week 0 (3 m old mice) up to 28w post DOX treatment. Ex-vivo immunohistochemistry and ELISA analysis was performed to assess the development of amyloid pathology. Functional Connectivity (FC) analysis demonstrated early abnormal hypersynchronisation in the TG mice compared to the controls at 8w post DOX treatment, particularly across regions of the default mode-like network, known to be affected in AD. Ex-vivo analyses performed at this time point confirmed a 20-fold increase in total sAβ levels preceding the apparition of Aβ plaques and inflammatory responses in the TG mice compared to the controls. On the contrary at week 28, TG mice showed an overall hypoconnectivity, coinciding with a widespread deposition of Aβ plaques in the brain. By preventing developmental influence of APP and/or sAβ during brain postnatal development, we demonstrated FC abnormalities potentially driven by sAβ neurotoxicity on resting-state neuronal networks in mature-induced TG mice. Thus, the Tet-Off APP mouse model could be a powerful tool while used as a mature-onset model to shed light into amyloidosis mechanisms in AD. [ABSTRACT FROM AUTHOR]

The establishment and validation of reliable induced pluripotent stem cell (iPSC)-derived in vitro models to study microglia and monocyte/macrophage immune function holds great potential for fundamental and translational neuro-immunology research. In this study, we first demonstrate that ramified CX 3 CR1+ iPSC-microglia (cultured within a neural environment) and round-shaped CX 3 CR1− iPSC-macrophages can easily be differentiated from newly established murine CX 3 CR1eGFP/+CCR2RFP/+ iPSC lines. Furthermore, we show that obtained murine iPSC-microglia and iPSC-macrophages are distinct cell populations, even though iPSC-macrophages may upregulate CX 3 CR1 expression when cultured within a neural environment. Next, we characterized the phenotypical and functional properties of murine iPSC-microglia and iPSC-macrophages following classical and alternative immune polarisation. While iPSC-macrophages could easily be triggered to adopt a classically-activated or alternatively-activated phenotype following, respectively, lipopolysaccharide + interferon γ or interleukin 13 (IL13) stimulation, iPSC-microglia and iPSC-macrophages cultured within a neural environment displayed a more moderate activation profile as characterised by the absence of MHCII expression upon classical immune polarisation and the absence of Ym1 expression upon alternative immune polarisation. Finally, extending our preceding in vivo studies, this striking phenotypical divergence was also observed for resident microglia and infiltrating monocytes within highly inflammatory cortical lesions in CX 3 CR1eGFP/+CCR2RFP/+ mice subjected to middle cerebral arterial occlusion (MCAO) stroke and following IL13-mediated therapeutic intervention thereon. In conclusion, our study demonstrates that the applied murine iPSC-microglia and iPSC-macrophage culture models are able to recapitulate in vivo microglia and monocyte/macrophage ontogeny and corresponding phenotypical/functional properties upon classical and alternative immune polarisation, and therefore represent a valuable in vitro platform to further study and modulate microglia and (infiltrating) monocyte immune responses under neuro-inflammatory conditions within a neural environment. [ABSTRACT FROM AUTHOR]

Background: Promoting the neuroprotective and repair-inducing effector functions of microglia and macrophages, by means of M2 polarisation or alternative activation, is expected to become a new therapeutic approach for central nervous system (CNS) disorders in which detrimental pro-inflammatory microglia and/or macrophages display a major contribution to the neuropathology. In this study, we present a novel in vivo approach using intracerebral grafting of mesenchymal stem cells (MSC) genetically engineered to secrete interleukin 13 (IL13-MSC). Methods: In the first experimental setup, control MSC and IL13-MSC were grafted in the CNS of eGFP+ bone marrow chimaeric C57BL/6 mice to histologically evaluate IL13-mediated expression of several markers associated with alternative activation, including arginase1 and Ym1, on MSC graft-recognising microglia and MSC graftinfiltrating macrophages. In the second experimental setup, IL13-MSC were grafted on the right side (or on both the right and left sides) of the splenium of the corpus callosum in wild-type C57BL/6 mice and in C57BL/6 CX3CR1eGFP/+CCR2RFP/+ transgenic mice. Next, CNS inflammation and demyelination was induced by means of a cuprizone-supplemented diet. The influence of IL13-MSC grafting on neuropathological alterations was monitored by non-invasive T2-weighted magnetic resonance imaging (MRI) and quantitative histological analyses, as compared to cuprizone-treated mice with control MSC grafts and/or cuprizone-treated mice without MSC injection. Results: In the first part of this study, we demonstrate that MSC graft-associated microglia and MSC graft-infiltrating macrophages are forced into alternative activation upon grafting of IL13-MSC, but not upon grafting of control MSC. In the second part of this study, we demonstrate that grafting of IL13-MSC, in addition to the recruitment of M2 polarised macrophages, limits cuprizone-induced microgliosis, oligodendrocyte death and demyelination. Furthermore, we here demonstrate that injection of IL13-MSC at both sides of the splenium leads to a superior protective effect as compared to a single injection at one side of the splenium. Conclusions: Controlled and localised production of IL13 by means of intracerebral MSC grafting has the potential to modulate cell graft- and pathology-associated microglial/macrophage responses, and to interfere with oligodendrocyte death and demyelinating events in the cuprizone mouse model. [ABSTRACT FROM AUTHOR]

