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Neural stem cell therapy for subacute and chronic ischemic stroke

Abstract

Neural stem cells (NSCs) play vital roles in brain homeostasis and exhibit a broad repertoire of potentially therapeutic actions following neurovascular injury. One such injury is stroke, a worldwide leading cause of death and disability. Clinically, extensive injury from ischemic stroke results from ischemia-reperfusion (IR), which is accompanied by inflammation, blood-brain barrier (BBB) damage, neural cell death, and extensive tissue loss. Tissue plasminogen activator (tPA) is still the only US Food and Drug Administration–approved clot-lysing agent. Whereas the thrombolytic role of tPA within the vasculature is beneficial, the effects of tPA (in a non-thrombolytic role) within the brain parenchyma have been reported as harmful. Thus, new therapies are needed to reduce the deleterious side effects of tPA and quickly facilitate vascular repair following stroke. The Stroke Treatment Academic Industry Roundtable (STAIR) recommends that stroke therapies “focus on drugs/devices/treatments with multiple mechanisms of action and that target multiple pathways”. Thus, based on multifactorial ischemic cascades in various stroke stages, effective stroke therapies need to focus on targeting and ameliorating early IR injury as well as facilitating angiogenesis, neurogenesis, and neurorestorative mechanisms following stroke. This review will discuss the preclinical perspectives of NSC transplantation as a promising treatment for neurovascular injury and will emphasize both the subacute and chronic phase of ischemic stroke.

Background

According to the Centers for Disease Control and Prevention, stroke is a leading cause of death in the United States [1], and ischemic stroke incidents comprise nearly 90% of all strokes [1]. Aging is a major risk factor, and overall, two thirds of strokes occur in patients over 65 years old [2]. Currently, we live in an aging nation with more than 45 million Americans who are at least 65 years old. By 2060, these numbers will likely have doubled.

Despite the high incidence of stroke and cerebrovascular disease, effective treatments and therapies are limited. Currently, therapy for ischemic stroke is limited to fast recanalization (thrombectomy) and tissue plasminogen activator (tPA), a compound that breaks apart thrombi in cerebral arteries to restore blood flow [3]. Major limitations to tPA treatment include a narrow effective therapeutic window of 4.5 h after initial stroke [4] and a high potential for hemorrhagic transformation [5]. Therefore, new treatment strategies are needed to increase the narrow therapeutic window of tPA, minimize detrimental side effects, and improve patient outcome following stroke. Of note, the pathophysiology of ischemic stroke is incredibly complex, and neural stem cells (NSCs) show pleiotropic effects that are potentially therapeutic for both the early (subacute) phase and chronic phase of stroke. These include functional neural replacement in multiple central nervous system (CNS) regions [6] or bystander effects that include delivery of NSC-synthesized therapeutic gene products, which could both directly protect the endangered host cells and inhibit toxic components of the microenvironment [7,8,9,10,11]. For example, a growing body of preclinical research suggests that NSC transplantation is an effective therapy for ischemic stroke through multiple mechanisms, such as preservation of the blood-brain barrier (BBB), alleviation of neuroinflammation, enhanced neurogenesis and angiogenesis, and ultimately functional neurological recovery [12, 13]. Furthermore, animal studies on NSC transplantation report different therapeutic effects depending on the stage of disease and route of administration. Therefore, this review is to highlight the therapeutic effects of NSC transplantation in various chronological stages of ischemic stroke. Stem cell delivery in subacute stroke may benefit more patients by ameliorating early-phase stroke injury, thus reducing later complications of secondary damage. This review will provide (1) an overview of NSC biology, including sources of NSCs and tracking of NSCs and their distribution, and (2) the complex pathophysiological cascades following ischemic stroke and impact of NSC delivery at these distinct stages of stroke.

Biology of neural stem cells

Endogenous neural stem cells

NSCs are the least committed cells of the nervous system and have the functional properties of self-renewal and multipotency to generate all three fundamental neuroectodermal lineages. NSCs generate neurons, astrocytes, and oligodendrocytes in a regional and developmental stage-appropriate manner throughout life. In mammals, neurogenesis occurs throughout life in localized brain regions called “niches” where NSCs are present [14]. These regions include the subgranular zone of the hippocampal dentate gyrus [15], the subventricular zone (SVZ) of the lateral ventricles [16,17,18], and the external germinal layer (EGL) of the cerebellum [19]. The EGL disappears once cerebellar development is completed, by 3 weeks postnatally in rodents [20] and by 2 years of age in humans [21]. In rodents, there is substantial evidence that neural progenitors in the SVZ proliferate throughout life, differentiate, and migrate through a track called the rostral migratory system to become new functional neurons in the olfactory bulb (OB) [14, 22]. However, recent studies suggest that humans and laboratory rodents differ in regard to the magnitude and duration of niche-specific adult neurogenesis. There is ample evidence that neurogenesis persists in the hippocampus [23, 24] and striatum [25] throughout aging in humans. Although the presence of NSCs in the adult human SVZ has been confirmed [26], postnatal migration of neuroblasts from this neurogenic niche to the OB may not occur in humans [27, 28]. Animal studies report that neurogenesis is very low by middle age [29], and human data support that hippocampal neurogenesis diminishes sharply over time [30].

In healthy adult mammals, neurogenesis occurs at a very limited rate [14, 31]. However, injury to the CNS stimulates neurogenesis. Pathological insults such as ischemia lead to increased proliferation, migration, and differentiation of NSCs and neural progenitors in the brain [32, 33]. Studies on adult humans have reported increased neural progenitor cells in brain parenchyma proximal to the ventricle wall and cells expressing Ki67, a marker of proliferation, in the ipsilateral SVZ following ischemic insult [34, 35]. Unfortunately, endogenous neurogenesis does not supply enough cells to repair neurological damage from major events like stroke [33]. Therefore, transplantation of cultured NSCs to aid endogenous neural progenitors could be an effective therapy to repair CNS damage from stroke.

Deriving neural stem cells in vitro

In order for NSC transplantation to be a viable therapy for stroke patients in the clinic, these cells must be able to be successfully cultured and expanded ex vivo. In fact, there are many ways to derive NSCs in vitro (Fig. 1). Since neurogenesis occurs throughout life in mammals, NSCs can be extracted directly from neural tissue, such as the neuroectoderm in developing fetuses or the subventricular or subgerminal zone in adults [36]. These NSCs can be propagated in vitro by using serum-free media with basic fibroblast growth factor (bFGF) [37,38,39] and epidermal growth factor (EGF) supplementation [36, 37, 40].

Fig. 1
figure 1

Schematic diagram of NSC generation via multiple methods. NSCs can be extracted directly from neurogenic niches, including the subgranular zone (SGZ) of the hippocampal dentate gyrus and subventricular zone (SVZ) of the lateral ventricles, from adult or fetal neural tissue and expanded in vitro. NSCs can be reprogrammed from patient-derived induced pluripotent stem cells by using combinations of specific transcription factors, small molecules, microRNAs, plasmids, and other morphogens. In addition, NSCs can be generated from blastocyst-derived embryonic stem cells by using specific combinations of differentiation factors, such as morphogens and extracellular matrix (ECM) proteins. Abbreviation: NSC neural stem cell

Isolation and propagation of NSCs can also be accomplished through other in vitro methods. For instance, NSCs can be derived from embryonic stem cells (ESCs) [41,42,43]. These cells originate from the inner cell mass of blastocysts and can give rise to progeny that can differentiate into any somatic cell type. One limitation of this approach is that ESCs require a great deal of manipulation to fully commit their fate toward differentiating into NSCs [41, 43]. Neuroinduction of ESCs can be accomplished by blocking transforming growth factor-beta/bone morphogenic protein (TGF-β/BMP) signaling pathways while promoting expansion with bFGF or EGF [44]. In order to minimize tumorigenic risk of undifferentiated cells, in vitro culturing time for ESC-derived NSCs is usually lengthened [45, 46].

NSCs can be similarly derived from human induced pluripotent stem cells (iPSCs) [44, 47]. Many types of somatic cells have been reprogrammed to generate iPSCs. These include fibroblasts, which can easily be obtained from human biopsies. Of note, the same method of dual-inhibiting SMAD signaling for ESCs can be used to transform iPSCs into NSCs [44]. Therefore, it is generally assumed that the same protocols for ESCs can be used to differentiate iPSCs into NSCs. However, generating iPSCs requires the extra, lengthy step of reprogramming already-differentiated somatic cells back to an undifferentiated state [48]. In vitro studies using microarray analysis have confirmed that iPSC-derived NSCs have very similar but not identical genetic expression compared with ESC-derived NSCs [49, 50]. Some advantages to using iPSCs are that they present fewer ethical concerns and fewer immune issues since they can be extracted and reprogrammed from a patient’s own tissue [47]. Therefore, iPSC-NSCs may have better potential as a treatment for CNS injury. NSCs derived in this manner have been tested in animal models of neurological disease and have proven to be therapeutic.

Also, methods have been developed to reprogram already-differentiated somatic cells into NSCs in a single step through the use of defined growth factors. For instance, experiments have successfully shown that adult fibroblasts can be successfully transformed into NSCs and neural progenitor cells by using the reprograming factors Oct4, Sox2, Klf4, and c-Myc [51]. The resulting induced NSCs exhibit morphology and molecular features similar to those of NSCs generated from other in vitro methods [52]. Similar results have been achieved with different combinations of transcription factors as well [53]. This method of generating NSCs in vitro is advantageous because the lengthy intermediate step of reprogramming somatic cells to iPSCs is skipped altogether. Therefore, direct differentiation of somatic cells to NSCs can save time without sacrificing the therapeutic quality of the manipulated cells. This technique also greatly reduces the risk of teratoma formation through the absence of undifferentiated iPSCs remaining in cell grafts following transplantation [52]. Additionally, direct differentiation of a patient’s own cells to NSCs can eliminate the risk of immune rejection and serve as a source of stem cells that can become neurons since other adult human stem cell sources have shown limited capabilities of fully differentiating into neural cell types [54]. For these reasons, the recent advancements in direct differentiation of stable and expandable NSCs from adult somatic cells are promising for therapeutic applications [55].

Labeling and tracking exogenous NSCs in vivo

Much of the preclinical research regarding NSC transplantation as a potential therapy for ischemic stroke relies on accurate identification and tracking of engrafted cells to assess their activity in vivo. There are a variety of different methods that investigators can use for labeling NSCs and tracking them after transplantation. One common method for pre-labeling NSCs involves the use of the compound bromodeoxyuridine (BrdU). This molecule incorporates into cellular DNA during the S phase of NSCs in vitro. Stem cells pre-labeled with BrdU can be identified via immunohistochemistry in fixed tissue by using anti-BrdU fluorescent-tagged antibodies or staining methods that use color-changing substrates [36]. Pre-labeling with BrdU is a useful technique for determining the number of cellular divisions that NSCs and their progenitors have undergone in vivo after transplantation [36]. However, this method of stem cell tracking is limited to short-term studies since the BrdU marker becomes increasingly diluted as transplanted stem cells continue to divide [36]. To monitor the migration and survival of engrafted NSCs in vivo non-invasively, human NSCs (hNSCs) can be labeled with super-paramagnetic iron oxide particles for magnetic resonance imaging (SPIO-MRI) [56].

Other methods for labeling and tracking stem cells in vivo include viral transduction of cells to express markers such as GFP and lacZ before transplantation. However, this method of introducing new genes through viral vectors raises concerns about changing the properties of stem cells and the risk of transgene inactivation [36]. When hNSCs are transplanted into rodent (e.g., species mismatch), hNSCs can be verified by human-specific immunomarkers such as human nuclear antigen or human mitochondria. Stem cells can also be identified by sex-mismatch if male stem cells containing Y chromosomes are transplanted into a female host or vice versa. This method can be especially useful if the graft cells are from the same species as the transplant recipient [36].

Stem cell migration in vivo

Despite the use of stereotactic transplantation equipment in some preclinical studies, stem cells must be able to navigate through the complex microenvironment of the stroke-damaged CNS in order to reach infarct tissue and maximize their therapeutic effects. In mammalian brains, NSCs demonstrate the ability to naturally migrate to areas of injury and neurodegeneration. NSCs from both humans and animals contain various chemokine receptors that facilitate their migration to areas of upregulated pro-inflammatory chemokine expression in the CNS [57,58,59]. Previous studies have demonstrated that chemokine stromal cell–derived factor-1 alpha (SDF-1α/CXCL12) interacts with NSC CXCR4 receptors as a key step in mediating migration to injury sites [60,61,62]. The significance of SDF-1α involvement in cell migration is also reflected in studies that have investigated stem cell homing in cardiovascular and renal disease [63,64,65]. In addition, in vitro analyses using the Transwell chemotaxis assay have shown that NSCs migrate to areas of higher SDF-1α concentrations [66]. SDF-1α levels increase significantly in the stroke-damaged region of the brain, and stem cells show increased migration to these areas of high SDF-1α concentration [67]. Since the SDF-1α/CRCX4 ligand-receptor interaction is heavily involved in stem cell migration in many forms of human disease and injury, manipulating this signaling pathway could also prove to be beneficial in maximizing the amount of NSCs that reach the infarct area of the brain following stroke.

Although the optimal route of NSC delivery remains unresolved, stem cells can be transplanted intracranially into the damaged or penumbral parenchyma [36, 68,69,70]. Also, NSCs can migrate from intravascular space to the injured brain following intravascular delivery via tail vein [71] or intra-arterial [72] injections. Intravenously administered stem cells can be trapped in filtering organs such as the liver, spleen, and lungs [73,74,75,76]. However, intra-arterial administration of stem cells may provide a more direct route to the lesion with better survival and engraftment of donor cells [77, 78].

Ischemic stroke pathophysiology

The pathophysiology of ischemic stroke is complex and still not completely understood. In addition to the initial ischemic insult, the CNS accumulates additional damage from reperfusion, BBB disruption, and inflammation as time from stroke onset progresses. In order to understand the therapeutic effects of NSC transplantation at the subacute or chronic stroke phase, it is crucial to first understand the pathophysiological cascades that occur with ischemic stroke.

