Takayuki Nakagomi

Endothelial Cells Support Survival, Proliferation, and Neuronal Differentiation of Transplanted Adult Ischemia‐Induced Neural Stem/Progenitor Cells After Cerebral Infarction

Transplantation of neural stem cells (NSCs) has been proposed as a therapy for a range of neurological disorders. To realize the potential of this approach, it is essential to control survival, proliferation, migration, and differentiation of NSCs after transplantation. NSCs are regulated in vivo, at least in part, by their specialized microenvironment or “niche.” In the adult central nervous system, neurogenic regions, such as the subventricular and subgranular zones, include NSCs residing in a vascular niche with endothelial cells. Although there is accumulating evidence that endothelial cells promote proliferation of NSCs in vitro, there is no description of their impact on transplanted NSCs. In this study, we grafted cortex‐derived stroke‐induced neural stem/progenitor cells, obtained from adult mice, onto poststroke cortex in the presence or absence of endothelial cells, and compared survival, proliferation, and neuronal differentiation of the neural precursors in vivo. Cotransplantation of endothelial cells and neural stem/progenitor cells increased survival and proliferation of ischemia‐induced neural stem/progenitor cells and also accelerated neuronal differentiation compared with transplantation of neural precursors alone. These data indicate that reconstitution of elements in the vascular niche enhances transplantation of adult neural progenitor cells. STEM CELLS 2009;27:2185–2195

Bone Marrow Mononuclear Cells Promote Proliferation of Endogenous Neural Stem Cells Through Vascular Niches After Cerebral Infarction

Increasing evidence shows that administration of bone marrow mononuclear cells (BMMCs) is a potential treatment for various ischemic diseases, such as ischemic stroke. Although angiogenesis has been considered primarily responsible for the effect of BMMCs, their direct contribution to endothelial cells (ECs) by being a functional elements of vascular niches for neural stem/progenitor cells (NSPCs) has not been considered. Herein, we examine whether BMMCs affected the properties of ECs and NSPCs, and whether they promoted neurogenesis and functional recovery after stroke. We compared i.v. transplantations 1 × 106 BMMCs and phosphate‐buffered saline in mice 2 days after cortical infarction. Systemically administered BMMCs preferentially accumulated at the postischemic cortex and peri‐infarct area in brains; cell proliferation of ECs (angiogenesis) at these regions was significantly increased in BMMCs‐treated mice compared with controls. We also found that endogenous NSPCs developed in close proximity to ECs in and around the poststroke cortex and that ECs were essential for proliferation of these ischemia‐induced NSPCs. Furthermore, BMMCs enhanced proliferation of NSPCs as well as ECs. Proliferation of NSPCs was suppressed by additional treatment with endostatin (known to inhibit proliferation of ECs) following BMMCs transplantation. Subsequently, neurogenesis and functional recovery were also promoted in BMMCs‐treated mice compared with controls. These results suggest that BMMCs can contribute to the proliferation of endogenous ischemia‐induced NSPCs through vascular niche regulation, which includes regulation of endothelial proliferation. In addition, these results suggest that BMMCs transplantation has potential as a novel therapeutic option in stroke treatment. STEM CELLS 2010;28:1292–1302

Injury-induced neural stem/progenitor cells in post-stroke human cerebral cortex.

Increasing evidence points to accelerated neurogenesis after stroke, and support of such endogenous neurogenesis has been shown to improve stroke outcome in experimental animal models. The present study analyses post‐stroke cerebral cortex after cardiogenic embolism in autoptic human brain. Induction of nestin‐ and musashi‐1‐positive cells, potential neural stem/progenitor cells, was observed at the site of ischemic lesions from day 1 after stroke. These two cell populations were present at distinct locations and displayed different temporal profiles of marker expression. However, no surviving differentiated mature neural cells were observed by 90 days after stroke in the previously ischemic region. Consistent with recent reports of neurogenesis in the cerebral cortex after induction of stroke in rodent models, the present current data indicate the presence of a regional regenerative response in human cerebral cortex. Furthermore, observations underline the potential importance of supporting survival and differentiation of endogenous neural stem/progenitor cells in post‐stroke human brain.

Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice

The CNS has the potential to marshal strong reparative mechanisms, including activation of endogenous neurogenesis, after a brain injury such as stroke. However, the response of neural stem/progenitor cells to stroke is poorly understood. Recently, neural stem/progenitor cells have been identified in the cerebral cortex, as well as previously recognized regions such as the subventricular or subgranular zones of the hippocampus, suggesting that a contribution of cortex‐derived neural stem/progenitor cells may repair ischemic lesions of the cerebral cortex. In the present study, using a highly reproducible murine model of cortical infarction, we have found nestin‐positive cells in the post‐stroke cerebral cortex, but not in the non‐ischemic cortex. Cells obtained from the ischemic core of the post‐stroke cerebral cortex formed neurosphere‐like cell clusters expressing nestin; such cells had the capacity for self‐renewal and differentiated into electrophysiologically functional neurons, astrocytes and myelin‐producing oligodendrocytes. Nestin‐positive cells from the stroke‐affected cortex migrated into the peri‐infarct area and differentiated into glial cells in vivo. Although we could not detect differentiation of nestin‐positive cells into neurons in vivo, our current observations indicate that endogenous neural stem/progenitors with the potential to become neurons can develop within post‐stroke cerebral cortex.

