Neural stem cell therapy for stroke: A multimechanistic approach to restoring neurological function

Abstract Introduction Neural stem cells (NSCs) have demonstrated multimodal therapeutic function for stroke, which is the leading cause of long‐term disability and the second leading cause of death worldwide. In preclinical stroke models, NSCs have been shown to modulate inflammation, foster neuroplasticity and neural reorganization, promote angiogenesis, and act as a cellular replacement by differentiating into mature neural cell types. However, there are several key technical questions to address before NSC therapy can be applied to the clinical setting on a large scale. Purpose of Review In this review, we will discuss the various sources of NSCs, their therapeutic modes of action to enhance stroke recovery, and considerations for the clinical translation of NSC therapies. Understanding the key factors involved in NSC‐mediated tissue recovery and addressing the current translational barriers may lead to clinical success of NSC therapy and a first‐in‐class restorative therapy for stroke patients.


| INTRODUC TI ON
Although stroke is the leading cause of long-term disability and the second leading cause of death worldwide, there are only two Food and Drug Administration (FDA)-approved therapies-tissue plasminogen activator and thrombectomy (Albers et al., 2018;Mozaffarian et al., 2015;Nogueira et al., 2018;Sharma et al., 2010). However, these therapies are significantly limited as they can only be utilized in acute patients resulting in a relatively small number of individuals being treated. Most therapies recently tested in clinical trials have focused on mitigating secondary injury mechanisms such as excitotoxicity (Clark, Wechsler, Sabounjian, & Schwiderski, 2001;Diener et al., 2000Diener et al., , 2008Mousavi, Saadatnia, Khorvash, Hoseini, & Sariaslani, 2011), immune and inflammatory responses (Enlimomab Acute Stroke Trial & I., 2001), or apoptosis (Franke et al., 1996), all of which have failed. Neural stem cells (NSCs) have garnered significant interest as a multimodel therapeutic capable of producing neuroprotective and regenerative growth factors, while also potentially serving as cell replacement for lost and damaged neural cell types (Andres et al., 2011;Baker et al., 2017;Chang et al., 2013;Eckert et al., 2015;Tornero et al., 2013;Watanabe et al., 2016;Zhang et al., 2011). Another potentially attractive advantage of NSC therapy over conventional drug therapies is NSCs can continually respond to environmental cues and secrete appropriate quantities and type of signaling factors, therefore providing a tailored response to individual stroke injuries. Due to the significant potential of NSCs, these cells have progressed from testing in preclinical models to clinical trials for stroke with promising results (Table 1; Andres et al., 2011;Kalladka et al., 2016;Watanabe et al., 2016;Zhang et al., 2011Zhang et al., , 2013. NSCs are multipotent and specifically differentiate into neural cell types (e.g., neurons, astrocytes and oligodendrocytes) and thus likely hold the greatest potential for cell replacement therapy after stroke. While significant progress has been made to understand NSC-mediated tissue recovery after stroke, key questions remain that must be resolved before NSC therapy can be utilized in the clinic at a large scale. In this review, we will discuss the sources of NSCs currently being studied, their mode of action in the context of stroke treatment, and clinical considerations to move NSC therapies from human trials to a standard of care for stroke patients.
TA B L E 1 Preclinical rodent ischemic stroke models testing human neural stem cell therapy NSC type

Route of administration
Cell dose

| SOURCE S OF NEUR AL S TEM CELL S
During the 1990s, novel protocols were developed to generate immortalized human neural cell lines capable of differentiating into mature neurons on a scale large enough to be therapeutically relevant (Carpenter et al., 1999;Storch et al., 2001;Svendsen et al., 1998;Villa, Snyder, Vescovi, & Martıńez-Serrano, A., 2000). Since then, multiple types of NSC lines that generate mature neural cell types have been developed and characterized including NSCs derived from fetal tissues, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), all of which have been shown to enhance recovery after stroke and comparable neurological disorders ( Figure 1).

