Transduction of Neural Precursor Cells with TAT-Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

Abstract

Novel therapeutic concepts against cerebral ischemia focus on cell-based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell-based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein-transduced with TAT-heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT-Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT-Hsp70-transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT-Hsp70-transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood-brain-barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long-term neuroprotection.

TAT-Hsp70 Induces Neuroprotection Against Stroke Via Anti-Inflammatory Actions Providing Appropriate Cellular Microenvironment for Transplantation of Neural Precursor Cells

“…12 Adult Subventricular Zone-Derived Neural Precursor Cells Neural precursor cells were isolated from the SVZ of 6 to 8-week old male transgenic green fluorescence protein positive (GFP þ ) animals (C57BL/6-Tg ACTB-EGFP, 1Osb/J; JAX Laboratory, Bar Harbor, ME, USA) as described. 14 The SVZ was microdissected under stereomicroscopic control (Zeiss, Jena, Germany) and minced into small pieces, followed by mechanical trituration and dissociation into a single-cell suspension. Thereafter, cells were cultured in serum-free basic DMEM (Dulbecco's Modified Eagle Medium)-F12 (PAA, Pasching, Austria) supplemented with epidermal growth factor (2 mg/mL), basic fibroblast growth factor (2 mg/mL), and penicillin-streptomycin (Invitrogen, Darmstadt, Germany).…”

Current Stage and Future Perspective of Stem Cell Therapy in Ischemic Stroke

“…According with this study in the first stage of stroke when we need to prevent BBB leakage and ROS formation systemic transplantation of NPCs can be the best route. Later in the subacute stage, when we need to enhance endogenous neurogenesis and promote long-term neuroprotection intracerebral transplantation seems to be the best route of delivery, which is followed by increased production of growth factors [24].…”

“…However, exogenous NSCs cells appear to have different mechanisms of action depending on delivery route. In this light, systemic delivery of NPCs in the acute phase (first 24h) of stroke in adult mice (11-13 weeks) has neuroprotective effect by preventing Blood-Brain-Barrier (BBB) damage, decrease matrix metalloprotease 9(MMP9) overexpression and Reactive Oxygen Species (ROS) [24].…”

Cell Therapy of Stroke and Spinal Cord Injury Complications

“…Хорошие результаты пока-зало применение дефинитивных стволовых и прогениторных клеток, полученных из раз-личных тканевых источников, которые были трансплантированы локально или системно животным, с моделированной фокальной це-ребральной ишемией [9]. Хотя прижившиеся клетки, как полагают, и не интегрировались в существующие нейронные сети, зато они спо-собствовали неврологическому восстановле-нию через непрямые паракринные механизмы, которые включают в себя стимуляцию эндо-генного ангионейрогенеза и пластичности ней-ронов, стабилизацию гематоэнцефалического барьера, а также модуляцию периферического и центрального иммунного ответа [10].…”

“…В частности, НПК, полученные из СВЗ боковых желудочков мозга, индуцируют мощный ней-ропротекторный эффект и ремоделирование головного мозга, как при системном, так и при локальном (интрацеребральном) введении. Этот аспект заслуживает особого внимания, так как по отношению к НПК до сих пор досто-верно не показана возможность их интеграции в нейронные сети, действие в основном проис-ходит через паракринные механизмы [9,10].…”