Ischemia-Induced Neural Stem/Progenitor Cells in the Pia Mater Following Cortical Infarction

Increasing evidence shows that neural stem/progenitor cells (NSPCs) can be activated in the nonconventional neurogenic zones such as the cortex following ischemic stroke. However, the precise origin, identity, and subtypes of the ischemia-induced NSPCs (iNSPCs), which can contribute to cortical neurogenesis, is currently still unclear. In our present study, using an adult mouse cortical infarction model, we found that the leptomeninges (pia mater), which is widely distributed within and closely associated with blood vessels as microvascular pericytes/perivascular cells throughout central nervous system (CNS), have NSPC activity in response to ischemia and can generate neurons. These observations indicate that microvascular pericytes residing near blood vessels that are distributed from the leptomeninges to the cortex are potential sources of iNSPCs for neurogenesis following cortical infarction. In addition, our results propose a novel concept that the leptomeninges, which cover the entire brain, have an important role in CNS restoration following brain injury such as stroke.

Immunodeficiency reduces neural stem/progenitor cell apoptosis and enhances neurogenesis in the cerebral cortex after stroke

Acute inflammation in the poststroke period exacerbates neuronal damage and stimulates reparative mechanisms, including neurogenesis. However, only a small fraction of neural stem/progenitor cells survives. In this report, by using a highly reproducible model of cortical infarction in SCID mice, we examined the effects of immunodeficiency on reduction of brain injury, survival of neural stem/progenitor cells, and functional recovery. Subsequently, the contribution of T lymphocytes to neurogenesis was evaluated in mice depleted for each subset of T lymphocyte. SCID mice revealed the reduced apoptosis and enhanced proliferation of neural stem/progenitor cells induced by cerebral cortex after stroke compared with the immunocompetent wild‐type mice. Removal of T lymphocytes, especially the CD4+ T‐cell population, enhanced generation of neural stem/progenitor cells, followed by accelerated functional recovery. In contrast, removal of CD25+ T cells, a cell population including regulatory T lymphocytes, impaired functional recovery through, at least in part, suppression of neurogenesis. Our findings demonstrate a key role of T lymphocytes in regulation of poststroke neurogenesis and indicate a potential novel strategy for cell therapy in repair of the central nervous system.

Brain Vascular Pericytes Following Ischemia Have Multipotential Stem Cell Activity to Differentiate Into Neural and Vascular Lineage Cells

Brain vascular pericytes (PCs) are a key component of the blood‐brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries. Stem Cells 2015;33:1962–1974

Granulocyte colony-stimulating factor has a negative effect on stroke outcome in a murine model

The administration of CD34‐positive cells after stroke has been shown to have a beneficial effect on functional recovery by accelerating angiogenesis and neurogenesis in rodent models. Granulocyte colony‐stimulating factor (G‐CSF) is known to mobilize CD34‐positive cells from bone marrow and has displayed neuroprotective properties after transient ischemic stress. This led us to investigate the effects of G‐CSF administration after stroke in mouse. We utilized permanent ligation of the M1 distal portion of the left middle cerebral artery to develop a reproducible focal cerebral ischemia model in CB‐17 mice. Animals treated with G‐CSF displayed cortical atrophy and impaired behavioral function compared with controls. The negative effect of G‐CSF on outcome was associated with G‐CSF induction of an exaggerated inflammatory response, based on infiltration of the peri‐infarction area with CD11b‐positive and F4/80‐positive cells. Although clinical trials with G‐CSF have been started for the treatment of myocardial and limb ischemia, our results indicate that caution should be exercised in applying these results to cerebral ischemia.

Telmisartan suppresses cerebral injury in a murine model of transient focal ischemia.

The beneficial effects of angiotensin II type 1 (AT1) receptor blockers (ARB) in cerebrovascular disease have been shown in clinical trials. However, the effects of ARBs vary based on their unique pharmacologic properties. In this study, we focused on telmisartan, a fat-soluble ARB with selective peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonist activity, and investigated its effects on ischemic injury in cerebral vasculature using murine models of both transient and permanent focal ischemia. Analysis by triphenyltetrazolium-staining revealed that pre-treatment of mice with telmisartan reduced stroke volume 72 h after the transient ischemic insult in a dose-dependent manner, though such treatment did not reduce stroke volume due to permanent ischemia. Transient ischemia induced pro-inflammatory adhesion molecules, such as ICAM-1 and P-selectin in the ischemic region, and treatment with telmisartan diminished the expression of these adhesion molecules with diminished infiltration of inflammatory cells. The beneficial effect of telmisartan was attenuated, in part, by administration of a PPAR gamma antagonist. Treatment with valsartan (an ARB without PPAR gamma agonist activity) also decreased ischemic injury after transient ischemia, though to a lesser extent than telmisartan. Our findings indicate that telmisartan has a beneficial effect in a murine model of ischemia/reperfusion injury through blockade of AT1 receptors, and, in addition, due to a positive effect via its specific anti-inflammatory PPAR gamma agonist activity.

Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke

BackgroundMicroglia are the resident macrophage population of the central nervous system (CNS) and play essential roles, particularly in inflammation-mediated pathological conditions such as ischemic stroke. Increasing evidence shows that the population of vascular cells located around the blood vessels, rather than circulating cells, harbor stem cells and that these resident vascular stem cells (VSCs) are the likely source of some microglia. However, the precise traits and origins of these cells under pathological CNS conditions remain unclear.MethodsIn this study, we used a mouse model of cerebral infarction to investigate whether reactive pericytes (PCs) acquire microglia-producing VSC activity following ischemia.ResultsWe demonstrated the localization of ionized calcium-binding adaptor molecule 1 (Iba1)-expressing microglia to perivascular regions within ischemic areas. These cells expressed platelet-derived growth factor receptor-β (PDGFRβ), a hallmark of vascular PCs. PDGFRβ+ PCs isolated from ischemic, but not non-ischemic, areas expressed stem/undifferentiated cell markers and subsequently differentiated into various cell types, including microglia-like cells with phagocytic capacity.ConclusionsThe study results suggest that vascular PCs acquire multipotent VSC activity under pathological conditions and may thus be a novel source of microglia.