• Intra-arterial transplantation of stem cells in large animals as a minimally-invasive strategy for the treatment of disseminated neurodegeneration

      Malysz-Cymborska, I.; Golubczyk, D.; Kalkowski, L.; Kwiatkowska, J.; Zawadzki, M.; Głodek, J.; Holak, P.; Sanford, J.; Milewska, K.; Adamiak, Z.; et al. (Nature Research, 2021-03-22)
      Stem cell transplantation proved promising in animal models of neurological diseases; however, in conditions with disseminated pathology such as ALS, delivery of cells and their broad distribution is challenging. To address this problem, we explored intra-arterial (IA) delivery route, of stem cells. The goal of this study was to investigate the feasibility and safety of MRI-guided transplantation of glial restricted precursors (GRPs) and mesenchymal stem cells (MSCs) in dogs suffering from ALS-like disease, degenerative myelopathy (DM). Canine GRP transplantation in dogs resulted in rather poor retention in the brain, so MSCs were used in subsequent experiments. To evaluate the safety of MSC intraarterial transplantation, naïve pigs (n = 3) were used as a pre-treatment control before transplantation in dogs. Cells were labeled with iron oxide nanoparticles. For IA transplantation a 1.2-French microcatheter was advanced into the middle cerebral artery under roadmap guidance. Then, the cells were transplanted under real-time MRI with the acquisition of dynamic T2*-weighted images. The procedure in pigs has proven to be safe and histopathology has demonstrated the successful and predictable placement of transplanted porcine MSCs. Transplantation of canine MSCs in DM dogs resulted in their accumulation in the brain. Interventional and follow-up MRI proved the procedure was feasible and safe. Analysis of gene expression after transplantation revealed a reduction of inflammatory factors, which may indicate a promising therapeutic strategy in the treatment of neurodegenerative diseases. Copyright 2021, The Author(s).
    • Mesenchymal stem cells injected into carotid artery to target focal brain injury home to perivascular space

      Andrzejewska, A.; Walczak, P.; Janowski, M. (Ivyspring International Publisher, 2020)
      Rationale: The groundbreaking discovery of mesenchymal stem cells (MSCs) with their multifaceted benefits led to their widespread application in experimental medicine, including neurology. Efficient delivery of MSCs to damaged regions of the central nervous system may be a critical factor in determining outcome. Integrin VLA-4 (α4β1) coded by ITGA4 and ITGB1 genes is an adhesion molecule expressed by leukocytes, which is responsible for initiation of their diapedesis through cell docking to the inflamed vessel wall expressing VCAM1 receptor. This function of VLA-4 has been recapitulated in neural stem cells and glial progenitors. Thus, it was prudent to investigate this tool as a vehicle driving extravasation of MSCs. Since MSCs naturally express ITGB1 subunit, we decided to supplement them with ITGA4 only. The purpose of our current study is to investigate the eventual fate of IA delivered ITGA4 engineered and naive MSCs. Methods: mRNA-ITGA4 transfected and naive MSCs were injected to right internal carotid artery of rats with focal brain injury. Through next three days MSC presence in animals' brain was navigated by magnetic resonance imaging. Transplanted cell location relative to the brain blood vessels and host immunological reaction were analyzed post-mortem by immunohistochemistry. The chemotaxis of modified and naive MSCs was additionally examined in in vitro transwell migration assay. Results: Both naïve and ITGA4-overexpressing cells remained inside the vascular lumen over the first two days after IA infusion. On the third day, 39% of mRNA-ITGA4 modified and 51% naïve MSCs homed to perivascular space in the injury region (p=NS). The gradual decrease of both naive and mRNA-ITGA4 transfected hBM-MSCs in the rat brain was observed. mRNA-ITGA4 transfected MSCs appeared to be more vulnerable to phagocytosis than naïve cells. Moreover, in vitro study revealed that homogenate from the injured brain repels migration of MSCs, corroborating the incomplete extravasation observed in vivo. Conclusions: In summary, IA transplanted MSCs are capable of homing to the perivascular space, an integral part of neurovascular unit, which might contribute to the replacement of injured pericytes, a critical element facilitating restoration of CNS function. The mRNA-ITGA4 transfection improves cell docking to vessel but this net benefit vanishes over the next two days due to fast clearance from cerebral vessels of the majority of transplanted cells, regardless of their engineering status. The drawbacks of mRNA-ITGA4 transfection become apparent on day 3 post transplantation due to the lower survival and higher vulnerability to host immune attack. Copyright The author(s).
    • Modeling human pediatric and adult gliomas in immunocompetent mice through costimulatory blockade