Blood-brain barrier and reperfusion injury

Integrity of the BBB is very important as it forms a physical barrier created by endothelial tight junction proteins that restrict molecular trafficking through a transcellular route [79, 80]. The BBB is formed from complex interactions between neurons, glial cells, vascular cells, and the extracellular matrix, which are collectively known as the neurovascular unit (NVU). This complex structure maintains brain homeostasis and proper neuronal signaling by regulating the transport of substances into and out of the CNS [81,82,83]. The BBB also regulates the entry of leukocytes into the CNS for routine immune surveillance and response to infections [84] or after tissue damage when debris needs to be cleared [85].

Ischemic stroke compromises the integrity of the BBB. Ischemia causes metabolic distress due to the deprivation of oxygen and glucose. Energy failure from the depletion of adenosine triphosphate (ATP) stores results in subsequent lactic acidosis, ion transport dysregulation, and the accumulation of extracellular glutamate [86]. These metabolic consequences contribute greatly to endothelial swelling and disruption of the BBB following ischemic insult [87].

Restoration of blood flow is critical to limit damage from ischemic stroke. However, reperfusion of the infarct tissue with oxygenated blood challenges the BBB with oxidative stress and contributes to disruption of the BBB through oxidative damage to cellular molecules, upregulation of inflammatory mediators and matrix metalloproteinases (MMPs), and modulation of tight junction proteins [88,89,90]. Reperfusion causes a biphasic opening of the BBB, which can cause further damage to the CNS. However, the exact mechanisms underlying increased BBB permeability in the early ischemia-reperfusion (IR) stages are not well understood. The initial opening of the BBB is reversible and occurs within several hours after reperfusion [91], but the second opening is irreversible and occurs 24–72 h after reperfusion [92]. The initial opening is associated with disruption of tight junction proteins from oxidative stress induced after reperfusion [93, 94]. MMPs are able to disrupt tight junction proteins and this makes the BBB leaky and facilitates transport of toxic substances into the ischemic tissue [95]. Later consequences of damage to tight junctions in the BBB include upregulation of pro-inflammatory cytokines and infiltration of peripheral immune cells into the CNS.

In general, the major stages of BBB disruption from reperfusion following ischemic stroke include endothelial swelling, followed by disruption of tight junctions between vascular cells, and eventually complete vascular disruption [96]. Given that the BBB is functionally important for protection against neurotoxic agents and inflammation, preservation of BBB integrity is an attractive therapeutic strategy for ischemic stroke.

Inflammatory and immune responses

Inflammation is a critical component of the pathophysiology of stroke [97, 98]. Increased permeability of the BBB from reperfusion injury allows peripheral innate and adaptive immune cells, including neutrophils, macrophages, and T cells, to infiltrate the CNS [99]. Although inflammation and immune cell activity both play important roles in wound healing, tissue remodeling, and recovery, a large body of evidence demonstrates that the inflammatory cascade following ischemic stroke can also exacerbate damage to the CNS [100].

Ischemic injury to the CNS triggers the release of inflammatory mediators from dying cells and stimulates an innate immune response [101]. Loss of contact with live neurons and the release of danger-associated molecular pattern molecules following stroke activate resident microglial cells [102,103,104], which regularly survey the brain for damage. These native, innate immune cells of the CNS exist in two functionally different phenotypic states. Loss of cell-cell contact with healthy neurons [102,103,104] and receptor activation in response to extracellular glutamate accumulation [105] during ischemic stroke polarize microglia toward the pro-inflammatory M1 phenotype, which secretes factors such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and reactive oxygen species [106]. Alternatively, microglia can polarize toward the M2 phenotype. M2 microglia are beneficial for resolving post-stroke inflammation by secreting anti-inflammatory cytokines such as IL-10 and TGF-β in addition to neurotrophic factors [106, 107].

Less than 24 h after ischemic stroke and subsequent reperfusion injury, peripheral innate immune cells begin to adhere to the damaged endothelium of the BBB and infiltrate the CNS [108]. Macrophages are a major initial infiltrate following stroke [109], and similar to microglia, these immune cells can exhibit functionally different phenotypes [110]. Macrophages with the M1 phenotype worsen damage to the CNS after stroke by secreting pro-inflammatory molecules such as TNF-α, IL-8, and IL-12 [98111]. In contrast, macrophages of the M2 phenotype are inflammation-resolving and secrete anti-inflammatory cytokines such as TFG-β and IL-10 [98, 112]. M2 macrophages also participate in the removal of ischemic debris and this phenotype is generally thought to be beneficial for stroke outcome [111]. Infiltrating M1 macrophages recruit neutrophils into the CNS via secretion of IL-8, which contribute to further inflammation and tissue damage by releasing nitric oxide, MMPs, and cathepsins [98]. Infiltration and secretion of factors by peripheral macrophages constitute the acute phase of the inflammatory cascade following ischemic stroke.

The adaptive immune response is also a key component of ischemic stroke pathophysiology. After the infiltration of monocyte-derived macrophages, peripheral CD4+ and CD8+ T lymphocytes begin to enter the CNS through the disrupted BBB [113, 114]. The accumulation of antigen-presenting cells that express major histocompatibility class II (MHC II) molecule and co-stimulatory molecule CD80 coincides with the height of lymphocyte infiltration in ischemic tissue [115, 116]. Depletion of lymphocytes in mice suggests that T cells, but not B cells, are responsible for further CNS damage in the acute stroke phase [117, 118]. For instance, CNS-specific type 1 T helper (Th1) cells secrete interferon gamma (IFNγ) following ischemic stroke to recruit immune cells into the CNS [119] and activate cytotoxic CD8+ T cells, which exacerbate CNS damage by inducing apoptosis via caspase activation and Fas ligand signaling [120]. In addition, γ δ T cells contribute to CNS injury following ischemia through the secretion of the pro-inflammatory cytokine IL-17 [121], while regulatory T cells (Treg) are protective through their secretion of IL-10 and TGF-β [122, 123].

Neutrophils are another type of immune infiltrate found in the ischemic hemisphere following reperfusion [124]. However, the arrival of these cells in brain parenchyma is preceded by previously discussed microglia, macrophages, and lymphocytes [115]. The presence of neutrophils relies upon intercellular adhesion molecule 1 (ICAM-1) expression [125] and is associated with increased secretion of proteolytic enzymes and worsened post-ischemic CNS injury in rodent models of stroke [98, 126]. In addition, neutrophil accumulation correlates with poor neurological outcome and severity of brain damage in human stroke patients [127]. Although there have been some reports that blocking neutrophil recruitment in hyperlipidemic mice reduces stroke damage [126], other preclinical studies report no significant effect of neutrophil depletion on ischemic stroke outcome [128].

Neuroinflammation after stroke is self-limited and eventually is resolved when pro-inflammatory mediators become further dampened by secretion of anti-inflammatory molecules, resolvins, and protectins [98]. Although much research has indicated that the inflammatory cascade further contributes to CNS damage, post-stroke neuroinflammation also plays important roles in structural and functional reorganization of the brain after ischemic injury [129]. Clearance of dead cells is completed by an inflammatory response comprised of activated resident microglia and infiltrated peripheral macrophage that gain access to the brain through the choroid plexus-cerebrospinal fluid route [98, 130, 131]. These innate immune cells are attracted to chemokines and purines released from damaged and dying brain cells [132, 133]. Phagocytosis of debris and dying cells promotes upregulation of anti-inflammatory mediators such as TGF-β and IL-10 in activated macrophages and monocytes [134], which help to dampen Th1 and Th2 responses, promote Treg development, and ultimately resolve post-stroke neuroinflammation [135]. Indeed, depletion of monocyte-derived macrophages during the acute post-stroke inflammatory response actually impairs long-term functional recovery in mice [129]. Therefore, immunomodulation to dampen inflammation associated with further CNS damage while preserving immune cell activity responsible for debris clearance and functional remodeling may serve as an excellent strategy for stem cell stroke therapy.

Tissue loss and behavioral dysfunction

Stroke ultimately leads to extreme tissue death within the CNS. Ischemia results in the depletion of cellular energy stores and this in turn leads to failure of neurons to maintain ionic balance or reuptake of neurotransmitters [136]. In particular, metabolic catastrophe from stroke results in the extracellular accumulation of glutamate, a main excitatory neurotransmitter. Excess extracellular glutamate binds to and activates NMDA and AMPA receptors, thereby promoting a high influx of calcium. Calcium overload in neurons activates proteases and lipases, which degrade cellular organelles and proteins and contribute to cell death [137]. For instance, calcium overload disrupts mitochondria, which house apoptotic proteins. This results in mitochondrial release of cytochrome c and subsequent activation of executioner caspases and downstream cell death pathways in neurons [138, 139].

Edema also contributes to cell death and tissue loss following ischemic stroke. Overactivation of glutamate receptors on neurons promotes the inappropriate influx of sodium and water, thereby causing pathogenic cell swelling [137]. In addition, a compromised integrity of the BBB allows entry of foreign proteins, fluid, and immune cells into the extracellular space within the CNS, which contributes to vasoactive edema and amplifies tissue damage [140]. Vasoactive edema is eventually resolved from post-stroke angiogenesis, but the generation of new blood vessels is too slow to prevent acute edema-associated CNS damage following stroke.

In association with cell death and tissue loss, neural circuitry is also disrupted following stroke. As mentioned above, ischemic insult results in excitotoxicity and sustained depression of inhibitory neurotransmission, primarily through the accumulation of extracellular glutamate [137, 141] and reduction in GABAA receptor expression [142]. This further contributes to behavioral dysfunction and disability after stroke.

Endogenous repair mechanisms

Angiogenesis

Although the brain has a limited repair capacity [143], spontaneous stroke recovery occurs [144, 145]. Angiogenesis, the generation of new blood vessels from pre-existing blood vessels, takes place after brain ischemia and is hypothesized to contribute to CNS plasticity and functional recovery [146,147,148,149]. Evidence demonstrates that endothelial cell proliferation begins in as little as 12 h following stroke and can persist for several weeks because of the upregulation of growth factors and angiogenic genes [150,151,152]. Specifically, angiogenesis is stimulated in the ischemic penumbra, which is comprised of tissue proximal to the infarct core and is where endogenous recovery mechanisms take place after ischemic stroke [146]. Higher densities of vessels in the ischemic penumbra are associated with prolonged survival following ischemic stroke [146, 152, 153], and further damage to the penumbra after stroke can have catastrophic consequences for recovery [154].

Angiogenesis has been associated with improved neurological function following stroke. For instance, growth factors that stimulate angiogenesis may also directly promote the survival of cells in the ischemic penumbra [155]. In addition, angiogenesis following ischemic stroke facilitates both increased blood supply and removal of necrotic tissue while establishing a vascular niche for NSC proliferation and migration [156, 157]. However, angiogenic potential can be limited by multiple factors, including aging [158]. Given that ischemic stroke usually occurs later in life, further research on stem cell therapies to improve vascular remodeling after stroke is greatly needed.

Endogenous neurogenesis and astrogenesis

Ischemic stroke increases endogenous neurogenesis [33], which includes the proliferation and differentiation of NSCs [32, 33, 144]. This process is important in the context of stroke because NSCs can replace damaged neuroblasts in response to stroke injury [159]. Angiogenesis and neurogenesis are coupled in the NVU in that endothelial cells participating in angiogenesis provide growth factors that regulate NSC self-renewal and neurogenesis [160]. The size and density of microvessels change after stroke [161], and neuroblasts from the SVZ migrate close to areas of vascular remodeling [162]. Additionally, NSCs improve angiogenesis via trophic support such as paracrine stimulation with vascular endothelial growth factor (VEGF) and can influence capillary blood flow in the CNS [163, 164]. This highlights a reciprocal relationship between neurogenesis and vascular remodeling after stroke.

Unfortunately, neurogenesis from endogenous NSCs does not supply enough cells to repair neurological damage and newly formed cells may not fully integrate into the neuronal network in ischemic stroke brains [33, 165, 166]. Therefore, transplantation of exogenous NSCs to aid endogenous neural progenitors may be an effective therapy to ameliorate CNS damage from stroke.

Astrocytes also play an important role in angiogenesis and neurogenesis by releasing neurotrophins and vascular growth factors [167, 168]. Astrocyte positioning can also mediate neurotransmission and maintain neuronal activity coupling with cerebral blood vessel activity [169]. In response to acute ischemic injury, astrocytes have neuroprotective effects, such as the formation of glial scars in the ischemic penumbra, uptake of excess glutamate, restriction of the spread of neurotoxic molecules, and increase of revascularization and stabilization of blood vessels in the CNS [170,171,172]. In response to ischemic injury, there is an increase in production of high thrombospondin-4–expressing (Thbs4hi) astrocytes from NSCs within the SVZ, which is mediated by direct Notch1 signaling and downstream Nfia transcription factor activity [173, 174]. These Thbs4hi astrocytes migrate to the injured cortex following ischemic injury in mice and help to mitigate microvascular hemorrhage into the brain parenchyma [173]. Moreover, Thbs4−/− mice exhibit dysfunctional SVZ astrogenesis and abnormal glial scar formation in addition to extensive, unresolved hemorrhaging in cortical regions following photothrombotic/ischemic injury [173]. Therefore, astrocytes generated from NSCs in the SVZ appear to play an important role in the resolution of BBB disruption in the early stages following stroke. However, in the chronic phase of stroke, the actions of astrocytes can be deleterious because they reduce neurological recovery through secretion of growth inhibitory molecules and glial scar formation, which result in poor connectivity of surviving neural pathways [175]. Thus, targeting early-phase stroke injury with stem cells to enhance endogenous neurogenesis and astrogenesis may reduce later complications of secondary stroke damage.

