Irene Bergwerf

Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice

BackgroundCell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.ResultsIn this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.ConclusionWe here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.

Allogeneic stromal cell implantation in brain tissue leads to robust microglial activation

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.

Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice

While neural stem cells (NSCs) are widely expected to become a therapeutic agent for treatment of severe injuries to the central nervous system (CNS), currently there are only few detailed preclinical studies linking cell fate with experimental outcome. In this study, we aimed to validate whether IV administration of allogeneic NSC can improve experimental autoimmune encephalomyelitis (EAE), a well-established animal model for human multiple sclerosis (MS). For this, we cultured adherently growing luciferase-expressing NSCs (NSC-Luc), which displayed a uniform morphology and expression profile of membrane and intracellular markers, and which displayed an in vitro differentiation potential into neurons and astrocytes. Following labeling with green fluorescent micron-sized iron oxide particles (f-MPIO-labeled NSC-Luc) or lentiviral transduction with the enhanced green fluorescent protein (eGFP) reporter gene (NSC-Luc/eGFP), cell implantation experiments demonstrated the intrinsic survival capacity of adherently cultured NSC in the CNS of syngeneic mice, as analyzed by real-time bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and histological analysis. Next, EAE was induced in C57BL/6 mice followed by IV administration of NSC-Luc/eGFP at day 7 postinduction with or without daily immunosuppressive therapy (cyclosporine A, CsA). During a follow-up period of 20 days, the observed clinical benefit could be attributed solely to CsA treatment. In addition, histological analysis demonstrated the absence of NSC-Luc/eGFP at sites of neuroinflammation. In order to investigate the absence of therapeutic potential, BLI biodistribution analysis of IV-administered NSC-Luc/eGFP revealed cell retention in lung capillaries as soon as 1-min postinjection, resulting in massive inflammation and apoptosis in lung tissue. In summary, we conclude that IV administration of NSCs currently has limited or no therapeutic potential for neuroinflammatory disease in mice, and presumably also for human MS. However, given the fact that grafted NSCs have an intrinsic survival capacity in the CNS, their therapeutic exploitation should be further investigated, and—in contrast to several other reports—will most likely be highly complex.

Cell type-associated differences in migration, survival, and immunogenicity following grafting in CNS tissue.

Cell transplantation has been suggested to display several neuroprotective and/or neuroregenerative effects in animal models of central nervous system (CNS) trauma. However, while most studies report on clinical observations, currently little is known regarding the actual fate of the cell populations grafted and whether or how the brains innate immune system, mainly directed by activated microglia and astrocytes, interacts with autologous cellular implants. In this study, we grafted well-characterized neural stem cell, mouse embryonic fibroblast, dendritic cell, bone marrow mononuclear cell, and splenocyte populations, all isolated or cultured from C57BL/6-eGFP transgenic mice, below the capsula externa (CE) of healthy C57BL/6 mice and below the inflamed/demyelinated CE of cuprizone-treated C57BL/6 mice. Two weeks postgrafting, an extensive quantitative multicolor histological analysis was performed in order (i) to quantify cell graft localization, migration, survival, and toxicity and (ii) to characterize endogenous CNS immune responses against the different cell grafts. Obtained results indicate dependence on the cell type grafted: (i) a different degree of cell graft migration, survival, and toxicity and (ii) a different organization of the endogenous immune response. Based on these observations, we warrant that further research should be undertaken to understand—and eventually control—cell graft-induced tissue damage and activation of the brains innate immune system. The latter will be inevitable before cell grafting in the CNS can be performed safely and successfully in clinical settings.

Recognition of cellular implants by the brain's innate immune system.

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 brains innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully.

Identification and characterization of Huntington related pathology: an in vivo DKI imaging study.

An important focus of Huntington Disease (HD) research is the identification of symptom-independent biomarkers of HD neuropathology. There is an urgent need for reproducible, sensitive and specific outcome measures, which can be used to track disease onset as well as progression. Neuroimaging studies, in particular diffusion-based MRI methods, are powerful probes for characterizing the effects of disease and aging on tissue microstructure. We report novel diffusional kurtosis imaging (DKI) findings in aged transgenic HD rats. We demonstrate altered diffusion metrics in the (pre)frontal cerebral cortex, external capsule and striatum. Presence of increased diffusion complexity and restriction in the striatum is confirmed by an increased fiber dispersion in this region. Immunostaining of the same specimens reveals decreased number of microglia in the (pre)frontal cortex, and increased numbers of oligodendrocytes in the striatum. We conclude that DKI allows sensitive and specific characterization of altered tissue integrity in this HD rat model, indicating a promising potential for diagnostic imaging of gray and white matter pathology.