      Lan, X.; Chu, C.; Jablonska, A.; Liang, Y.; Janowski, M.; Walczak, P. (Taylor and Francis Inc., 2020)
      Currently, human glioma tumors are mostly modeled in immunodeficient recipients; however, lack of interactions with adaptive immune system is a serious flaw, particularly in the era when immunotherapies dominate treatment strategies. Our group was the first to successfully establish the orthotopic transplantation of human glioblastoma (GBM) in immunocompetent mice by inducing immunological tolerance using a short-term, systemic costimulation blockade strategy (CTLA-4-Ig and MR1). In this study, we further validated the feasibility of this method by modeling pediatric diffuse intrinsic pontine glioma (DIPG) and two types of adult GBM (GBM1, GBM551), in mice with intact immune systems and immunodeficient mice. We found that all three glioma models were successfully established, with distinct difference in tumor growth patterns and morphologies, after orthotopic xenotransplantation in tolerance-induced immunocompetent mice. Long-lasting tolerance that is maintained for up to nearly 200 d in GBM551 confirmed the robustness of this model. Moreover, we found that tumors in immunocompetent mice displayed features more similar to the clinical pathophysiology found in glioma patients, characterized by inflammatory infiltration and strong neovascularization, as compared with tumors in immunodeficient mice. In summary, we have validated the robustness of the costimulatory blockade strategy for tumor modeling and successfully established three human glioma models including the pediatric DIPG whose preclinical study is particularly thwarted by the lack of proper animal models. Copyright 2020 the Author(s).
    • Optimization of osmotic blood-brain barrier opening to enable intravital microscopy studies on drug delivery in mouse cortex

      Jablonska, A.; Lan, X.; Janowski, M.; Walczak, P. (Elsevier B.V., 2020)
      Intra-arterial (IA) infusion of mannitol induces osmotic blood-brain barrier opening (OBBBO) and that method has been used for decades to improve drug delivery to the brain. However, high variability of outcomes prevented vast clinical adoption. Studies on dynamic multi-scale imaging of OBBBO as well as extravasation of IA injected therapeutic agents are essential to develop strategies assuring precision and reproducibility of drug delivery. Intravital microscopy is increasingly used to capture the dynamics of biological processes at the molecular level in convenient mouse models. However, until now OBBBO has been achieved safely in subcortical structures, which prevented direct insight into the process of extravasation through the skull window. Here, we used our previously developed real-time MRI to adjust the procedure to achieve robust cortical OBBBO. We found that catheter-mediated delivery to the cortex from the ipsilateral carotid artery can be improved by temporarily occluding the contralateral carotid artery. The reproducibility and safety of the method were validated by MRI and histology. This experimental platform was further exploited for studying with intravital microscopy the extravasation of 0.58 kDa rhodamine and 153 kDa anti-VEGF monoclonal antibody (bevacizumab) upon IA injection. Dynamic imaging during IA infusion captured the spatiotemporal dynamic of infiltration for each molecule into the brain parenchyma upon OBBBO. Small-sized rhodamine exhibited faster and higher penetration than the antibody. Histological analysis showed some uptake of the monoclonal antibody after IA delivery, and OBBBO significantly amplified the extent of its uptake. For quantitative assessment of cortical uptake, bevacizumab was radiolabeled with zirconium-89 and infused intraarterially. As expected, OBBBO potentiated brain accumulation, providing 33.90 ± 9.06% of injected dose per gram of brain tissue (%ID/g) in the cortex and 17.09 ± 7.22%ID/g in subcortical structures. In contrast IA infusion with an intact BBB resulted in 3.56 ± 1.06%ID/g and 3.57 ± 0.59%ID/g in the same brain regions, respectively. This study established reproducible cortical OBBBO in mice, which enabled multi-photon microscopy studies on OBBBO and drug targeting. This approach helped demonstrate in a dynamic fashion extravasation of fluorescently-tagged antibodies and their effective delivery into the brain across an osmotically opened BBB.