Neural stem cell transplantation for ischemic stroke

The environment within the CNS changes dramatically with time following initial ischemic injury. Owing to the complex pathophysiological cascades in ischemic stroke, the timing of NSC transplantation greatly dictates therapeutic mechanisms. Overall, exogenous NSCs may improve neurological outcome following stroke through the replacement of dead or damaged cells or through bystander effects in which NSCs secrete neurotrophic and anti-inflammatory factors to protect brain cells and promote repair.

Replacement of functional neurons and other brain cells is an obvious therapeutic goal of exogenous stem cell transplantation in the stroke-damaged brain. Indeed, grafted NSCs are able to eventually make synaptic connections with host neurons and display electrophysiological properties of mature neurons [176]. However, preclinical research now indicates that bystander effects of exogenous NSCs may be equally or more effective at improving neurological outcome following ischemic insult. It is well established that NSCs provide therapeutic gene products to modify the extracellular microenvironment and promote neuronal circuit plasticity [69, 177, 178]. For example, brain-derived neurotrophic factor (BDNF) is secreted by NSCs. BDNF is a major neurotrophin and promotes neuroprotection, angiogenesis, neurogenesis, and functional recovery after stroke [179,180,181,182]. Also, molecular neurotrophic factors (NTFs) help maintain healthy neurons and play important roles in extracellular matrix remodeling, cell generation, and proliferation. NTFs also protect sensitive neural tissue from damage [183]. An important neurotrophic factor that helps promote angiogenesis and also protects and repairs neural tissue is VEGF [155, 184]. VEGF acts as a potent mitogen and survival factor for endothelial cells [185, 186] and has a neuroprotective effect against ischemic injury [155, 187, 188]. When VEGF is inhibited, NSC-mediated protection is significantly decreased [189]. For instance, recent work has demonstrated that NSC-secreted VEGF upregulates glutamate transporter 1 (GLT-1) through PI3-K/Akt pathway activation in astrocytes to facilitate removal of peri-ischemic extracellular glutamate and thereby improve structural and functional plasticity following ischemic insult [190]. Ciliary neurotrophic factor, glial cell line–derived neurotrophic factor, and neural growth factor are other neurotrophic factors that play important roles in maintenance and protection of neural tissue [191,192,193,194,195,196]. Overall, the therapeutic mechanisms of exogenous NSCs may shift more toward functional cell replacement or bystander effects (or both) depending on whether they are transplanted during the early or chronic stroke phase.

Transplantation of NSCs at early stroke phase

As mentioned above, reperfusion injury, BBB disruption, and infiltration of immune cells cause further damage to the CNS in the early phase of stroke [88,89,90, 100]. Most commonly, pharmacological treatments have been employed to reduce acute/subacute stroke damage. For example, minocycline has shown potential to serve as a neuroprotective pharmacological agent for clinical treatment of stroke [197, 198] because of its anti-inflammatory and anti-apoptotic effects and ability to reduce BBB damage [199,200,201,202,203]. However, in some clinical scenarios where ischemic stroke is still in the acute/subacute phase, NSC transplantation may be used as a multifaceted neuroprotective strategy to limit the severity of damage and cell loss from ischemic insult. For instance, studies using the middle cerebral artery occlusion (MCAO) mouse model of ischemic stroke have demonstrated that NSC-engrafted brains 48 h post-stroke (24 h post-transplantation) show decreased inflammation, decreased BBB damage, and consequent improvement of long-term stroke outcome [69, 70, 177, 204,205,206,207]. Below are multiple actions of NSCs transplanted in the subacute phase of stroke.

BBB support

Early transplantation of NSCs helps to protect against damage to the BBB after ischemic reperfusion. Transplantation of hNSCs into the hippocampus of mice 24 h after MCAO resulted in extensive migration of NSCs to the lesions, reduction in infarct volume, and decreased damage to the BBB compared with untreated MCAO controls [69]. This rapid response suggests that the therapeutic mechanisms of NSCs were anti-inflammatory. In support of this hypothesis, hNSC-transplanted mice showed decreased microglia activation as well as decreased expression of inflammatory factors such as IL-6, IL-1β, and macrophage inflammatory protein-1α [69]. Similarly, transplantation of human iPSC (hiPSC)-NSCs into a stroke rodent model 24 h after ischemic reperfusion resulted in decreased levels of IgG, which is known to cross the compromised rodent BBB and leak into the brain parenchyma [70]. MMP activity, which is associated with dysfunction of tight junctions between endothelial cells of the BBB [70], was also decreased in rodents that received NSC transplants following stroke [70]. In another study, transplantation of adjudin-preconditioned mouse embryonic NSCs into the ipsilateral striatum at 24 h after MCAO in adult mice resulted in decreased infarct volume and amelioration of BBB disruption, which was marked by significantly decreased Evans Blue dye and IgG leakage into the brain parenchyma [204]. These results suggest that NSCs are able to preserve the integrity of the BBB and help prevent further damage to the CNS following stroke [70, 204]. Since tPA is known to exacerbate neural cell death and BBB damage beyond the 4.5 h window, using NSC transplantation to preserve the integrity of the BBB could have immense clinical applications for extending the narrow therapeutic window of tPA to benefit a greater population of stroke patients.

Inflammation

As mentioned above, ischemic stroke results in increased CNS inflammation as a result of activated astrocytes and microglia in addition to peripheral immune cell infiltration through the compromised BBB. Transplantation of NSCs in the subacute phase of ischemic stroke may also improve outcome by dampening this inflammatory cascade [177, 205, 206] (Fig. 2). Specifically, NSCs protect against further CNS damage by downregulating inflammatory regulator molecules such as TNF-α, monocyte chemoattractant protein-1 (MCP-1), IL-1β, IL-6, and Iba-1 [177, 206]. For example, hiPSC-NSC transplantation results in significantly lower amounts of Iba-1+ and ED1+ cells within and around the ischemic core in the stroke mouse model [207]. Animals in the transplanted group also had lower levels of glial fibrillary acidic protein-positive (GFAP+) astrocytes [207]. Similarly, NSC-treated mice had significantly reduced numbers of CD45+ and Iba-1+/MHC II immune cells within the brain post-stroke [206]. These results support the notion that NSCs can aid in functional recovery from ischemic insult by manipulating the extracellular microenvironment and decreasing inflammation.

Fig. 2
figure 2

Schematic diagram of NSC transplantation illustrating multiple therapeutic benefits in ischemic stroke. Exogenous NSCs can be transplanted intravenously or directly into the damaged or penumbral parenchyma. NSCs can replace damaged neural cells in ischemic brain tissue. NSCs may also help to prevent further tissue damage in ischemic stroke through their bystander effects. These bystander effects include support of the blood-brain barrier (BBB), neurons, astrocytes, and microglia, as well as increased angiogenesis, and modulation of immune responses that result from ischemic insult. Abbreviations: EC endothelial cell, NSC neural stem cell

Long-term outcome of NSC transplantation at the early (subacute) stroke phase

The goal for transplantation of NSCs in the early subacute phases of stroke is neuroprotection from the cytotoxic microenvironment following ischemia. However, early NSC transplantation is also able to elicit neurorestorative mechanisms to improve long-term outcome in the days and weeks following initial ischemic insult.

Improved neurological outcome

NSC transplantation in the early stroke phase has also been demonstrated to improve behavior: an indication of functional recovery and the ultimate readout of any stroke therapy. Evaluation of neurological function using modified Neurologic Severity Score (mNSS) tests showed that when hNSCs were transplanted early into the rat brain (1 d after stroke), hNSC-transplanted rats showed significantly improved behavioral scores and performance 2 weeks after transplantation [208]. Also, studies showed that, by 48 h post-injury, neurological function rapidly improved in mice that received hNSC transplantation at 24 h after MCAO compared with non-transplanted stroke controls [69]. Transplanted mice showed improvement in the forepaw adhesive removal test (which assesses sensorimotor deficits), the beam walk test for assessing balance, and the rotarod test for evaluating balance and motor coordination. Recovery of behavioral function persisted through a month of monitoring [69], suggesting that early intervention is paramount for achieving lasting positive outcomes in stroke patients. In support of these results, other studies have demonstrated that engrafting iPSC-NSCs into mouse brains 24 h after MCAO improves long-term neurological outcome [70] and that transplantation of exogenous primary NSCs [69] or iPSC-derived NSCs [70] into the striatum and cortex of stroke animals results in improved behavioral tests and recovery [69, 70, 209]. Also, in a rodent stroke model, intracranial transplantation of NSCs directly into the hippocampus (where neurogenesis is ongoing) facilitated rapid migration to damaged areas and improved recovery after stroke [69, 70]. Another study showed that conditionally immortalized NSCs, murine NSC line clone 36 (MHP36), promoted functional recovery following intracranial transplantation 2 d after MCAO in mice [210]. When analyzed at various time points until 28 d post-stroke, engrafted animals from this study showed improved foot-fault function assessed by the ladder rung test and also improved asymmetry scores in the spontaneous forelimb (cylinder) test [210]. In other studies, transplantation of NSCs at later time points in the subacute phase of stroke (3–4 d after MCAO) also resulted in reduced infarct volume and improved neurological outcome compared with controls [211,212,213]. Taken together, these preclinical studies clearly demonstrate that NSC transplantation in the subacute phase of stroke significantly improves neurological and behavioral recovery.

Angiogenesis

NSC transplantation may help to facilitate recovery from stroke by promoting angiogenesis, which is coupled to neurogenesis [160]. In one study, early transplantation of hNSCs (1 d) following stroke enhanced angiogenesis in rats [208]. At day 14 after NSC transplantation, the number of BrdU+/vWF+ (von Willebrand factor-positive) proliferating endothelial cells was increased in the ischemic region of NSC-engrafted rats, suggesting increased angiogenesis [208]. Another study showed that early transplantation (6 h after MCAO) of mouse NSCs (preconditioned with IL-6) into the cortex promoted angiogenesis marked by increased density of lectin-perfused blood vessels at 14 d after transplantation [209]. In another study, intravenous administration of magnetically tagged, human fetal NSCs 24 h after MCAO also resulted in an increased number of cells positive for vWF and augmented angiogenesis when brains were analyzed at 28 d post-injury [214]. In aged rats, transplantation of mouse embryonic NSCs into the ipsilateral striatum 24 h after MCAO was able to reduce infarct volume, at least partially through increased VEGF expression and enhanced angiogenesis [215]. These studies clearly indicate that NSC therapy during the early stroke phase is able to have long-lasting beneficial effects on neovascularization.

Neurogenesis

In addition to angiogenesis, studies indicate that transplantation of NSCs in the early stroke phase may benefit long-term recovery through functional neuronal replacement, either directly or through augmentation of the endogenous neurogenic response [70, 214,215,216,217,218,219,220,221,222]. For instance, implantation of exogenous human fetal NSCs into the ipsilateral striatum 48 h after MCAO in rats resulted in subpopulations of engrafted cells that differentiated to neuroblasts and mature neurons at 6 and 14 weeks in addition to increased amounts of proliferating and migrating neuroblasts from the SVZ [216].

Other studies have reported modest neuronal replacement following NSC transplantation in the early phases of stroke as well. When hNSCs were injected on the same day (D0) following transient forebrain ischemia in rats, intravenously engrafted hNSCs differentiated into mature neurons to replace lost neural cells in the adult hippocampus of human–rat neural chimeras [219, 220]. Similar results have been observed with intravenously transplanted, magnetically targeted hNSCs at 24 h after MCAO in rats [214]. Transplanted hNSCs also give rise to astrocytes in the rodent brain following transient forebrain ischemia [214, 219, 220] and intracranial hemorrhage [221]. Transplantation of mouse NSCs into the ipsilateral striatum of rats 24 h after MCAO also augmented endogenous neurogenesis and reduced infarct volume [215, 222]. Of note, even though limited cell replacement has been observed in studies, a considerable amount of time is required for NSCs to differentiate into functional neurons, and there is still not enough evidence that cell replacement is vital for NSC-mediated recovery. For instance, a different study demonstrated that intracranial iPSC-NSC engraftment at 24 h post-stroke resulted in survival of engrafted cells at 30 d post-transplant, but the vast majority of donor cells remained as nestin-positive NSCs, and only a small percentage of the exogenous hiPSC-NSCs co-expressed the neuronal marker TuJ-1 [70]. Thus, these findings clearly suggest that neural replacement may not be a main contributor to beneficial stroke outcome after NSC transplantation in the subacute stroke phase.

NSC transplantation at chronic stroke phase

Current clinical trials focus on the effect of stem cells on neurorestoration by administering cells during the chronic stroke stage when inflammatory signals are diminished. In rodent stroke models, pro-inflammatory cytokines were highly expressed in the acute/subacute stages of MCAO but decreased over time and returned to almost normal baseline levels 12 d after MCAO [223]. Stem cell transplantation at delayed time points (e.g., 7 d after stroke) in rodent models can avoid the cytotoxic environment in the acute/subacute phases of IR [224, 225]. Thus, transplantation of NSCs in this chronic phase of stroke may be an attractive model to test “neurorestorative” strategies to repair the damaged CNS. Indeed, preclinical research suggests that multiple actions of NSCs offer great promise in treating the chronic phase of stroke through multiple mechanisms, including enhancement of neurogenesis and angiogenesis, mediated at least in part by paracrine signaling through growth factors and neurotrophins.

