Different Contacted Cell Types Contribute to Acquiring Different Properties in Brain Microglial Cells upon Intercellular Interaction.

Microglial cells (MGs), originally derived from progenitor cells in a yolk sac during early development, are glial cells located in a physiological and pathological brain. Since the brain contains various cell types, MGs could frequently interact with different cells, such as astrocytes (ACs), pericytes (PCs), and endothelial cells (ECs). However, how microglial traits are regulated via cell-cell interactions by ACs, PCs, or ECs and how they are different depending on the contacted cell types is unclear. This study aimed to clarify these questions by coculturing MGs with ACs, PCs, or ECs using mouse brain-derived cells, and microglial phenotypic changes were investigated under culture conditions that enabled direct cell-cell contact. Our results showed that ACs or PCs dose-dependently increased the number of MG, while ECs decreased it. Microarray and gene ontology analysis showed that cell fate-related genes (e.g., cell cycle, proliferation, growth, death, and apoptosis) of MGs were altered after a cell-cell contact with ACs, PCs, and ECs. Notably, microarray analysis showed that several genes, such as gap junction protein alpha 1 (Gja1), were prominently upregulated in MGs after coincubation with ACs, PCs, or ECs, regardless of cell types. Similarly, immunohistochemistry showed that an increased Gja1 expression was observed in MGs after coincubation with ACs, PCs, or ECs. Immunofluorescent and fluorescence-activated cell sorting analysis also showed that calcein-AM was transferred into MGs after coincubation with ACs, PCs, or ECs, confirming that intercellular interactions occurred between these cells. However, while Gja1 inhibition reduced the number of MGs after coincubation with ACs and PCs, this was increased after coincubation with ECs; this indicates that ACs and PCs positively regulate microglial numbers via Gja1, while ECs decrease it. Results show that ACs, PCs, or ECs exert both common and specific cell type-dependent effects on MGs through intercellular interactions. These findings also suggest that brain microglial phenotypes are different depending on their surrounding cell types, such as ACs, PCs, or ECs.

Bifidobacterium breve during infancy attenuates mobility in low birthweight rats.

BACKGROUND: Children with low birthweight (LBW) have a higher risk for developing attention-deficit/hyperactivity disorder, for which no prophylactic measure exists. The gut microbiota in infants with LBW is different from that in infants with normal birthweight and is associated with attention-deficit/hyperactivity disorder. Oral supplementation with Bifidobacterium has several health benefits, such as suppressing inflammation. METHODS: We examined the effect of gavage supplementation with Bifidobacterium breve M-16V from postnatal days 1-21 in a rat model of intrauterine hypoperfusion. RESULTS: The open-field test at 5 weeks of age (equivalent to human pubertal age) showed that rats in the LBW-vehicle group were marginally hyperactive compared with rats in the sham group, while rats in the LBW-B.breve group were significantly hypoactive compared with rats in the LBW-vehicle group. The gut microbiota in the LBW-vehicle group exhibited a profile significantly different from that in the sham group, whereas the gut microbiota in the LBW-B.breve group did not exhibit a significant difference from that in the sham group. Anatomical/histological evaluation at 6 weeks of age demonstrated that the brain weight and the cerebral areas on coronal sections were reduced in the LBW groups compared with the sham group. Probiotic supplementation did not ameliorate these morphological brain anomalies in LBW animals. The percentage of Iba-1+ cells in the brain was not different among the LBW-B.breve, LBW-vehicle, and sham groups. CONCLUSION: Bifidobacterium breve supplementation during early life is suggested to have the potential to help children with LBW attenuate hypermobility in adolescence.

Establishment of a Reproducible Ischemic Stroke Model in Nestin-GFP Mice with High Survival Rates.

An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

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

BACKGROUND: Microglia 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. METHODS: In this study, we used a mouse model of cerebral infarction to investigate whether reactive pericytes (PCs) acquire microglia-producing VSC activity following ischemia. RESULTS: We 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-ß (PDGFRß), a hallmark of vascular PCs. PDGFRß(+) 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. CONCLUSIONS: The study results suggest that vascular PCs acquire multipotent VSC activity under pathological conditions and may thus be a novel source of microglia.