Multimodal in vivo imaging reveals limited allograft survival, intrapulmonary cell trapping and minimal evidence for ischemia-directed BMSC homing.

BackgroundDespite positive reports on the efficacy of stem cell therapy for the treatment of cardiovascular disease, the nature of stem cell homing to ischemic tissues remains elusive.ResultsWe used a mouse model of peripheral tissue ischemia to study the survival and homing capacity of dual reporter gene (eGFP/Luciferase) expressing bone marrow-derived stromal cells (BMSC). Cell homing and survival were studied in the presence and absence of ciclosporin A (CsA) immunosuppression using bioluminescence imaging (BLI) together with confocal endomicroscopy. Different injection strategies were applied: central venous (CV), intra-arterial (IA) and intramuscular (IM). BLI and confocal endomicroscopy evidenced complete rejection of the IM injected allogeneic BMSC transplant within 5 to 10 days. Immunosuppression with CsA could only marginally prolong graft survival. IM injected BMSC did not migrate to the site of the arterial ligation. CV injection of BMSC resulted in massive pulmonary infarction, leading to respiratory failure and death. Intrapulmonary cell trapping was evidenced by confocal endomicroscopy, BLI and fluorescence microscopy. IA injection of BMSC proved to be a feasible and safe strategy to bypass the lung circulation. During the follow-up period, neither BLI nor confocal endomicroscopy revealed any convincing ischemia-directed homing of BMSC.ConclusionsBLI and confocal endomicroscopy are complementary imaging techniques for studying the in vivo biology of dual reporter gene-expressing BMSC. Allogeneic BMSC survival is limited in an immunocompetent host and cannot be preserved by CsA immunosuppression alone. We did not find substantial evidence for ischemia-directed BMSC homing and caution against CV injection of BMSC, which can lead to massive pulmonary infarction.

Labeling of Luciferase/eGFP-Expressing Bone Marrow-Derived Stromal Cells with Fluorescent Micron-Sized Iron Oxide Particles Improves Quantitative and Qualitative Multimodal Imaging of Cellular Grafts In Vivo

PurposeDevelopment of multimodal imaging strategies is currently of utmost importance for the validation of preclinical stem cell therapy studies.ProceduresWe 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.ResultsUsing 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.ConclusionsThis study describes an optimized labeling strategy for multimodal stem cell imaging resulting in improved quantitative and qualitative detection of cellular grafts.

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).

Distinct in vitro properties of embryonic and extraembryonic fibroblast-like cells are reflected in their in vivo behavior following grafting in the adult mouse brain.

Although intracerebral transplantation of various fibroblast(-like) cell populations has been shown feasible, little is known about the actual in vivo remodeling of these cellular grafts and their environment. In this study, we aimed to compare the in vitro and in vivo behavior of two phenotypically similar—but developmentally distinct—fibroblast-like cell populations, namely, mouse embryonic fibroblasts (mEFs) and mouse fetal membrane-derived stromal cells (mFMSCs). While both mEFs and mFMSCs are readily able to reduce TNF-α secretion by LPS/IFN-γ-activated BV-2 microglia, mFMSCs and mEFs display strikingly opposite behavior with regard to VEGF production under normal and inflammatory conditions. Whereas mFMSCs downregulate VEGF production upon coculture with LPS/IFN-γ-activated BV-2 microglia, mEFs upregulate VEGF production in the presence of LPS/IFN-γ-activated BV-2 microglia. Subsequently, in vivo grafting of mFMSCs and mEFs revealed no difference in microglial and astroglial responses toward the cellular grafts. However, mFMSC grafts displayed a lower degree of neoangiogenesis compared to mEF grafts, thereby potentially explaining the lower cell number able to survive in mFMSC grafts. In summary, our results suggest that physiological differences between fibroblast-like cell populations might lie at the basis of variations in histopathological and/or clinical outcome following cell grafting in mouse brain.