Angiogenesis

In addition to enhancing the endogenous neurogenic response, several lines of evidence indicate that exogenous NSC transplantation during the chronic phase of stroke facilitates angiogenesis and neovascularization. For example, transplanting iPSC-NSCs into the penumbra intracranially 7 d after stroke in neonatal rat brains resulted in greater expression of VEGF in the peri-infarct region [226]. At 21 d post-stroke (14 d post-transplant), iPSC-NSC engraftment increased angiogenesis, as shown by the increased number of BrdU/collagen IV co-labeled cells in the vessel basement membrane in the peri-infarct area compared with stroke controls [226]. In support of these results, a study that modeled ischemic stroke in adult nude male rats with distal MCAO demonstrated that transplantation of NSCs at 7 d post-injury resulted in delayed “spatio-temporal” enhancement of neovascularization, which was dependent upon NSC expression of human VEGF [227]. Transplantation of IL-6 preconditioned fetal murine NSCs into the peri-infarct cortex 7 d after MCAO also enhances angiogenesis through STAT3-mediated upregulation of VEGF [209], and transplantation of SDF-1α–overexpressing iPSC-derived NSCs in the same brain region at 7 d post-focal ischemia also displayed significantly increased angiogenesis and recovery [228]. In another study using the microsphere-induced cerebral embolism rat model, animals that received intravenous injection of NSCs on day 7 after cerebral ischemia showed improved angiogenesis by day 28 compared with controls [229]. Moreover, animals that received exogenous NSCs displayed greater expression levels of VEGF, and its receptor VEGFR2 in brain capillaries [229]. Similar results have been observed with the CTX0E03 human immortalized NSC cell line [230], whereby stroke-damaged mouse brains that received CTX0E03 engraftments demonstrated significant increases in VWF+ microvessels compared with vehicle-treated brains 7 d after transplant [231]. Taken together, these studies suggest that exogenous NSCs largely facilitate recovery in the chronic phase of stroke through augmentation of angiogenesis and neovascularization, which is likely a result of increased VEGF paracrine signaling.

Neurogenesis

Disability from stroke is ultimately a result of mass cell death and tissue loss in the CNS. Therefore, direct replacement of lost brain cells through neurogenesis is an obvious therapeutic mechanism of NSC transplantation in the chronic phase of stroke. Several preclinical studies have demonstrated that transplanted NSCs differentiated into mature neurons and integrated with host neuronal circuitry in the chronic phase of stroke and engrafted animals showed improved functional and behavioral recovery [176, 226, 232,233,234,235]. For instance, engrafted iPSC-NSCs at 7 d after stroke in neonatal rat brains differentiated into neurons showing significantly greater numbers of BrdU/NeuN+ cells at 21 d post-stroke [226]. Rats that received the iPSC-NSC engraftments also performed better on the vibrissae-elicited forelimb placement test [226], which examines forepaw motor function and whisker somatosensation as proxies for neurological recovery following stroke [236, 237]. Another study demonstrated that hiPSC-derived neuroepithelial-like stem cells transplanted 9 d after MCAO were able to differentiate into mature neurons with electrophysiological activity in stroke brains of rats and mice [176]. Moreover, at 4 weeks post-transplantation, engrafted cells were receiving synaptic input from surrounding endogenous neurons, extended their axonal projections, and improved motor recovery in behavioral tests [176]. Transplantation of human ESC-NSCs 14 d after MCAO enhanced endogenous neurogenesis, measured by expression of doublecortin (Dcx), a marker for immature neurons, at 60 d in the ipsilateral SVZ in both young and aged rats [232]. Delayed transplantation of human ESC-derived neural precursor cells also reduced infarct volume and improved behavioral outcome in rats [233]. Transplantation of CTX0E03 in rats 4 weeks after MCAO significantly increases endogenous proliferation of Dcx+ neuroblasts in the striatum of the ischemic host brain [234] and leads to dose-related improved behavioral and functional improvements [235].

Of note, not all transplanted NSCs are able to survive and differentiate into mature neurons within the stroke-damaged brain [238]. Transplantation of NSCs after 7 d showed that grafted NSCs made synaptic connections with host neurons and displayed electrophysiological properties of mature neurons [176]. However, the number of NSCs that successfully differentiated and survived in the host brain was lower than expected [176]. Some studies have reported that NSC-engrafted brains from adult rats showed a significantly decreased number of donor cells from 4 to 12 weeks after stroke [177]. As mentioned above, differentiation of NSCs into functional CNS cell types within the stroke-damaged brain is a feat that requires significant time in a hostile microenvironment. Even though modest replacement of functional neurons has been observed in some NSC transplantation studies, it is still unclear whether cell replacement mechanisms are largely responsible for improved outcome. In fact, reports that low graft survival is still associated with improved recovery in the chronic stroke phase suggest that the therapeutic potential of NSC transplantation may place greater emphasis on augmentation of host neurogenesis and angiogenesis.

Conclusions

Our ability to address neurovascular diseases will require multifaceted solutions, including pharmacological, cell replacement, genetic, and tissue engineering. The fundamental biological attributes of NSCs may likely be harnessed to circumvent some of the obstacles that currently exist in treating stroke. NSCs can be used adjunctively with other interventions such as tPA. These interventions may indeed work synergistically with each other. Specifically, NSCs show anti-inflammatory, anti-apoptotic, pro-angiogenic, and pro-regenerative effects, which can ameliorate the adverse side effects associated with tPA treatment in stroke patients. NSC transplantation in preclinical models also clearly improves functional recovery after stroke. However, more research is still needed to contribute to successful clinical trials and expand patient inclusion criteria for stroke treatment. For example, diabetes not only increases the risk of having a stroke but increases post-stroke mortality by three to four times [239, 240]. Owing to enhanced risk of BBB damage, patients with diabetes are also not able to receive tPA treatment [239]. In rodent models of stroke, diabetic mice have a more rapid increase in MMP-9 mRNA and protein activity post-stroke compared with controls [241]. Another mechanism by which diabetes increases risk of damage is through increased inflammation, as diabetic mouse models have shown increased inflammatory markers [241]. Therefore, more therapies are needed in order to treat stroke in patients with comorbidities.

Although much of the preclinical research on stem cell transplantation for stroke therapy has yielded promising results, certain safety concerns must also be properly addressed. For instance, one study that transplanted iPSCs into the ipsilateral striatum 24 h after MCAO in mice reported tumorigenesis from the transplanted cells, which was exacerbated in ischemia-damaged brains versus sham-operated brains [242]. Even though iPSC transplantation was able to supply Dcx+ neuroblasts and some mature neurons, recovery was still delayed in MCAO mice that received iPSCs compared with MCAO control mice that received only phosphate-buffered saline [242]. Nevertheless, these results highlight the importance of minimizing tumorigenic potential of stem cells for treatment of ischemic stroke.

Clinically, stem cell therapy is currently aiming for stroke rehabilitation by transplanting stem cells during the later recovery periods following stroke, and NSCs are beginning to gain attention for human trials [243,244,245]. For example, a phase 1 clinical trial with hNSCs showed no safety concerns and some promising signs of efficacy [244]. However, stem cell therapy for the subacute phase may benefit more stroke patients by ameliorating early-phase stroke injury and subsequently improving chronic-phase stroke outcome. Therefore, there is a major need for additional studies on early-phase post-stroke BBB disruption, how tPA contributes to it, and where to therapeutically target this pathological mechanism to increase the therapeutic window of tPA and ultimately improve stroke outcome.

Abbreviations

BBB:

Blood-brain barrier

BDNF:

Brain-derived neurotrophic factor

CNS:

Central nervous system

CXCR4:

C-X-C chemokine type 4 receptor

EGF:

Epidermal growth factor

ESC:

Embryonic stem cell

FGF:

Fibroblast growth factor

hiPSC:

Human induced pluripotent stem cell

hNSC:

Human neural stem cell

iPSC:

Induced pluripotent stem cell

IR:

Ischemia-reperfusion

MCAO:

Middle cerebral artery occlusion

MMP-9:

Matrix metalloproteinase-9

NSC:

Neural stem cell

NTF:

Neurotrophic factor

NVU:

Neurovascular unit

SDF-1α:

Stromal cell–derived factor 1-alpha

STAIR:

Stroke Treatment Academic Industry Roundtable

SVZ:

Subventricular zone

TGF-β:

Transforming growth factor-beta

Th1:

Type 1 T helper

Th2:

Type 2 T helper

TNF-α:

Tumor necrosis factor-alpha

tPA:

Tissue plasminogen activator

VEGF:

Vascular endothelial growth factor

References

  1. Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, de Ferranti SD, Floyd J, Fornage M, Gillespie C, et al. Heart disease and stroke Statistics-2017 update: a report from the American Heart Association. Circulation. 2017;135(10):e146–603.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Feigin VL, Forouzanfar MH, Krishnamurthi R, Mensah GA, Connor M, Bennett DA, Moran AE, Sacco RL, Anderson L, Truelsen T, et al. Global and regional burden of stroke during 1990-2010: findings from the global burden of disease study 2010. Lancet. 2014;383(9913):245–54.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Yamashita T, Deguchi K, Nagotani S, Abe K. Vascular protection and restorative therapy in ischemic stroke. Cell Transplant. 2011;20(1):95–7.

    Article  PubMed  Google Scholar 

  4. Roth JM. Recombinant tissue plasminogen activator for the treatment of acute ischemic stroke. Proc (Baylor Univ Med Cent). 2011;24(3):257–9.

    Article  Google Scholar 

  5. Kilic E, Bahr M, Hermann DM. Effects of recombinant tissue plasminogen activator after intraluminal thread occlusion in mice: role of hemodynamic alterations. Stroke. 2001;32(11):2641–7.

    Article  PubMed  CAS  Google Scholar 

  6. Yandava BD, Billinghurst LL, Snyder EY. “Global” cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci U S A. 1999;96(12):7029–34.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Lee JP, Jeyakumar M, Gonzalez R, Takahashi H, Lee PJ, Baek RC, Clark D, Rose H, Fu G, Clarke J, et al. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med. 2007;13(4):439–47.

    Article  PubMed  CAS  Google Scholar 

  8. Park KI, Teng YD, Snyder EY. The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol. 2002;20(11):1111–7.

    Article  PubMed  CAS  Google Scholar 

  9. Jeyakumar M, Lee JP, Sibson NR, Lowe JP, Stuckey DJ, Tester K, Fu G, Newlin R, Smith DA, Snyder EY, et al. Neural stem cell transplantation benefits a monogenic Neurometabolic disorder during the symptomatic phase of disease. Stem Cells. 2009;27(9):2362–70.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Ourednik J, Ourednik V, Lynch WP, Schachner M, Snyder EY. Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol. 2002;20(11):1103–10.

    Article  PubMed  CAS  Google Scholar 

  11. Redmond DE Jr, Bjugstad KB, Teng YD, Ourednik V, Ourednik J, Wakeman DR, Parsons XH, Gonzalez R, Blanchard BC, Kim SU, et al. Behavioral improvement in a primate Parkinson's model is associated with multiple homeostatic effects of human neural stem cells. Proc Natl Acad Sci U S A. 2007;104(29):12175–80.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Sinden JD, Vishnubhatla I, Muir KW. Prospects for stem cell-derived therapy in stroke. Prog Brain Res. 2012;201:119–67.

    Article  PubMed  Google Scholar 

  13. Miljan EA, Sinden JD. Stem cell treatment of ischemic brain injury. Curr Opin Mol Ther. 2009;11(4):394–403.

    PubMed  CAS  Google Scholar 

  14. Elder GA, De Gasperi R, Sosa MAG. Research update: neurogenesis in adult brain and neuropsychiatric disorders. Mt Sinai J Med. 2006;73(7):931–40.

    PubMed  Google Scholar 

  15. Djavadian RL. Serotonin and neurogenesis in the hippocampal dentate gyrus of adult mammals. Acta Neurobiol Exp (Wars). 2004;64(2):189–200.

    Google Scholar 

  16. Alvarez-Buylla A, Garcia-Verdugo JM. Neurogenesis in adult subventricular zone. J Neurosci. 2002;22(3):629–34.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. Alvarez-Buylla A, Lois C. Neuronal stem cells in the brain of adult vertebrates. Stem Cells. 1995;13(3):263–72.

    Article  PubMed  CAS  Google Scholar 

  18. Lledo PM, Alonso M, Grubb MS. Adult neurogenesis and functional plasticity in neuronal circuits. Nat Rev Neurosci. 2006;7(3):179–93.

    Article  PubMed  CAS  Google Scholar 

  19. Hatten ME, Heintz N. Mechanisms of neural patterning and specification in the developing cerebellum. Annu Rev Neurosci. 1995;18:385–408.

    Article  PubMed  CAS  Google Scholar 

  20. Miale IL, Sidman RL. An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp Neurol. 1961;4:277–96.

    Article  PubMed  CAS  Google Scholar 

  21. Marzban H, Del Bigio MR, Alizadeh J, Ghavami S, Zachariah RM, Rastegar M. Cellular commitment in the developing cerebellum. Front Cell Neurosci. 2014;8:450.

    PubMed  Google Scholar 

  22. Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adult neurogenesis. Cell. 2008;132(4):645–60.

    Article  PubMed  CAS  Google Scholar 

  23. Spalding KL, Bergmann O, Alkass K, Bernard S, Salehpour M, Huttner HB, Bostrom E, Westerlund I, Vial C, Buchholz BA, et al. Dynamics of hippocampal neurogenesis in adult humans. Cell. 2013;153(6):1219–27.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Boldrini M, Fulmore CA, Tartt AN, Simeon LR, Pavlova I, Poposka V, Rosoklija GB, Stankov A, Arango V, Dwork AJ, et al. Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell. 2018;22(4):589–599.e585.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. Ernst A, Alkass K, Bernard S, Salehpour M, Perl S, Tisdale J, Possnert G, Druid H, Frisen J. Neurogenesis in the striatum of the adult human brain. Cell. 2014;156(5):1072–83.

    Article  PubMed  CAS  Google Scholar 

  26. Sanai N, Tramontin AD, Quinones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S, Lawton MT, McDermott MW, Parsa AT, Manuel-Garcia Verdugo J, et al. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature. 2004;427(6976):740–4.

    Article  PubMed  CAS  Google Scholar 

  27. Bergmann O, Liebl J, Bernard S, Alkass K, Yeung MS, Steier P, Kutschera W, Johnson L, Landen M, Druid H, et al. The age of olfactory bulb neurons in humans. Neuron. 2012;74(4):634–9.