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.

Neonatal Brains Exhibit Higher Neural Reparative Activities than Adult Brains in a Mouse Model of Ischemic Stroke.

The neonatal brain is substantially more resistant to various forms of injury than the mature brain. For instance, the prognosis following ischemic stroke is generally poor in the elderly but favorable in neonates. Identifying the cellular and molecular mechanisms underlying reparative activities in the neonatal brain after ischemic injury may provide feasible targets for therapeutic interventions in adults. To this end, we compared the reparative activities in postnatal day 13 and adult (8-12-week-old) mouse brain following middle cerebral artery occlusion. Immunohistochemistry revealed considerably greater generation of ischemia-induced neural stem/progenitor cells (iNSPCs) expressing nestin or Sox2 in ischemic areas of the neonatal brain. The iNSPCs isolated from the neonatal brain also demonstrated greater proliferative activity than those isolated from adult mice. In addition, genes associated with neuronal differentiation were enriched in iNSPCs isolated from the neonatal brain according to microarray and gene ontogeny analyses. Immunohistochemistry further revealed considerably greater production of newborn doublecortin+ neurons at the sites of ischemic injury in the neonatal brain compared to the adult brain. These findings suggest that greater iNSPC generation and neurogenic differentiation capacities contribute to the superior regeneration of the neonatal brain following ischemia. Together, our findings may help identify therapeutic targets for enhancing the reparative potential of the adult brain following stroke.

Administration of Human-Derived Mesenchymal Stem Cells Activates Locally Stimulated Endogenous Neural Progenitors and Reduces Neurological Dysfunction in Mice after Ischemic Stroke.

Increasing evidence shows that the administration of mesenchymal stem cells (MSCs) is a promising option for various brain diseases, including ischemic stroke. Studies have demonstrated that MSC transplantation after ischemic stroke provides beneficial effects, such as neural regeneration, partially by activating endogenous neural stem/progenitor cells (NSPCs) in conventional neurogenic zones, such as the subventricular and subgranular zones. However, whether MSC transplantation regulates the fate of injury-induced NSPCs (iNSPCs) regionally activated at injured regions after ischemic stroke remains unclear. Therefore, mice were subjected to ischemic stroke, and mCherry-labeled human MSCs (h-MSCs) were transplanted around the injured sites of nestin-GFP transgenic mice. Immunohistochemistry of brain sections revealed that many GFP+ cells were observed around the grafted sites rather than in the regions in the subventricular zone, suggesting that transplanted mCherry+ h-MSCs stimulated GFP+ locally activated endogenous iNSPCs. In support of these findings, coculture studies have shown that h-MSCs promoted the proliferation and neural differentiation of iNSPCs extracted from ischemic areas. Furthermore, pathway analysis and gene ontology analysis using microarray data showed that the expression patterns of various genes related to self-renewal, neural differentiation, and synapse formation were changed in iNSPCs cocultured with h-MSCs. We also transplanted h-MSCs (5.0 × 104 cells/µL) transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion. Compared with phosphate-buffered saline-injected controls, h-MSC transplantation displayed significantly improved neurological functions. These results suggest that h-MSC transplantation improves neurological function after ischemic stroke in part by regulating the fate of iNSPCs.

Voluntary running exercise modifies astrocytic population and features in the peri-infarct cortex.

Rehabilitative exercise following a brain stroke has beneficial effects on the morphological plasticity of neurons. Particularly, voluntary running exercise after focal cerebral ischemia promotes functional recovery and ameliorates ischemia-induced dendritic spine loss in the peri-infarct motor cortex layer 5. Moreover, neuronal morphology is affected by changes in the perineuronal environment. Glial cells, whose phenotypes may be altered by exercise, are known to play a pivotal role in the formation of this perineuronal environment. Herein, we investigated the effects of voluntary running exercise on glial cells after middle cerebral artery occlusion. Voluntary running exercise increased the population of glial fibrillary acidic protein-positive astrocytes born between post-operative days (POD) 0 and 3 on POD15 in the peri-infarct cortex. After exercise, transcriptomic analysis of post-ischemic astrocytes revealed 10 upregulated and 70 downregulated genes. Furthermore, gene ontology analysis showed that the 70 downregulated genes were significantly associated with neuronal morphology. In addition, exercise reduced the number of astrocytes expressing lipocalin 2, a regulator of dendritic spine density, on POD15. Our results suggest that exercise modifies the composition of astrocytic population and their phenotype.

