Debbie Le Blon

Quantitative and phenotypic analysis of mesenchymal stromal cell graft survival and recognition by microglia and astrocytes in mouse brain

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.

Spatiotemporal evolution of early innate immune responses triggered by neural stem cell grafting

IntroductionTransplantation 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.MethodsHere, 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.ResultsAn 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.ConclusionsThe 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.

Distinct spatial distribution of microglia and macrophages following mesenchymal stem cell implantation in mouse brain

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.

Tackling the physiological barriers for successful mesenchymal stem cell transplantation into the central nervous system

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.

Interleukin-13 immune gene therapy prevents CNS inflammation and demyelination via alternative activation of microglia and macrophages

Detrimental inflammatory responses in the central nervous system are a hallmark of various brain injuries and diseases. With this study we provide evidence that lentiviral vector‐mediated expression of the immune‐modulating cytokine interleukin 13 (IL‐13) induces an alternative activation program in both microglia and macrophages conferring protection against severe oligodendrocyte loss and demyelination in the cuprizone mouse model for multiple sclerosis (MS). First, IL‐13 mediated modulation of cuprizone induced lesions was monitored using T2‐weighted magnetic resonance imaging and magnetization transfer imaging, and further correlated with quantitative histological analyses for inflammatory cell influx, oligodendrocyte death, and demyelination. Second, following IL‐13 immune gene therapy in cuprizone‐treated eGFP+ bone marrow chimeric mice, we provide evidence that IL‐13 directs the polarization of both brain‐resident microglia and infiltrating macrophages towards an alternatively activated phenotype, thereby promoting the conversion of a pro‐inflammatory environment toward an anti‐inflammatory environment, as further evidenced by gene expression analyses. Finally, we show that IL‐13 immune gene therapy is also able to limit lesion severity in a pre‐existing inflammatory environment. In conclusion, these results highlight the potential of IL‐13 to modulate microglia/macrophage responses and to improve disease outcome in a mouse model for MS. GLIA 2016;64:2181–2200

Multimodal imaging of stem cell implantation in the central nervous system of mice.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

Cell-Based Delivery of Interleukin-13 Directs Alternative Activation of Macrophages Resulting in Improved Functional Outcome after Spinal Cord Injury

Summary The therapeutic effects of mesenchymal stem cell (MSC) transplantation following spinal cord injury (SCI) to date have been limited. Therefore, we aimed to enhance the immunomodulatory properties of MSCs via continuous secretion of the anti-inflammatory cytokine interleukin-13 (IL-13). By using MSCs as carriers of IL-13 (MSC/IL-13), we investigated their therapeutic potential, compared with non-engineered MSCs, in a mouse model of SCI. We show that transplanted MSC/IL-13 significantly improve functional recovery following SCI, and also decrease lesion size and demyelinated area by more than 40%. Further histological analyses in CX3CR1EGFP/+ CCR2RFP/+ transgenic mice indicated that MSC/IL-13 significantly decrease the number of resident microglia and increase the number of alternatively activated macrophages. In addition, the number of macrophage-axon contacts in MSC/IL-13-treated mice was decreased by 50%, suggesting a reduction in axonal dieback. Our data provide evidence that transplantation of MSC/IL-13 leads to improved functional and histopathological recovery in a mouse model of SCI.

In Vivo Interleukin-13-Primed Macrophages Contribute to Reduced Alloantigen-Specific T Cell Activation and Prolong Immunological Survival of Allogeneic Mesenchymal Stem Cell Implants

Transplantation of mesenchymal stem cells (MSCs) into injured or diseased tissue—for the in situ delivery of a wide variety of MSC‐secreted therapeutic proteins—is an emerging approach for the modulation of the clinical course of several diseases and traumata. From an emergency point‐of‐view, allogeneic MSCs have numerous advantages over patient‐specific autologous MSCs since “off‐the‐shelf” cell preparations could be readily available for instant therapeutic intervention following acute injury. Although we confirmed the in vitro immunomodulatory capacity of allogeneic MSCs on antigen‐presenting cells with standard coculture experiments, allogeneic MSC grafts were irrevocably rejected by the hosts immune system upon either intramuscular or intracerebral transplantation. In an attempt to modulate MSC allograft rejection in vivo, we transduced MSCs with an interleukin‐13 (IL13)‐expressing lentiviral vector. Our data clearly indicate that prolonged survival of IL13‐expressing allogeneic MSC grafts in muscle tissue coincided with the induction of an alternatively activated macrophage phenotype in vivo and a reduced number of alloantigen‐reactive IFNγ‐ and/or IL2‐producing CD8+ T cells compared to nonmodified allografts. Similarly, intracerebral IL13‐expressing MSC allografts also exhibited prolonged survival and induction of an alternatively activated macrophage phenotype, although a peripheral T cell component was absent. In summary, this study demonstrates that both innate and adaptive immune responses are effectively modulated in vivo by locally secreted IL13, ultimately resulting in prolonged MSC allograft survival in both muscle and brain tissue. Stem Cells 2016;34:1971–1984

Intracerebral transplantation of interleukin 13-producing mesenchymal stem cells limits microgliosis, oligodendrocyte loss and demyelination in the cuprizone mouse model

BackgroundPromoting 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).MethodsIn 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 graft-infiltrating 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.ResultsIn 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.ConclusionsControlled 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.

Early Inflammatory Responses Following Cell Grafting in the CNS Trigger Activation of the Subventricular Zone: A Proposed Model of Sequential Cellular Events

While multiple rodent preclinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety, and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP mouse embryonic fibroblasts (mEFs) in the CNS of immunocompetent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neoangiogenesis, microglia/macrophage recruitment, astrogliosis, and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the subventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses and will eventually allow for successful manipulation of this intervention.