    Article  PubMed  CAS  Google Scholar 

  28. Sanai N, Nguyen T, Ihrie RA, Mirzadeh Z, Tsai HH, Wong M, Gupta N, Berger MS, Huang E, Garcia-Verdugo JM, et al. Corridors of migrating neurons in the human brain and their decline during infancy. Nature. 2011;478(7369):382–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Altman J, Das GD. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol. 1965;124(3):319–35.

    Article  PubMed  CAS  Google Scholar 

  30. Sorrells SF, Paredes MF, Cebrian-Silla A, Sandoval K, Qi D, Kelley KW, James D, Mayer S, Chang J, Auguste KI, et al. Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature. 2018;555(7696):377–81.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  31. Liu YP, Lang BT, Baskaya MK, Dempsey RJ, Vemuganti R. The potential of neural stem cells to repair stroke-induced brain damage. Acta Neuropathol. 2009;117(5):469–80.

    Article  PubMed  Google Scholar 

  32. Kernie SG, Parent JM. Forebrain neurogenesis after focal ischemic and traumatic brain injury. Neurobiol Dis. 2010;37(2):267–74.

    Article  PubMed  Google Scholar 

  33. Kokaia Z, Lindvall O. Neurogenesis after ischaemic brain insults. Curr Opin Neurobiol. 2003;13(1):127–32.

    Article  PubMed  CAS  Google Scholar 

  34. Macas J, Nern C, Plate KH, Momma S. Increased generation of neuronal progenitors after ischemic injury in the aged adult human forebrain. J Neurosci. 2006;26(50):13114–9.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  35. Marti-Fabregas J, Romaguera-Ros M, Gomez-Pinedo U, Martinez-Ramirez S, Jimenez-Xarrie E, Marin R, Marti-Vilalta JL, Garcia-Verdugo JM. Proliferation in the human ipsilateral subventricular zone after ischemic stroke. Neurology. 2010;74(5):357–65.

    Article  PubMed  CAS  Google Scholar 

  36. Lee JP, McKercher S, Muller FJ, Snyder EY. Neural stem cell transplantation in mouse brain. Curr Protoc Neurosci. 2008, Chapter 3:Unit 3 10.

  37. Carpenter MK, Cui X, Hu ZY, Jackson J, Sherman S, Seiger A, Wahlberg LU. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol. 1999;158(2):265–78.

    Article  PubMed  CAS  Google Scholar 

  38. Gensburger C, Labourdette G, Sensenbrenner M. Brain basic fibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro. FEBS Lett. 1987;217(1):1–5.

    Article  PubMed  CAS  Google Scholar 

  39. Palmer TD, Ray J, Gage FH. FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci. 1995;6(5):474–86.

    Article  PubMed  CAS  Google Scholar 

  40. Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol. 1996;175(1):1–13.

    Article  PubMed  CAS  Google Scholar 

  41. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T. Neural progenitors from human embryonic stem cells. Nat Biotechnol. 2001;19(12):1134–40.

    Article  PubMed  CAS  Google Scholar 

  42. Daadi MM, Maag AL, Steinberg GK. Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model. PLoS One. 2008;3(2):e1644.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol. 2001;19(12):1129–33.

    Article  PubMed  CAS  Google Scholar 

  44. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27(3):275–80.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Newman MB, Misiuta I, Willing AE, Zigova T, Karl RC, Borlongan CV, Sanberg PR. Tumorigenicity issues of embryonic carcinoma-derived stem cells: relevance to surgical trials using NT2 and hNT neural cells. Stem Cells Dev. 2005;14(1):29–43.

    Article  PubMed  CAS  Google Scholar 

  46. Daadi MM, Steinberg GK. Manufacturing neurons from human embryonic stem cells: biological and regulatory aspects to develop a safe cellular product for stroke cell therapy. Regen Med. 2009;4(2):251–63.

    Article  PubMed  CAS  Google Scholar 

  47. Liu J. Induced pluripotent stem cell-derived neural stem cells: new hope for stroke? Stem Cell Res Ther. 2013;4(5):115.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Park IH, Lerou PH, Zhao R, Huo H, Daley GQ. Generation of human-induced pluripotent stem cells. Nat Protoc. 2008;3(7):1180–6.

    Article  PubMed  CAS  Google Scholar 

  49. Newman AM, Cooper JB. Lab-specific gene expression signatures in pluripotent stem cells. Cell Stem Cell. 2010;7(2):258–62.

    Article  PubMed  CAS  Google Scholar 

  50. Chin MH, Mason MJ, Xie W, Volinia S, Singer M, Peterson C, Ambartsumyan G, Aimiuwu O, Richter L, Zhang J, et al. Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell. 2009;5(1):111–23.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Kim J, Efe JA, Zhu S, Talantova M, Yuan X, Wang S, Lipton SA, Zhang K, Ding S. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc Natl Acad Sci U S A. 2011;108(19):7838–43.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Thier M, Worsdorfer P, Lakes YB, Gorris R, Herms S, Opitz T, Seiferling D, Quandel T, Hoffmann P, Nothen MM, et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell. 2012;10(4):473–9.

    Article  PubMed  CAS  Google Scholar 

  53. Han DW, Tapia N, Hermann A, Hemmer K, Hoing S, Arauzo-Bravo MJ, Zaehres H, Wu G, Frank S, Moritz S, et al. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell. 2012;10(4):465–72.

    Article  PubMed  CAS  Google Scholar 

  54. Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol. 2002;174(1):11–20.

    Article  PubMed  Google Scholar 

  55. Pluchino S, Peruzzotti-Jametti L. Rewiring the ischaemic brain with human-induced pluripotent stem cell-derived cortical neurons. Brain. 2013;136(Pt 12):3525–7.

    Article  PubMed  Google Scholar 

  56. Politi LS, Bacigaluppi M, Brambilla E, Cadioli M, Falini A, Comi G, Scotti G, Martino G, Pluchino S. Magnetic-resonance-based tracking and quantification of intravenously injected neural stem cell accumulation in the brains of mice with experimental multiple sclerosis. Stem Cells. 2007;25(10):2583–92.

    Article  PubMed  Google Scholar 

  57. Tran PB, Banisadr G, Ren D, Chenn A, Miller RJ. Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain. J Comp Neurol. 2007;500(6):1007–33.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Tran PB, Ren D, Veldhouse TJ, Miller RJ. Chemokine receptors are expressed widely by embryonic and adult neural progenitor cells. J Neurosci Res. 2004;76(1):20–34.

    Article  PubMed  CAS  Google Scholar 

  59. Tran PB, Miller RJ. Chemokine receptors: signposts to brain development and disease. Nat Rev Neurosci. 2003;4(6):444–55.

    Article  PubMed  CAS  Google Scholar 

  60. Carbajal KS, Schaumburg C, Strieter R, Kane J, Lane TE. Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis. Proc Natl Acad Sci U S A. 2010;107(24):11068–73.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Imitola J, Raddassi K, Park KI, Mueller FJ, Nieto M, Teng YD, Frenkel D, Li J, Sidman RL, Walsh CA, et al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1α/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A. 2004;101(52):18117–22.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Itoh T, Satou T, Ishida H, Nishida S, Tsubaki M, Hashimoto S, Ito H. The relationship between SDF-1alpha/CXCR4 and neural stem cells appearing in damaged area after traumatic brain injury in rats. Neurol Res. 2009;31(1):90–102.

    Article  PubMed  CAS  Google Scholar 

  63. Schober A, Karshovska E, Zernecke A, Weber C. SDF-1alpha-mediated tissue repair by stem cells: a promising tool in cardiovascular medicine? Trends Cardiovasc Med. 2006;16(4):103–8.

    Article  PubMed  CAS  Google Scholar 

  64. Togel F, Isaac J, Hu Z, Weiss K, Westenfelder C. Renal SDF-1 signals mobilization and homing of CXCR4-positive cells to the kidney after ischemic injury. Kidney Int. 2005;67(5):1772–84.

    Article  PubMed  Google Scholar 

  65. Vasyutina E, Stebler J, Brand-Saberi B, Schulz S, Raz E, Birchmeier C. CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells. Genes Dev. 2005;19(18):2187–98.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Xue L, Wang J, Wang W, Yang Z, Hu Z, Hu M, Ding P. The effect of stromal cell-derived factor 1 in the migration of neural stem cells. Cell Biochem Biophys. 2014;70(3):1609–16.

    Article  PubMed  CAS  Google Scholar 

  67. Robin AM, Zhang ZG, Wang L, Zhang RL, Katakowski M, Zhang L, Wang Y, Zhang C, Chopp M. Stromal cell-derived factor 1alpha mediates neural progenitor cell motility after focal cerebral ischemia. J Cereb Blood Flow Metab. 2006;26(1):125–34.

    Article  PubMed  CAS  Google Scholar 

  68. Smith EJ, Stroemer RP, Gorenkova N, Nakajima M, Crum WR, Tang E, Stevanato L, Sinden JD, Modo M. Implantation site and lesion topology determine efficacy of a human neural stem cell line in a rat model of chronic stroke. Stem Cells. 2012;30(4):785–96.

    Article  PubMed  CAS  Google Scholar 

  69. Huang L, Wong S, Snyder EY, Hamblin MH, Lee JP. Human neural stem cells rapidly ameliorate symptomatic inflammation in early-stage ischemic-reperfusion cerebral injury. Stem Cell Res Ther. 2014;5(6):129.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Eckert A, Huang L, Gonzalez R, Kim HS, Hamblin MH, Lee JP. Bystander effect fuels human induced pluripotent stem cell-derived neural stem cells to quickly attenuate early stage neurological deficits after stroke. Stem Cells Transl Med. 2015;4(7):841–51.

    Article  PubMed  PubMed Central  Google Scholar 

  71. Song M, Kim Y, Kim Y, Ryu S, Song I, Kim SU, Yoon BW. MRI tracking of intravenously transplanted human neural stem cells in rat focal ischemia model. Neurosci Res. 2009;64(2):235–9.

    Article  PubMed  Google Scholar 

  72. Chua JY, Pendharkar AV, Wang N, Choi R, Andres RH, Gaeta X, Zhang J, Moseley ME, Guzman R. Intra-arterial injection of neural stem cells using a microneedle technique does not cause microembolic strokes. J Cereb Blood Flow Metab. 2011;31(5):1263–71.

    Article  PubMed  Google Scholar 

  73. Lee ST, Chu K, Jung KH, Kim SJ, Kim DH, Kang KM, Hong NH, Kim JH, Ban JJ, Park HK, et al. Anti-inflammatory mechanism of intravascular neural stem cell transplantation in haemorrhagic stroke. Brain. 2008;131:616–29.

    Article  PubMed  Google Scholar 

  74. Fischer UM, Harting MT, Jimenez F, Monzon-Posadas WO, Xue H, Savitz SI, Laine GA, Cox CS Jr. Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect. Stem Cells Dev. 2009;18(5):683–92.

    Article  PubMed  CAS  Google Scholar 

  75. Hauger O, Frost EE, van Heeswijk R, Deminiere C, Xue R, Delmas Y, Combe C, Moonen CT, Grenier N, Bulte JW. MR evaluation of the glomerular homing of magnetically labeled mesenchymal stem cells in a rat model of nephropathy. Radiology. 2006;238(1):200–10.

    Article  PubMed  Google Scholar 

  76. Kraitchman DL, Tatsumi M, Gilson WD, Ishimori T, Kedziorek D, Walczak P, Segars WP, Chen HH, Fritzges D, Izbudak I, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation. 2005;112(10):1451–61.

    Article  PubMed  PubMed Central  Google Scholar 

  77. Bliss T, Guzman R, Daadi M, Steinberg GK. Cell transplantation therapy for stroke. Stroke. 2007;38(2):817–26.

    Article  PubMed  Google Scholar 

  78. Guzman R, De Los Angeles A, Cheshier S, Choi R, Hoang S, Liauw J, Schaar B, Steinberg G. Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model. Stroke. 2008;39(4):1300–6.

    Article  PubMed  Google Scholar 

  79. Begley DJ, Brightman MW. Structural and functional aspects of the blood-brain barrier. Prog Drug Res. 2003;61:39–78.

    PubMed  CAS  Google Scholar 

  80. Abbott NJ. Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat. 2002;200(6):629–38.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  81. Obermeier B, Daneman R, Ransohoff RM. Development, maintenance and disruption of the blood-brain barrier. Nat Med. 2013;19(12):1584–96.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  82. Schoknecht K, David Y, Heinemann U. The blood-brain barrier-gatekeeper to neuronal homeostasis: clinical implications in the setting of stroke. Semin Cell Dev Biol. 2015;38:35–42.

    Article  PubMed  Google Scholar 

  83. Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7(1):41–53.

    Article  PubMed  CAS  Google Scholar 

  84. Ransohoff RM, Engelhardt B. The anatomical and cellular basis of immune surveillance in the central nervous system. Nat Rev Immunol. 2012;12(9):623–35.

    Article  PubMed  CAS  Google Scholar 

  85. Shechter R, Schwartz M. Harnessing monocyte-derived macrophages to control central nervous system pathologies: no longer ‘if’ but ‘how’. J Pathol. 2013;229(2):332–46.

    Article  PubMed  CAS  Google Scholar 

  86. White BC, Sullivan JM, DeGracia DJ, O'Neil BJ, Neumar RW, Grossman LI, Rafols JA, Krause GS. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J Neurol Sci. 2000;179(S 1–2):1–33.

    Article  PubMed  CAS  Google Scholar 

  87. Kulik T, Kusano Y, Aronhime S, Sandler AL, Winn HR. Regulation of cerebral vasculature in normal and ischemic brain. Neuropharmacology. 2008;55(3):281–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  88. Yang GY, Betz AL. Reperfusion-induced injury to the blood-brain barrier after middle cerebral artery occlusion in rats. Stroke. 1994;25(8):1658–64. discussion 1664-1655

    Article  PubMed  CAS  Google Scholar 

  89. Yemisci M, Gursoy-Ozdemir Y, Vural A, Can A, Topalkara K, Dalkara T. Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat Med. 2009;15(9):1031–7.