Microglia Negatively Regulate the Proliferation and Neuronal Differentiation of Neural Stem/Progenitor Cells Isolated from Poststroke Mouse Brains.

We previously demonstrated that neural stem/progenitor cells (NSPCs) were induced within and around the ischemic areas in a mouse model of ischemic stroke. These injury/ischemia-induced NSPCs (iNSPCs) differentiated to electrophysiologically functional neurons in vitro, indicating the presence of a self-repair system following injury. However, during the healing process after stroke, ischemic areas were gradually occupied by inflammatory cells, mainly microglial cells/macrophages (MGs/MΦs), and neurogenesis rarely occurred within and around the ischemic areas. Therefore, to achieve neural regeneration by utilizing endogenous iNSPCs, regulation of MGs/MΦs after an ischemic stroke might be necessary. To test this hypothesis, we used iNSPCs isolated from the ischemic areas after a stroke in our mouse model to investigate the role of MGs/MΦs in iNSPC regulation. In coculture experiments, we show that the presence of MGs/MΦs significantly reduces not only the proliferation but also the differentiation of iNSPCs toward neuronal cells, thereby preventing neurogenesis. These effects, however, are mitigated by MG/MΦ depletion using clodronate encapsulated in liposomes. Additionally, gene ontology analysis reveals that proliferation and neuronal differentiation are negatively regulated in iNSPCs cocultured with MGs/MΦs. These results indicate that MGs/MΦs negatively impact neurogenesis via iNSPCs, suggesting that the regulation of MGs/MΦs is essential to achieve iNSPC-based neural regeneration following an ischemic stroke.

Transplantation of Human Brain-Derived Ischemia-Induced Multipotent Stem Cells Ameliorates Neurological Dysfunction in Mice After Stroke.

We recently demonstrated that injury/ischemia-induced multipotent stem cells (iSCs) develop within post-stroke human brains. Because iSCs are stem cells induced under pathological conditions, such as ischemic stroke, the use of human brain-derived iSCs (h-iSCs) may represent a novel therapy for stroke patients. We performed a preclinical study by transplanting h-iSCs transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion (MCAO). Compared with PBS-treated controls, h-iSC transplantation significantly improved neurological function. To identify the underlying mechanism, green fluorescent protein (GFP)-labeled h-iSCs were transplanted into post-stroke mouse brains. Immunohistochemistry revealed that GFP+ h-iSCs survived around the ischemic areas and some differentiated into mature neuronal cells. To determine the effect on endogenous neural stem/progenitor cells (NSPCs) by h-iSC transplantation, mCherry-labeled h-iSCs were administered to Nestin-GFP transgenic mice which were subjected to MCAO. As a result, many GFP+ NSPCs were observed around the injured sites compared with controls, indicating that mCherry+ h-iSCs activate GFP+ endogenous NSPCs. In support of these findings, coculture studies revealed that the presence of h-iSCs promotes the proliferation of endogenous NSPCs and increases neurogenesis. In addition, coculture experiments indicated neuronal network formation between h-iSC- and NSPC-derived neurons. These results suggest that h-iSCs exert positive effects on neural regeneration through not only neural replacement by grafted cells but also neurogenesis by activated endogenous NSPCs. Thus, h-iSCs have the potential to be a novel source of cell therapy for stroke patients.

Enhanced angiogenic properties of umbilical cord blood primed by OP9 stromal cells ameliorates neurological deficits in cerebral infarction mouse model.