Over the past decade a lot of research has been performed towards the therapeutic use of mesenchymal stem cells (MSCs) in neurodegenerative and neuroinflammatory diseases. MSCs have shown to be beneficial in different preclinical studies of central nervous system (CNS) disorders due to their immunomodulatory properties and their capacity to secrete various growth factors. Nevertheless, most of the transplanted cells die within the first hours after transplantation and induce a neuroinflammatory response. In order to increase the efficacy of MSC transplantation, it is thus imperative to completely characterise the mechanisms mediating neuroinflammation and cell death following MSC transplantation into the CNS. Consequently, different components of these cell death- and neuroinflammation-inducing pathways can be targeted in an attempt to improve the therapeutic potential of MSCs for CNS disorders [ABSTRACT FROM AUTHOR]

Abstract: Although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (CNS) transplantation immunology. In this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (MSC) in the CNS of immune competent mice. First, the survival of grafted Luciferase/eGFP-expressing MSC (MSC-Luc/eGFP) was demonstrated to be stable from on day 3 post implantation using in vivo bioluminescence imaging (BLI), which was further confirmed by quantitative histological analysis of MSC-Luc/eGFP graft survival. Additional histological analyses at week 1 and week 2 post grafting revealed the appearance of (i) graft-surrounding/-invading Iba1+ microglia and (ii) graft-surrounding GFAP+ astrocytes, as compared to day 0 post grafting. While the density of graft-surrounding astrocytes and microglia did not change between week 1 and week 2 post grafting, the density of graft-invading microglia significantly decreased between week 1 and week 2 post implantation. However, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graft-invading microglia, based on CD11b- and MHCII-expression, revealed >50% of graft-invading microglia at week 2 post implantation to display an activated status. Although microglial expression of CD11b and MHCII is already suggestive for a pro-inflammatory M1-oriented phenotype, the latter was further confirmed by: (i) the expression of NOS2 by microglia within the graft site, and (ii) the absence of arginase 1-expression, an enzyme known to suppress NO activity in M2-oriented microglia, on graft-surrounding and -invading microglia. In summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the CNS of mice, and warrant that such intervention is associated with an M1-oriented microglia response and severe astrogliosis. [Copyright &y& Elsevier]

Introduction: Transplantation of neural stem cells (NSCs) is increasingly suggested to become part of future therapeutic approaches to improve functional outcome of various central nervous system disorders. However, recently it has become clear that only a small fraction of grafted NSCs display long-term survival in the (injured) adult mouse brain. Given the clinical invasiveness of NSC grafting into brain tissue, profound characterisation and understanding of early post-transplantation events is imperative to claim safety and efficacy of cell-based interventions. Methods: Here, we applied in vivo bioluminescence imaging (BLI) and post-mortem quantitative histological analysis to determine the localisation and survival of grafted NSCs at early time points post-transplantation. Results: An initial dramatic cell loss (up to 80% of grafted cells) due to apoptosis could be observed within the first 24 hours post-implantation, coinciding with a highly hypoxic NSC graft environment. Subsequently, strong spatiotemporal microglial and astroglial cell responses were initiated, which stabilised by day 5 post-implantation and remained present during the whole observation period. Moreover, the increase in astrocyte density was associated with a high degree of astroglial scarring within and surrounding the graft site. During the two-week follow up in this study, the NSC graft site underwent extensive remodelling with NSC graft survival further declining to around 1% of the initial number of grafted cells. Conclusions: The present study quantitatively describes the early post-transplantation events following NSC grafting in the adult mouse brain and warrants that such intervention is directly associated with a high degree of cell loss, subsequently followed by strong glial cell responses. [ABSTRACT FROM AUTHOR]