    Article  PubMed  CAS  Google Scholar 

  90. Daneman R. The blood-brain barrier in health and disease. Ann Neurol. 2012;72(5):648–72.

    Article  PubMed  CAS  Google Scholar 

  91. Shi Y, Zhang L, Pu H, Mao L, Hu X, Jiang X, Xu N, Stetler RA, Zhang F, Liu X, et al. Rapid endothelial cytoskeletal reorganization enables early blood-brain barrier disruption and long-term ischaemic reperfusion brain injury. Nat Commun. 2016;7:10523.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  92. Yang Y, Rosenberg GA. Blood-brain barrier breakdown in acute and chronic cerebrovascular disease. Stroke. 2011;42(11):3323–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  93. Pop V, Badaut J. A neurovascular perspective for long-term changes after brain trauma. Transl Stroke Res. 2011;2(4):533–45.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  94. Jiao H, Wang Z, Liu Y, Wang P, Xue Y. Specific role of tight junction proteins claudin-5, occludin, and ZO-1 of the blood-brain barrier in a focal cerebral ischemic insult. J Mol Neurosci. 2011;44(2):130–9.

    Article  PubMed  CAS  Google Scholar 

  95. Gu Y, Zheng G, Xu M, Li Y, Chen X, Zhu W, Tong Y, Chung SK, Liu KJ, Shen J. Caveolin-1 regulates nitric oxide-mediated matrix metalloproteinases activity and blood-brain barrier permeability in focal cerebral ischemia and reperfusion injury. J Neurochem. 2012;120(1):147–56.

    Article  PubMed  CAS  Google Scholar 

  96. Krueger M, Bechmann I, Immig K, Reichenbach A, Hartig W, Michalski D. Blood-brain barrier breakdown involves four distinct stages of vascular damage in various models of experimental focal cerebral ischemia. J Cereb Blood Flow Metab. 2015;35(2):292–303.

    Article  PubMed  CAS  Google Scholar 

  97. Mena H, Cadavid D, Rushing EJ. Human cerebral infarct: a proposed histopathologic classification based on 137 cases. Acta Neuropathol. 2004;108(6):524–30.

    Article  PubMed  Google Scholar 

  98. Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med. 2011;17(7):796–808.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  99. Aktas O, Ullrich O, Infante-Duarte C, Nitsch R, Zipp F. Neuronal damage in brain inflammation. Arch Neurol. 2007;64(2):185–9.

    Article  PubMed  Google Scholar 

  100. Gendron A, Teitelbaum J, Cossette C, Nuara S, Dumont M, Geadah D, du Souich P, Kouassi E. Temporal effects of left versus right middle cerebral artery occlusion on spleen lymphocyte subsets and mitogenic response in Wistar rats. Brain Res. 2002;955(1–2):85–97.

    Article  PubMed  CAS  Google Scholar 

  101. Kono H, Rock KL. How dying cells alert the immune system to danger. Nat Rev Immunol. 2008;8(4):279–89.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  102. Marsh BJ, Williams-Karnesky RL, Stenzel-Poore MP. Toll-like receptor signaling in endogenous neuroprotection and stroke. Neuroscience. 2009;158(3):1007–20.

    Article  PubMed  CAS  Google Scholar 

  103. Wang XJ, Ye M, Zhang YH, Chen SD. CD200-CD200R regulation of microglia activation in the pathogenesis of Parkinson's disease. J Neuroimmune Pharmacol. 2007;2(3):259–64.

    Article  PubMed  Google Scholar 

  104. Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, Blom B, Homola ME, Streit WJ, Brown MH, et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science. 2000;290(5497):1768–71.

    Article  PubMed  CAS  Google Scholar 

  105. Kaindl AM, Degos V, Peineau S, Gouadon E, Chhor V, Loron G, Le Charpentier T, Josserand J, Ali C, Vivien D, et al. Activation of microglial N-methyl-D-aspartate receptors triggers inflammation and neuronal cell death in the developing and mature brain. Ann Neurol. 2012;72(4):536–49.

    Article  PubMed  CAS  Google Scholar 

  106. Taylor RA, Sansing LH. Microglial responses after ischemic stroke and intracerebral hemorrhage. Clin Dev Immunol. 2013;2013:746068.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  107. Wang J, Yang Z, Liu C, Zhao Y, Chen Y. Activated microglia provide a neuroprotective role by balancing glial cell-line derived neurotrophic factor and tumor necrosis factor-alpha secretion after subacute cerebral ischemia. Int J Mol Med. 2013;31(1):172–8.

    Article  PubMed  CAS  Google Scholar 

  108. Rezai-Zadeh K, Gate D, Town T. CNS infiltration of peripheral immune cells: D-day for neurodegenerative disease? J Neuroimmune Pharmacol. 2009;4(4):462–75.

    Article  PubMed  PubMed Central  Google Scholar 

  109. Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, McCullough LD. Functional differences between microglia and monocytes after ischemic stroke. J Neuroinflammation. 2015;12:106.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  110. Kim E, Cho S. Microglia and monocyte-derived macrophages in stroke. Neurotherapeutics. 2016;13(4):702–18.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  111. Xiong XY, Liu L, Yang QW. Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke. Prog Neurobiol. 2016;142:23–44.

    Article  PubMed  CAS  Google Scholar 

  112. Vivien D, Ali C. Transforming growth factor-beta signalling in brain disorders. Cytokine Growth Factor Rev. 2006;17(1–2):121–8.

    Article  PubMed  CAS  Google Scholar 

  113. Stevens SL, Bao J, Hollis J, Lessov NS, Clark WM, Stenzel-Poore MP. The use of flow cytometry to evaluate temporal changes in inflammatory cells following focal cerebral ischemia in mice. Brain Res. 2002;932(1–2):110–9.

    Article  PubMed  CAS  Google Scholar 

  114. Yilmaz G, Granger DN. Leukocyte recruitment and ischemic brain injury. NeuroMolecular Med. 2010;12(2):193–204.

    Article  PubMed  CAS  Google Scholar 

  115. Gelderblom M, Leypoldt F, Steinbach K, Behrens D, Choe CU, Siler DA, Arumugam TV, Orthey E, Gerloff C, Tolosa E, et al. Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke. 2009;40(5):1849–57.

    Article  PubMed  Google Scholar 

  116. Felger JC, Abe T, Kaunzner UW, Gottfried-Blackmore A, Gal-Toth J, McEwen BS, Iadecola C, Bulloch K. Brain dendritic cells in ischemic stroke: time course, activation state, and origin. Brain Behav Immun. 2010;24(5):724–37.

    Article  PubMed  CAS  Google Scholar 

  117. Schuhmann MK, Langhauser F, Kraft P, Kleinschnitz C. B cells do not have a major pathophysiologic role in acute ischemic stroke in mice. J Neuroinflammation. 2017;14:112.

    Article  PubMed  PubMed Central  Google Scholar 

  118. Hurn PD, Subramanian S, Parker SM, Afentoulis ME, Kaler LJ, Vandenbark AA, Offner H. T- and B-cell-deficient mice with experimental stroke have reduced lesion size and inflammation. J Cereb Blood Flow Metab. 2007;27(11):1798–805.

    Article  PubMed  CAS  Google Scholar 

  119. Kunis G, Baruch K, Rosenzweig N, Kertser A, Miller O, Berkutzki T, Schwartz M. IFN-gamma-dependent activation of the brain's choroid plexus for CNS immune surveillance and repair. Brain. 2013;136(Pt 11):3427–40.

    Article  PubMed  Google Scholar 

  120. Niu FN, Zhang X, Hu XM, Chen J, Chang LL, Li JW, Liu Z, Cao W, Xu Y. Targeted mutation of Fas ligand gene attenuates brain inflammation in experimental stroke. Brain Behav Immun. 2012;26(1):61–71.

    Article  PubMed  CAS  Google Scholar 

  121. Shichita T, Sugiyama Y, Ooboshi H, Sugimori H, Nakagawa R, Takada I, Iwaki T, Okada Y, Iida M, Cua DJ, et al. Pivotal role of cerebral interleukin-17-producing gammadeltaT cells in the delayed phase of ischemic brain injury. Nat Med. 2009;15(8):946–50.

    Article  PubMed  CAS  Google Scholar 

  122. Liesz A, Suri-Payer E, Veltkamp C, Doerr H, Sommer C, Rivest S, Giese T, Veltkamp R. Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med. 2009;15(2):192–9.

    Article  PubMed  CAS  Google Scholar 

  123. Zhang L, Yi H, Xia XP, Zhao Y. Transforming growth factor-beta: an important role in CD4+CD25+ regulatory T cells and immune tolerance. Autoimmunity. 2006;39(4):269–76.

    Article  PubMed  CAS  Google Scholar 

  124. Barone FC, Schmidt DB, Hillegass LM, Price WJ, White RF, Feuerstein GZ, Clark RK, Lee EV, Griswold DE, Sarau HM. Reperfusion increases neutrophils and leukotriene B4 receptor binding in rat focal ischemia. Stroke. 1992;23(9):1337–47. discussion 1347–1338

    Article  PubMed  CAS  Google Scholar 

  125. Connolly ES Jr, Winfree CJ, Springer TA, Naka Y, Liao H, Yan SD, Stern DM, Solomon RA, Gutierrez-Ramos JC, Pinsky DJ. Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke. J Clin Invest. 1996;97(1):209–16.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  126. Herz J, Sabellek P, Lane TE, Gunzer M, Hermann DM, Doeppner TR. Role of neutrophils in exacerbation of brain injury after focal cerebral ischemia in hyperlipidemic mice. Stroke. 2015;46(10):2916–25.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  127. Akopov SE, Simonian NA, Grigorian GS. Dynamics of polymorphonuclear leukocyte accumulation in acute cerebral infarction and their correlation with brain tissue damage. Stroke. 1996;27(10):1739–43.

    Article  PubMed  CAS  Google Scholar 

  128. Harris AK, Ergul A, Kozak A, Machado LS, Johnson MH, Fagan SC. Effect of neutrophil depletion on gelatinase expression, edema formation and hemorrhagic transformation after focal ischemic stroke. BMC Neurosci. 2005;6:49.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  129. Wattananit S, Tornero D, Graubardt N, Memanishvili T, Monni E, Tatarishvili J, Miskinyte G, Ge R, Ahlenius H, Lindvall O, et al. Monocyte-derived macrophages contribute to spontaneous long-term functional recovery after stroke in mice. J Neurosci. 2016;36(15):4182.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  130. Schilling M, Besselmann M, Muller M, Strecker JK, Ringelstein EB, Kiefer R. Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice. Exp Neurol. 2005;196(2):290–7.

    Article  PubMed  CAS  Google Scholar 

  131. Ge R, Tornero D, Hirota M, Monni E, Laterza C, Lindvall O, Kokaia Z. Choroid plexus-cerebrospinal fluid route for monocyte-derived macrophages after stroke. J Neuroinflammation. 2017;14(1):153.

    Article  PubMed  PubMed Central  Google Scholar 

  132. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci. 2005;8(6):752–8.

    Article  PubMed  CAS  Google Scholar 

  133. Prinz M, Priller J. Tickets to the brain: role of CCR2 and CX3CR1 in myeloid cell entry in the CNS. J Neuroimmunol. 2010;224(1–2):80–4.

    Article  PubMed  CAS  Google Scholar 

  134. Nathan C, Ding A. Nonresolving inflammation. Cell. 2010;140(6):871–82.

    Article  PubMed  CAS  Google Scholar 

  135. Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: the role of T regulatory cells. Immunology. 2006;117(4):433–42.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  136. Besancon E, Guo S, Lok J, Tymianski M, Lo EH. Beyond NMDA and AMPA glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke. Trends Pharmacol Sci. 2008;29(5):268–75.

    Article  PubMed  CAS  Google Scholar 

  137. Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999;79(4):1431–568.

    Article  PubMed  CAS  Google Scholar 

  138. Zhao H, Yenari MA, Cheng D, Sapolsky RM, Steinberg GK. Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity. J Neurochem. 2003;85(4):1026–36.

    Article  PubMed  CAS  Google Scholar 

  139. Andreyev AY, Fahy B, Fiskum G. Cytochrome c release from brain mitochondria is independent of the mitochondrial permeability transition. FEBS Lett. 1998;439(3):373–6.

    Article  PubMed  CAS  Google Scholar 

  140. Petrovic-Djergovic D, Goonewardena SN, Pinsky DJ. Inflammatory disequilibrium in stroke. Circ Res. 2016;119(1):142–58.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  141. Domercq M, Etxebarria E, Perez-Samartin A, Matute C. Excitotoxic oligodendrocyte death and axonal damage induced by glutamate transporter inhibition. Glia. 2005;52(1):36–46.

    Article  PubMed  Google Scholar 

  142. Schiene K, Bruehl C, Zilles K, Qu M, Hagemann G, Kraemer M, Witte OW. Neuronal hyperexcitability and reduction of GABAA-receptor expression in the surround of cerebral photothrombosis. J Cereb Blood Flow Metab. 1996;16(5):906–14.

    Article  PubMed  CAS  Google Scholar 

  143. Nakagomi T, Molnar Z, Nakano-Doi A, Taguchi A, Saino O, Kubo S, Clausen M, Yoshikawa H, Nakagomi N, Matsuyama T. Ischemia-induced neural stem/progenitor cells in the pia mater following cortical infarction. Stem Cells Dev. 2011;20(12):2037–51.

    Article  PubMed  CAS  Google Scholar 

  144. Jin KL, Wang XM, Xie L, Mao XO, Zhu W, Wang Y, Shen JF, Mao Y, Banwait S, Greenberg DA. Evidence for stroke-induced neurogenesis in the human brain. Proc Natl Acad Sci U S A. 2006;103(35):13198–202.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  145. Yu TS, Washington PM, Kernie SG. Injury-induced neurogenesis: mechanisms and relevance. Neuroscientist. 2016;22(1):61–71.