Umbilical cord blood (UCB) transplantation shows proangiogenic effects and contributes to symptom amelioration in animal models of cerebral infarction. However, the effect of specific cell types within a heterogeneous UCB population are still controversial. OP9 is a stromal cell line used as feeder cells to promote the hematoendothelial differentiation of embryonic stem cells. Hence, we investigated the changes in angiogenic properties, underlying mechanisms, and impact on behavioral deficiencies caused by cerebral infarction in UCB co-cultured with OP9 for up to 24 h. In the network formation assay, only OP9 pre-conditioned UCB formed network structures. Single-cell RNA sequencing and flow cytometry analysis showed a prominent phenotypic shift toward M2 in the monocytic fraction of OP9 pre-conditioned UCB. Further, OP9 pre-conditioned UCB transplantation in mice models of cerebral infarction facilitated angiogenesis in the peri-infarct lesions and ameliorated the associated symptoms. In this study, we developed a strong, fast, and feasible method to augment the M2, tissue-protecting, pro-angiogenic features of UCB using OP9. The ameliorative effect of OP9-pre-conditioned UCB in vivo could be partly due to promotion of innate angiogenesis in peri-infarct lesions.

A Bone Morphogenetic Protein Signaling Inhibitor, LDN193189, Converts Ischemia-Induced Multipotent Stem Cells into Neural Stem/Progenitor Cell-Like Cells.

Stem cell therapy is used to restore neurological function in stroke patients. We have previously reported that ischemia-induced multipotent stem cells (iSCs), which are likely derived from brain pericytes, develop in poststroke human and mouse brains. Although we have demonstrated that iSCs can differentiate into neural lineage cells, the factors responsible for inducing this differentiation remain unclear. In this study, we found that LDN193189, a bone morphogenetic protein (BMP) inhibitor, caused irreversible changes in the shape of iSCs. In addition, compared with iSCs incubated without LDN193189, the iSCs incubated with LDN193189 (LDN-iSCs) showed upregulated expression of neural lineage-related genes and proteins, including those expressed in neural stem/progenitor cells (NSPCs), and downregulated expression of mesenchymal and pericytic-related genes and proteins. Moreover, microarray analysis revealed that LDN-iSCs and NSPCs had similar gene expression profiles. Furthermore, LDN-iSCs differentiated into electrophysiologically functional neurons. These results indicate that LDN193189 induces NSPC-like cells from iSCs, suggesting that bioactive molecules regulating BMP signaling are potential targets for promoting neurogenesis from iSCs in the pathological brain, such as during ischemic stroke. We believe that our findings will bring us one step closer to the clinical application of iSCs.

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 x 10(6) 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.

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.

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.

CD44 expression in stem cells and niche microglia/macrophages following ischemic stroke.

BACKGROUND: CD44, an adhesion molecule in the hyaluronate receptor family, plays diverse and important roles in multiple cell types and organs. Increasing evidence is mounting for CD44 expression in various types of stem cells and niche cells surrounding stem cells. However, the precise phenotypes of CD44+ cells in the brain under pathologic conditions, such as after ischemic stroke, remain unclear. METHODS: In the present study, using a mouse model for cerebral infarction by middle cerebral artery (MCA) occlusion, we examined the localization and traits of CD44+ cells. RESULTS: In sham-mice operations, CD44 was rarely observed in the cortex of MCA regions. Following ischemic stroke, CD44+ cells emerged in ischemic areas of the MCA cortex during the acute phase. Although CD44 at ischemic areas was, in part, expressed in stem cells, it was also expressed in hematopoietic lineages, including activated microglia/macrophages, surrounding the stem cells. CD44 expression in microglia/macrophages persisted through the chronic phase following ischemic stroke. CONCLUSIONS: These data demonstrate that CD44 is expressed in stem cells and cells in the niches surrounding them, including inflammatory cells, suggesting that CD44 may play an important role in reparative processes within ischemic areas under neuroinflammatory conditions; in particular, strokes.

Early Reperfusion Following Ischemic Stroke Provides Beneficial Effects, Even After Lethal Ischemia with Mature Neural Cell Death.