Purpose: Development of multimodal imaging strategies is currently of utmost importance for the validation of preclinical stem cell therapy studies.Procedures: We performed a combined labeling strategy for bone marrow-derived stromal cells (BMSC) based on genetic modification with the reporter genes Luciferase and eGFP (BMSC-Luc/eGFP) and physical labeling with blue fluorescent micron-sized iron oxide particles (MPIO) in order to unambiguously identify BMSC localization, survival, and differentiation following engraftment in the central nervous system of mice by in vivo bioluminescence (BLI) and magnetic resonance imaging and postmortem histological analysis.Results: Using this combination, a significant increase of in vivo BLI signal was observed for MPIO-labeled BMSC-Luc/eGFP. Moreover, MPIO labeling of BMSC-Luc/eGFP allows for the improved identification of implanted cells within host tissue during histological observation.Conclusions: This study describes an optimized labeling strategy for multimodal stem cell imaging resulting in improved quantitative and qualitative detection of cellular grafts. [ABSTRACT FROM AUTHOR]

Currently, much attention is given to the development of cellular therapies for treatment of central nervous system (CNS) injuries. Diverse cell implantation strategies, either to directly replace damaged neural tissue or to create a neuroregenerative environment, are proposed to restore impaired brain function. However, because of the complexity of the CNS, it is now becoming clear that the contribution of cell implantation into the brain will mainly act in a supportive manner. In addition, given the time dependence of neural development during embryonic and post-natal life, cellular implants, either self or non-self, will most likely have to interact for a sustained period of time with both healthy and injured neural tissue. The latter also implies potential recognition of cellular implants by the innate immune system of the brain. In this review, we will emphasize on preclinical observations in rodents, regarding the recognition and immunogenicity of autologous, allogeneic and xenogeneic cellular implants in the CNS of immune-competent hosts. Taken together, we here suggest that a profound study of the interaction between cellular grafts and the brain's innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully. [ABSTRACT FROM AUTHOR]

Abstract: SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells. [ABSTRACT FROM AUTHOR]

Although adult and embryonic stem cell-based therapy for central nervous system (CNS) injury is being developed worldwide, less attention is given to the immunological aspects of allogeneic cell implantation in the CNS. The latter is of major importance because, from a practical point of view, future stem cell-based therapy for CNS injury will likely be performed using well-characterised allogeneic stem cell populations. In this study, we aimed to further describe the immunological mechanism leading to rejection of allogeneic bone marrow-derived stromal cells (BM-SC) after implantation in murine CNS. For this, we first investigated the impact of autologous and allogeneic BM-SC on microglia activation in vitro. Although the results indicate that both autologous and allogeneic BM-SC do not activate microglia themselves in vitro, they also do not inhibit activation of microglia after exogenous stimuli in vitro. Next, we investigated the impact of allogeneic BM-SC on microglia activation in vivo. In contrast to the in vitro observations, microglia become highly activated in vivo after implantation of allogeneic BM-SC in the CNS of immune-competent mice. Moreover, our results suggest that microglia, rather than T-cells, are the major contributors to allograft rejection in the CNS.Immunology and Cell Biology (2009) 87, 267–273; doi:10.1038/icb.2009.12; published online 17 March 2009 [ABSTRACT FROM AUTHOR]

Background: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes.Methods: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load.Results: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score.Conclusions: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS. [ABSTRACT FROM AUTHOR]

Background: Subtle adjustment of the activation status of CNS resident microglia and peripheral macrophages, to promote their neuroprotective and neuroregenerative functions, may facilitate research towards curing neurodegenerative disorders. In the present study, we investigated whether targeted intracerebral delivery of the anti-inflammatory cytokine interleukin (IL)13, by means of transplanting IL13-expressing mesenchymal stem cells (IL13-MSCs), can promote a phenotypic switch in both microglia and macrophages during the pro-inflammatory phase in a mouse model of ischemic stroke.Methods: We used the CX3CR1eGFP/+ CCR2RFP/+ transgenic mouse model to separately recognize brain-resident microglia from infiltrated macrophages. Quantitative immunohistochemical analyses were applied to characterize polarization phenotypes of both cell types.Results: Distinct behaviors of both cell populations were noted dependent on the anatomical site of the lesion. Immunohistochemistry revealed that mice grafted with IL13-MSCs, in contrast to non-grafted and MSC-grafted control mice, were able to drive recruited microglia and macrophages into an alternative activation state, as visualized by a significant increase of Arg-1 and a noticeable decrease of MHC-II expression at day 14 after ischemic stroke. Interestingly, both Arg-1 and MHC-II were expressed more abundantly in macrophages than in microglia, further confirming the distinct behavior of both cell populations.Conclusions: The current data highlight the importance of controlled and localized delivery of the anti-inflammatory cytokine IL13 for modulation of both microglia and macrophage responses after ischemic stroke, thereby providing pre-clinical rationale for the application of L13-MSCs in future investigations of neurodegenerative disorders. [ABSTRACT FROM AUTHOR]