    Article  PubMed  Google Scholar 

  146. Krupinski J, Kaluza J, Kumar P, Kumar S, Wang JM. Role of angiogenesis in patients with cerebral ischemic stroke. Stroke. 1994;25(9):1794–8.

    Article  PubMed  CAS  Google Scholar 

  147. Plate KH, Beck H, Danner S, Allegrini PR, Wiessner C. Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct. J Neuropathol Exp Neurol. 1999;58(6):654–66.

    Article  PubMed  CAS  Google Scholar 

  148. Wei L, Erinjeri JP, Rovainen CM, Woolsey TA. Collateral growth and angiogenesis around cortical stroke. Stroke. 2001;32(9):2179–84.

    Article  PubMed  CAS  Google Scholar 

  149. Yu SW, Friedman B, Cheng Q, Lyden PD. Stroke-evoked angiogenesis results in a transient population of microvessels. J Cereb Blood Flow Metab. 2007;27(4):755–63.

    Article  PubMed  Google Scholar 

  150. Marti HJ, Bernaudin M, Bellail A, Schoch H, Euler M, Petit E, Risau W. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol. 2000;156(3):965–76.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  151. Hayashi T, Noshita N, Sugawara T, Chan PH. Temporal profile of angiogenesis and expression of related genes in the brain after ischemia. J Cereb Blood Flow Metab. 2003;23(2):166–80.

    Article  PubMed  CAS  Google Scholar 

  152. Liu XS, Zhang ZG, Zhang RL, Gregg S, Morris DC, Wang Y, Chopp M. Stroke induces gene profile changes associated with neurogenesis and angiogenesis in adult subventricular zone progenitor cells. J Cereb Blood Flow Metab. 2007;27(3):564–74.

    Article  PubMed  CAS  Google Scholar 

  153. Chen J, Li Y, Katakowski M, Chen X, Wang L, Lu D, Lu M, Gautam SC, Chopp M. Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat. J Neurosci Res. 2003;73(6):778–86.

    Article  PubMed  CAS  Google Scholar 

  154. Krupinski J, Stroemer P, Slevin M, Marti E, Kumar P, Rubio F. Three-dimensional structure and survival of newly formed blood vessels after focal cerebral ischemia. Neuroreport. 2003;14(8):1171–6.

    Article  PubMed  Google Scholar 

  155. Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, Greenberg DA. VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest. 2003;111(12):1843–51.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  156. Arenillas JF, Sobrino T, Castillo J, Davalos A. The role of angiogenesis in damage and recovery from ischemic stroke. Curr Treat Options Cardiovasc Med. 2007;9(3):205–12.

    Article  PubMed  Google Scholar 

  157. Wang YQ, Cui HR, Yang SZ, Sun HP, Qiu MH, Feng XY, Sun FY. VEGF enhance cortical newborn neurons and their neurite development in adult rat brain after cerebral ischemia. Neurochem Int. 2009;55(7):629–36.

    Article  PubMed  CAS  Google Scholar 

  158. Petcu EB, Smith RA, Miroiu RI, Opris MM. Angiogenesis in old-aged subjects after ischemic stroke: a cautionary note for investigators. J Angiogenes Res. 2010;2:26.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  159. Zhang R, Zhang Z, Wang L, Wang Y, Gousev A, Zhang L, Ho KL, Morshead C, Chopp M. Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats. J Cereb Blood Flow Metab. 2004;24(4):441–8.

    Article  PubMed  Google Scholar 

  160. Shen Q, Goderie SK, Jin L, Karanth N, Sun Y, Abramova N, Vincent P, Pumiglia K, Temple S. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science. 2004;304(5675):1338–40.

    Article  PubMed  CAS  Google Scholar 

  161. Lin CY, Chang C, Cheung WM, Lin MH, Chen JJ, Hsu CY, Chen JH, Lin TN. Dynamic changes in vascular permeability, cerebral blood volume, vascular density, and size after transient focal cerebral ischemia in rats: evaluation with contrast-enhanced magnetic resonance imaging. J Cereb Blood Flow Metab. 2008;28(8):1491–501.

    Article  PubMed  Google Scholar 

  162. Thored P, Wood J, Arvidsson A, Cammenga J, Kokaia Z, Lindvall O. Long-term neuroblast migration along blood vessels in an area with transient angiogenesis and increased vascularization after stroke. Stroke. 2007;38(11):3032–9.

    Article  PubMed  Google Scholar 

  163. Lacar B, Herman P, Platel JC, Kubera C, Hyder F, Bordey A. Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone. J Neurosci. 2012;32(46):16435–48.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  164. Teng H, Zhang ZG, Wang L, Zhang RL, Zhang L, Morris D, Gregg SR, Wu Z, Jiang A, Lu M, et al. Coupling of angiogenesis and neurogenesis in cultured endothelial cells and neural progenitor cells after stroke. J Cereb Blood Flow Metab. 2008;28(4):764–71.

    Article  PubMed  CAS  Google Scholar 

  165. Thored P, Arvidsson A, Cacci E, Ahlenius H, Kallur T, Darsalia V, Ekdahl CT, Kokaia Z, Lindvall O. Persistent production of neurons from adult brain stem cells during recovery after stroke. Stem Cells. 2006;24(3):739–47.

    Article  PubMed  CAS  Google Scholar 

  166. Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med. 2002;8(9):963–70.

    Article  PubMed  CAS  Google Scholar 

  167. Goss JR, O'Malley ME, Zou L, Styren SD, Kochanek PM, DeKosky ST. Astrocytes are the major source of nerve growth factor upregulation following traumatic brain injury in the rat. Exp Neurol. 1998;149(2):301–9.

    Article  PubMed  CAS  Google Scholar 

  168. Weidemann A, Krohne TU, Aguilar E, Kurihara T, Takeda N, Dorrell MI, Simon MC, Haase VH, Friedlander M, Johnson RS. Astrocyte hypoxic response is essential for pathological but not developmental angiogenesis of the retina. Glia. 2010;58(10):1177–85.

    PubMed  PubMed Central  Google Scholar 

  169. Venkat P, Chopp M, Chen J. New insights into coupling and uncoupling of cerebral blood flow and metabolism in the brain. Croat Med J. 2016;57(3):223–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  170. Huang L, Wu ZB, Zhuge Q, Zheng W, Shao B, Wang B, Sun F, Jin K. Glial scar formation occurs in the human brain after ischemic stroke. Int J Med Sci. 2014;11(4):344–8.

    Article  PubMed  PubMed Central  Google Scholar 

  171. Roitbak T, Sykova E. Diffusion barriers evoked in the rat cortex by reactive astrogliosis. Glia. 1999;28(1):40–8.

    Article  PubMed  CAS  Google Scholar 

  172. Becerra-Calixto A, Cardona-Gomez GP. The role of astrocytes in neuroprotection after brain stroke: potential in cell therapy. Front Mol Neurosci. 2017;10:88.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  173. Benner EJ, Luciano D, Jo R, Abdi K, Paez-Gonzalez P, Sheng H, Warner DS, Liu C, Eroglu C, Kuo CT. Protective astrogenesis from the SVZ niche after injury is controlled by notch modulator Thbs4. Nature. 2013;497(7449):369–73.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  174. Deneen B, Ho R, Lukaszewicz A, Hochstim CJ, Gronostajski RM, Anderson DJ. The transcription factor NFIA controls the onset of gliogenesis in the developing spinal cord. Neuron. 2006;52(6):953–68.

    Article  PubMed  CAS  Google Scholar 

  175. Abeysinghe HC, Phillips EL, Chin-Cheng H, Beart PM, Roulston CL. Modulating astrocyte transition after stroke to promote brain rescue and functional recovery: emerging targets include rho kinase. Int J Mol Sci. 2016;17(3):288.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  176. Oki K, Tatarishvili J, Wood J, Koch P, Wattananit S, Mine Y, Monni E, Tornero D, Ahlenius H, Ladewig J, et al. Human-induced pluripotent stem cells form functional neurons and improve recovery after grafting in stroke-damaged brain. Stem Cells. 2012;30(6):1120–33.

    Article  PubMed  CAS  Google Scholar 

  177. De Feo D, Merlini A, Laterza C, Martino G. Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection. Curr Opin Neurol. 2012;25(3):322–33.

    Article  PubMed  CAS  Google Scholar 

  178. Andres RH, Horie N, Slikker W, Keren-Gill H, Zhan K, Sun G, Manley NC, Pereira MP, Sheikh LA, McMillan EL, et al. Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain. Brain. 2011;134(Pt 6):1777–89.

    Article  PubMed  PubMed Central  Google Scholar 

  179. Emanueli C, Schratzberger P, Kirchmair R, Madeddu P. Paracrine control of vascularization and neurogenesis by neurotrophins. Br J Pharmacol. 2003;140(4):614–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  180. Lee HJ, Lim IJ, Lee MC, Kim SU. Human neural stem cells genetically modified to overexpress brain-derived neurotrophic factor promote functional recovery and neuroprotection in a mouse stroke model. J Neurosci Res. 2010;88(15):3282–94.

    Article  PubMed  CAS  Google Scholar 

  181. Schabitz WR, Steigleder T, Cooper-Kuhn CM, Schwab S, Sommer C, Schneider A, Kuhn HG. Intravenous brain-derived neurotrophic factor enhances poststroke sensorimotor recovery and stimulates neurogenesis. Stroke. 2007;38(7):2165–72.

    Article  PubMed  CAS  Google Scholar 

  182. Lim JY, Park SI, Oh JH, Kim SM, Jeong CH, Jun JA, Lee KS, Oh W, Lee JK, Jeun SS. Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways. J Neurosci Res. 2008;86(10):2168–78.

    Article  PubMed  CAS  Google Scholar 

  183. Abe K. Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury. J Cereb Blood Flow Metab. 2000;20(10):1393–408.

    Article  PubMed  CAS  Google Scholar 

  184. Sondell M, Sundler F, Kanje M. Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor. Eur J Neurosci. 2000;12(12):4243–54.

    Article  PubMed  CAS  Google Scholar 

  185. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989;246(4935):1306–9.

    Article  PubMed  CAS  Google Scholar 

  186. Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem. 1998;273(46):30336–43.

    Article  PubMed  CAS  Google Scholar 

  187. Hayashi T, Abe K, Itoyama Y. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. J Cereb Blood Flow Metab. 1998;18(8):887–95.

    Article  PubMed  CAS  Google Scholar 

  188. Kaya D, Gursoy-Ozdemir Y, Yemisci M, Tuncer N, Aktan S, Dalkara T. VEGF protects brain against focal ischemia without increasing blood--brain permeability when administered intracerebroventricularly. J Cereb Blood Flow Metab. 2005;25(9):1111–8.

    Article  PubMed  CAS  Google Scholar 

  189. Harms KM, Li L, Cunningham LA. Murine neural stem/progenitor cells protect neurons against ischemia by HIF-1 alpha-regulated VEGF signaling. PLoS One. 2010;5(3):e9767.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  190. Bacigaluppi M, Russo GL, Peruzzotti-Jametti L, Rossi S, Sandrone S, Butti E, De Ceglia R, Bergamaschi A, Motta C, Gallizioli M, et al. Neural stem cell transplantation induces stroke recovery by upregulating glutamate transporter GLT-1 in astrocytes. J Neurosci. 2016;36(41):10529–44.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  191. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci. 2001;24:677–736.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  192. French Research Group on Multiple Sclerosis. Multiple sclerosis in 54 twinships: concordance rate is independent of zygosity. French Research Group on Multiple Sclerosis. Ann Neurol. 1992;32(6):724–7.

  193. Kang SS, Keasey MP, Arnold SA, Reid R, Geralds J, Hagg T. Endogenous CNTF mediates stroke-induced adult CNS neurogenesis in mice. Neurobiol Dis. 2013;49:68–78.

    Article  PubMed  CAS  Google Scholar 

  194. Sofroniew MV, Howe CL, Mobley WC. Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci. 2001;24:1217–81.

    Article  PubMed  CAS  Google Scholar 

  195. Cattaneo E, McKay R. Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor. Nature. 1990;347(6295):762–5.

    Article  PubMed  CAS  Google Scholar 

  196. Yuan M, Wen SJ, Yang CX, Pang YG, Gao XQ, Liu XQ, Huang L, Yuan QL. Transplantation of neural stem cells overexpressing glial cell line-derived neurotrophic factor enhances Akt and Erk1/2 signaling and neurogenesis in rats after stroke. Chin Med J. 2013;126(7):1302–9.

    PubMed  CAS  Google Scholar 

  197. Lampl Y, Boaz M, Gilad R, Lorberboym M, Dabby R, Rapoport A, Anca-Hershkowitz M, Sadeh M. Minocycline treatment in acute stroke: an open-label, evaluator-blinded study. Neurology. 2007;69(14):1404–10.

    Article  PubMed  CAS  Google Scholar 

  198. Yang F, Zhou L, Wang D, Wang Z, Huang QY. Minocycline ameliorates hypoxia-induced blood-brain barrier damage by inhibition of HIF-1alpha through SIRT-3/PHD-2 degradation pathway. Neuroscience. 2015;304:250–9.

    Article  PubMed  CAS  Google Scholar 

  199. Garrido-Mesa N, Camuesco D, Arribas B, Comalada M, Bailon E, Cueto-Sola M, Utrilla P, Nieto A, Zarzuelo A, Rodriguez-Cabezas ME, et al. The intestinal anti-inflammatory effect of minocycline in experimental colitis involves both its immunomodulatory and antimicrobial properties. Pharmacol Res. 2011;63(4):308–19.

    Article  PubMed  CAS  Google Scholar 

  200. Metz LM, Li DKB, Traboulsee AL, Duquette P, Eliasziw M, Cerchiaro G, Greenfield J, Riddehough A, Yeung M, Kremenchutzky M, et al. Trial of minocycline in a clinically isolated syndrome of multiple sclerosis. N Engl J Med. 2017;376(22):2122–33.