Ischemic stroke is a critical disease caused by cerebral artery occlusion in the central nervous system (CNS). Recent therapeutic advances, such as neuroendovascular intervention and thrombolytic therapy, have allowed recanalization of occluded brain arteries in an increasing number of stroke patients. Although previous studies have focused on rescuing neural cells that still survive despite decreased blood flow, expanding the therapeutic time window may allow more patients to undergo reperfusion in the near future, even after lethal ischemia, which is characterized by death of mature neural cells, such as neurons and glia. However, it remains unclear whether early reperfusion following lethal ischemia results in positive outcomes. The present study used two ischemic mouse models-90-min transient middle cerebral artery occlusion (t-MCAO) paired with reperfusion to induce lethal ischemia and permanent middle cerebral artery occlusion (p-MCAO)-to investigate the effect of early reperfusion up to 8 w following MCAO. Although early reperfusion following 90-min t-MCAO did not rescue mature neural cells, it preserved the vascular cells within the ischemic areas at 1 d following 90-min t-MCAO compared to that following p-MCAO. In addition, early reperfusion facilitated the healing processes, including not only vascular but also neural repair, during acute and chronic periods and improved recovery. Furthermore, compared with p-MCAO, early reperfusion after t-MCAO prevented behavioral symptoms of neurological deficits without increasing negative complications, including hemorrhagic transformation and mortality. These results indicate that early reperfusion provides beneficial effects presumably via cytoprotective and regenerative mechanisms in the CNS, suggesting that it may be useful for stroke patients that experienced lethal ischemia.

Ischemia-Induced Multipotent Stem Cells Isolated from Stroke Patients Exhibit Higher Neurogenic Differentiation Potential than Bone Marrow-Derived Mesenchymal Stem Cells.

Perivascular areas of the brain harbor multipotent stem cells. We recently demonstrated that after a stroke, brain pericytes exhibit features of multipotent stem cells. Moreover, these ischemia-induced multipotent stem cells (iSCs) are present within ischemic areas of the brain of patients diagnosed with stroke. Although increasing evidence shows that iSCs have traits similar to those of mesenchymal stem cells (MSCs), the phenotypic similarities and differences between iSCs and MSCs remain unclear. In this study, we used iSCs extracted from stroke patients (h-iSCs) and compared their neurogenic potential with that of human MSCs (h-MSCs) in vitro. Microarray analysis, fluorescence-activated cell sorting, immunohistochemistry, and multielectrode array were performed to compare the characteristics of h-iSCs and h-MSCs. Although h-iSCs and h-MSCs had similar gene expression profiles, the percentage expressing the neural stem/progenitor cell marker nestin was significantly higher in h-iSCs than in h-MSCs. Consistent with these findings, h-iSCs, but not h-MSCs, differentiated into electrophysiologically functional neurons. In contrast, although both h-iSCs and h-MSCs were able to differentiate into several mesodermal lineages, including adipocytes, osteocytes, and chondrocytes, the potential of h-iSCs to differentiate into adipocytes and osteocytes was relatively low. These results suggest that compared with h-MSCs, h-iSCs predominantly exhibit neural rather than mesenchymal lineages. In addition, these results indicate that h-iSCs have the potential to repair the injured brain of patients with stroke by directly differentiating into neuronal lineages.

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.

Isolation and Characterization of Cerebellum-Derived Stem Cells in Poststroke Human Brain.

There is compelling evidence that the mature central nervous system (CNS) harbors stem cell populations outside conventional neurogenic regions. We previously demonstrated that brain pericytes (PCs) in both mouse and human exhibit multipotency to differentiate into various neural lineages following cerebral ischemia. PCs are found throughout the CNS, including cerebellum, but it remains unclear whether cerebellar PCs also form ischemia-induced multipotent stem cells (iSCs). In this study, we demonstrate that putative iSCs can be isolated from poststroke human cerebellum (cerebellar iSCs [cl-iSCs]). These cl-iSCs exhibited multipotency and differentiated into electrophysiologically active neurons. Neurogenic potential was also confirmed in single-cell suspensions. DNA microarray analysis revealed highly similar gene expression patterns between PCs and cl-iSCs, suggesting PC origin. Global gene expression comparison with cerebral iSCs revealed general similarity, but cl-iSCs differentially expressed certain cerebellum-specific genes. Thus, putative iSCs are present in poststroke cerebellum and possess region-specific traits, suggesting potential capacity to regenerate functional cerebellar neurons following ischemic stroke.