    Article  PubMed  CAS  Google Scholar 

  201. Ohshima S, Fujimoto S, Petrov A, Nakagami H, Haider N, Zhou J, Tahara N, Osako MK, Fujimoto A, Zhu J, et al. Effect of an antimicrobial agent on atherosclerotic plaques: assessment of metalloproteinase activity by molecular imaging. J Am Coll Cardiol. 2010;55(12):1240–9.

    Article  PubMed  CAS  Google Scholar 

  202. Machado LS, Kozak A, Ergul A, Hess DC, Borlongan CV, Fagan SC. Delayed minocycline inhibits ischemia-activated matrix metalloproteinases 2 and 9 after experimental stroke. BMC Neurosci. 2006;7:56.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  203. Yang Y, Salayandia VM, Thompson JF, Yang LY, Estrada EY, Yang Y. Attenuation of acute stroke injury in rat brain by minocycline promotes blood-brain barrier remodeling and alternative microglia/macrophage activation during recovery. J Neuroinflammation. 2015;12:26.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  204. Zhang T, Yang X, Liu T, Shao J, Fu N, Yan A, Geng K, Xia W. Adjudin-preconditioned neural stem cells enhance neuroprotection after ischemia reperfusion in mice. Stem Cell Res Ther. 2017;8(1):248.

    Article  PubMed  PubMed Central  Google Scholar 

  205. Smith HK, Gavins FN. The potential of stem cell therapy for stroke: is PISCES the sign? FASEB J. 2012;26(6):2239–52.

    Article  PubMed  CAS  Google Scholar 

  206. Bacigaluppi M, Pluchino S, Peruzzotti-Jametti L, Kilic E, Kilic U, Salani G, Brambilla E, West MJ, Comi G, Martino G, et al. Delayed post-ischaemic neuroprotection following systemic neural stem cell transplantation involves multiple mechanisms. Brain. 2009;132(Pt 8):2239–51.

    Article  PubMed  Google Scholar 

  207. Chang DJ, Lee N, Park IH, Choi C, Jeon I, Kwon J, Oh SH, Shin DA, Do JT, Lee DR, et al. Therapeutic potential of human induced pluripotent stem cells in experimental stroke. Cell Transplant. 2013;22(8):1427–40.

    Article  PubMed  Google Scholar 

  208. Ryu S, Lee SH, Kim SU, Yoon BW. Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain. Neural Regen Res. 2016;11(2):298–304.

    Article  PubMed  PubMed Central  Google Scholar 

  209. Sakata H, Narasimhan P, Niizuma K, Maier CM, Wakai T, Chan PH. Interleukin 6-preconditioned neural stem cells reduce ischaemic injury in stroke mice. Brain. 2012;135:3298–310.

    Article  PubMed  PubMed Central  Google Scholar 

  210. Patkar S, Tate R, Modo M, Plevin R, Carswell HV. Conditionally immortalised neural stem cells promote functional recovery and brain plasticity after transient focal cerebral ischaemia in mice. Stem Cell Res. 2012;8(1):14–25.

    Article  PubMed  CAS  Google Scholar 

  211. Ziaee SM, Tabeshmehr P, Haider KH, Farrokhi M, Shariat A, Amiri A, Hosseini SM. Optimization of time for neural stem cells transplantation for brain stroke in rats. Stem Cell Investig. 2017;4:29.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  212. Yao Y, Zheng XR, Zhang SS, Wang X, Yu XH, Tan JL, Yang YJ. Transplantation of vascular endothelial growth factor-modified neural stem/progenitor cells promotes the recovery of neurological function following hypoxic-ischemic brain damage. Neural Regen Res. 2016;11(9):1456–63.

    PubMed  PubMed Central  Google Scholar 

  213. Lu Y, Jiang L, Li W, Qu M, Song Y, He X, Zhang Z, Yang GY, Wang Y. Optogenetic inhibition of striatal neuronal activity improves the survival of transplanted neural stem cells and neurological outcomes after ischemic stroke in mice. Stem Cells Int. 2017;2017:4364302.

    PubMed  PubMed Central  Google Scholar 

  214. Song M, Kim YJ, Kim YH, Roh J, Kim EC, Lee HJ, Kim SU, Yoon BW. Long-term effects of magnetically targeted ferumoxide-labeled human neural stem cells in focal cerebral ischemia. Cell Transplant. 2015;24(2):183–90.

    Article  PubMed  Google Scholar 

  215. Tang Y, Wang J, Lin X, Wang L, Shao B, Jin K, Wang Y, Yang GY. Neural stem cell protects aged rat brain from ischemia-reperfusion injury through neurogenesis and angiogenesis. J Cereb Blood Flow Metab. 2014;34(7):1138–47.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  216. Mine Y, Tatarishvili J, Oki K, Monni E, Kokaia Z, Lindvall O. Grafted human neural stem cells enhance several steps of endogenous neurogenesis and improve behavioral recovery after middle cerebral artery occlusion in rats. Neurobiol Dis. 2013;52:191–203.

    Article  PubMed  CAS  Google Scholar 

  217. Cho T, Bae JH, Choi HB, Kim SS, McLarnon JG, Suh-Kim H, Kim SU, Min CK. Human neural stem cells: electrophysiological properties of voltage-gated ion channels. Neuroreport. 2002;13(11):1447–52.

    Article  PubMed  CAS  Google Scholar 

  218. Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, et al. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol. 1998;16(11):1033–9.

    Article  PubMed  CAS  Google Scholar 

  219. Chu K, Kim M, Jeong SW, Kim SU, Yoon BW. Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia. Neurosci Lett. 2003;343(2):129–33.

    Article  PubMed  CAS  Google Scholar 

  220. Chu K, Kim M, Chae SH, Jeong SW, Kang KS, Jung KH, Kim J, Kim YJ, Kang L, Kim SU, et al. Distribution and in situ proliferation patterns of intravenously injected immortalized human neural stem-like cells in rats with focal cerebral ischemia. Neurosci Res. 2004;50(4):459–65.

    Article  PubMed  CAS  Google Scholar 

  221. Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke. 2003;34(9):2258–63.

    Article  PubMed  Google Scholar 

  222. Zhang JJ, Zhu JJ, Hu YB, Xiang GH, Deng LC, Wu FZ, Wei XJ, Wang YH, Sun LY, Lou XQ, et al. Transplantation of bFGF-expressing neural stem cells promotes cell migration and functional recovery in rat brain after transient ischemic stroke. Oncotarget. 2017;8(60):102067–77.

    PubMed  PubMed Central  Google Scholar 

  223. Kang SS, Keasey MP, Arnold SA, Reid R, Geralds J, Hagg T. Endogenous CNTF mediates stroke-induced adult CNS neurogenesis in mice. Neurobiol Dis. 2012;49C:68–78.

    Google Scholar 

  224. Mohamad O, Drury-Stewart D, Song M, Faulkner B, Chen D, Yu SP, Wei L. Vector-free and transgene-free human iPS cells differentiate into functional neurons and enhance functional recovery after ischemic stroke in mice. PLoS One. 2013;8(5):e64160.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  225. Drury-Stewart D, Song M, Mohamad O, Guo Y, Gu X, Chen D, Wei L. Highly efficient differentiation of neural precursors from human embryonic stem cells and benefits of transplantation after ischemic stroke in mice. Stem Cell Res Ther. 2013;4(4):93.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  226. Chau M, Deveau TC, Song M, Gu X, Chen D, Wei L. iPS cell transplantation increases regeneration and functional recovery after ischemic stroke in neonatal rats. Stem Cells. 2014;32(12):3075–87.

    Article  PubMed  CAS  Google Scholar 

  227. Horie N, Pereira MP, Niizuma K, Sun G, Keren-Gill H, Encarnacion A, Shamloo M, Hamilton SA, Jiang K, Huhn S, et al. Transplanted stem cell-secreted vascular endothelial growth factor effects poststroke recovery, inflammation, and vascular repair. Stem Cells. 2011;29(2):274–85.

    Article  PubMed  CAS  Google Scholar 

  228. Chau M, Deveau TC, Song M, Wei ZZ, Gu X, Yu SP, Wei L. Transplantation of iPS cell-derived neural progenitors overexpressing SDF-1alpha increases regeneration and functional recovery after ischemic stroke. Oncotarget. 2017;8(57):97537–53.

    Article  PubMed  PubMed Central  Google Scholar 

  229. Moriyama Y, Takagi N, Hashimura K, Itokawa C, Tanonaka K. Intravenous injection of neural progenitor cells facilitates angiogenesis after cerebral ischemia. Brain Behav. 2013;3(2):43–53.

    Article  PubMed  Google Scholar 

  230. Pollock K, Stroemer P, Patel S, Stevanato L, Hope A, Miljan E, Dong Z, Hodges H, Price J, Sinden JD. A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol. 2006;199(1):143–55.

    Article  PubMed  Google Scholar 

  231. Hicks C, Stevanato L, Stroemer RP, Tang E, Richardson S, Sinden JD. In vivo and in vitro characterization of the angiogenic effect of CTX0E03 human neural stem cells. Cell Transplant. 2013;22(9):1541–52.

    Article  PubMed  Google Scholar 

  232. Jin K, Xie L, Mao X, Greenberg MB, Moore A, Peng B, Greenberg RB, Greenberg DA. Effect of human neural precursor cell transplantation on endogenous neurogenesis after focal cerebral ischemia in the rat. Brain Res. 2011;1374:56–62.

    Article  PubMed  CAS  Google Scholar 

  233. Jin K, Mao X, Xie L, Greenberg RB, Peng B, Moore A, Greenberg MB, Greenberg DA. Delayed transplantation of human neural precursor cells improves outcome from focal cerebral ischemia in aged rats. Aging Cell. 2010;9(6):1076–83.

    Article  PubMed  CAS  Google Scholar 

  234. Hassani Z, O'Reilly J, Pearse Y, Stroemer P, Tang E, Sinden J, Price J, Thuret S. Human neural progenitor cell engraftment increases neurogenesis and microglial recruitment in the brain of rats with stroke. PLoS One. 2012;7(11):e50444.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  235. Stroemer P, Patel S, Hope A, Oliveira C, Pollock K, Sinden J. The neural stem cell line CTX0E03 promotes behavioral recovery and endogenous neurogenesis after experimental stroke in a dose-dependent fashion. Neurorehabil Neural Repair. 2009;23(9):895–909.

    Article  PubMed  Google Scholar 

  236. Schaar KL, Brenneman MM, Savitz SI. Functional assessments in the rodent stroke model. Exp Transl Stroke Med. 2010;2(1):13.

    Article  PubMed  PubMed Central  Google Scholar 

  237. Hua Y, Schallert T, Keep RF, Wu J, Hoff JT, Xi G. Behavioral tests after intracerebral hemorrhage in the rat. Stroke. 2002;33(10):2478–84.

    Article  PubMed  Google Scholar 

  238. Darsalia V, Allison SJ, Cusulin C, Monni E, Kuzdas D, Kallur T, Lindvall O, Kokaia Z. Cell number and timing of transplantation determine survival of human neural stem cell grafts in stroke-damaged rat brain. J Cereb Blood Flow Metab. 2011;31(1):235–42.

    Article  PubMed  Google Scholar 

  239. Ergul A, Kelly-Cobbs A, Abdalla M, Fagan SC. Cerebrovascular complications of diabetes: focus on stroke. Endocr Metab Immune Disord Drug Targets. 2012;12(2):148–58.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  240. Ergul A, Li W, Elgebaly MM, Bruno A, Fagan SC. Hyperglycemia, diabetes and stroke: focus on the cerebrovasculature. Vasc Pharmacol. 2009;51(1):44–9.

    Article  CAS  Google Scholar 

  241. Tureyen K, Bowen K, Liang J, Dempsey RJ, Vemuganti R. Exacerbated brain damage, edema and inflammation in type-2 diabetic mice subjected to focal ischemia. J Neurochem. 2011;116(4):499–507.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  242. Kawai H, Yamashita T, Ohta Y, Deguchi K, Nagotani S, Zhang X, Ikeda Y, Matsuura T, Abe K. Tridermal tumorigenesis of induced pluripotent stem cells transplanted in ischemic brain. J Cereb Blood Flow Metab. 2010;30(8):1487–93.

    Article  PubMed  PubMed Central  Google Scholar 

  243. Steinberg GK, Kondziolka D, Wechsler LR, Lunsford LD, Coburn ML, Billigen JB, Kim AS, Johnson JN, Bates D, King B, et al. Clinical Outcomes of Transplanted Modified Bone Marrow-Derived Mesenchymal Stem Cells in Stroke: A Phase 1/2a Study. Stroke. 2016;47(7):1817–24.

    Article  PubMed  PubMed Central  Google Scholar 

  244. Kalladka D, Sinden J, Pollock K, Haig C, McLean J, Smith W, McConnachie A, Santosh C, Bath PM, Dunn L, et al. Human neural stem cells in patients with chronic ischaemic stroke (PISCES): a phase 1, first-in-man study. Lancet. 2016;388(10046):787–96.

    Article  PubMed  Google Scholar 

  245. Muir KW. Clinical trial design for stem cell therapies in stroke: what have we learned? Neurochem Int. 2017;106:108–13.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported by a Louisiana Clinical and Translational Science Center Grant, a Tulane Carol Lavin Bernick Faculty Grant, and the Tulane School of Medicine Faculty Research Pilot Fund to J-PL.

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ACB prepared figures. ACB, Q-SEL, DP, MHH, and J-PL drafted the manuscript. ACB, J-PL, and MHH edited and revised the manuscript. All authors read and approved the final version of the manuscript.

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Boese, A.C., Le, QS.E., Pham, D. et al. Neural stem cell therapy for subacute and chronic ischemic stroke. Stem Cell Res Ther 9, 154 (2018). https://doi.org/10.1186/s13287-018-0913-2

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