| Fetal-derived neural stem cells
Fetal-derived NSC lines (fetal-NSC) were one of the first cell sources developed that had significant potential as a stroke cell therapy. Fetal-NSCs were generated by dissociating human fetal cortex, mesencephalon, or spinal cord tissues between 7 and 21 days postconception (Pollock et al., 2006;Svendsen et al., 1998).
These cells proved to be capable of long-term expansion when cultured in mitogens such as epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2), even without genetic modification, while maintaining their neurogenic and gliogenic multipotent differentiation potential. Fetal-NSCs have shown therapeutic potential for a number of neurological diseases such as stroke (Kalladka et al., 2016;Pollock et al., 2006), traumatic brain injury (Shin et al., 2015;Skardelly et al., 2011), spinal cord injury (Cheng et al., 2012;Shin et al., 2015), and amyotrophic lateral sclerosis (Glass et al., 2012;Xu et al., 2006). However, previous studies have shown that fetal-NSCs undergo senescence earlier than other NSC lines, which would make large-scale manufacturing more difficult (Wright, Prowse, Wallace, Linskens, & Svendsen, 2006). To address this, some fetal-NSC lines have undergone genetic modification leading to immortalization and enhanced expandability. A prominent example of this is the CTX0E03 F I G U R E 1 Multifunctional therapeutic action of transplanted neural stem cells. Transplanted NSCs derived from ESCs, iPSCs, or fetal brain have demonstrated multimodal therapeutic function after intravenous, intraparenchymal, or intracerebroventricular administration Lau et al., 2015). NSCs demonstrate immunomodulatory function through the expression of cytokines and chemokines in response pro-inflammatory signaling from activated microglia and infiltrating circulatory immune cells (Huang et al., 2014;Watanabe et al., 2016). NSCs also promote angiogenesis and stimulate neural repair mechanisms including synaptic reorganization and neurogenesis (Andres et al., 2011;Zhang et al., 2011). Transplanted NSCs can act as a cell replacement therapy by differentiating to mature neural cell types (neurons, astrocytes, and oligodendrocytes) and integrating into the host brain tissue (Baker et al., 2017;Kelly et al., 2004;Oki et al., 2012;Tornero et al., 2013). The prepotency of one mechanism to promote tissue repair over another is not well known. Regardless of therapeutic mechanism, the final outcome after NSC transplantation is improved tissue and functional recovery  cell line, which is derived from fetal cortical tissue and transduced with a c-mycER TAM construct (Pollock et al., 2006). Studies have demonstrated that CTX0E03 has multimodal therapeutic function in preclinical animal models including angiogenic, neurogenic, and immunomodulation effects leading to improvements in functional recovery (Hassani et al., 2012;Hicks et al., 2013;Stroemer et al., 2009). CTX0E03 was recently evaluated in phase I clinical trials for stroke and demonstrated no cell-related adverse events (Hassani et al., 2012;Hicks et al., 2013;Kalladka et al., 2016;Stroemer et al., 2009). However, these cells have shown limited long-term engraftment suggesting that the effect of these cells is mostly due to paracrine signaling and not cell replacement (Hicks et al., 2013). Limited cell replacement is a common challenge in NSC therapy regardless of cell source or method of delivery (e.g., intraparenchymal, intravenous). This suggests that an increased level of efficacy could be achieved with NSC therapy if cells could be maintained long term and successfully integrate into damaged tissues. The cause of limited NSC engraftment is still unclear, but is likely a combination of a number of factors including the hostile stroke environment (e.g., high levels of reactive oxygen species and inflammatory cytokines) and the lack of appropriately orchestrated differentiation and integration signaling.

| Induced pluripotent stem cell-derived neural stem cells
More recently, the research of Yamanaka and others have shown that adult somatic cells can be reprogrammed to a pluripotent state through the overexpression of transcription factors, and these reprogrammed cells have similar plasticity and neural differentiation potency as ESCs (Denham & Dottori, 2011;Takahashi et al., 2007;Warren et al., 2010). These cells have been deemed induced pluripotent stem cells (iPSCs), and their NSC progeny (iPSC-NSCs). iPSC-NSCs possess unprecedented therapeutic potential for neurological disease as they can be generated from the patient's own somatic cells, avoiding the risk of immune rejection associated with allogeneic transplants (Araki et al., 2013;Guha, Morgan, Mostoslavsky, Rodrigues, & Boyd, 2013

| NEUR AL S TEM CELL MODE S OF AC TI O N
NSCs have demonstrated multimodal therapeutic function after transplantation into preclinical animal models of stroke (documented NSC modes of action in preclinical animal models are summarized in Table 1). Depending on the treatment protocol, NSCs are able to protect at-risk neural cells, promote endogenous NSC proliferation and migration, foster synaptic remodeling, stimulate new vessel formation, and/or integrate into host neural circuits, which have been associated with improvements in cognitive and sensorimotor function (Mine et al., 2013;Zhang et al., 2013; Figure 1). However, further studies are needed to determine which tissue recovery mechanism is most effective in restoring neurological function.

| Immunomodulation
Stroke injury is propagated by a strong immune and inflammatory response through the activation of microglia, the resident immune cells of the brain, which produce pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) in response to damage-associated molecular patterns (DAMPS) within minutes after onset of ischemia (Hossmann, 2006;Jin, Yang, & Li, 2010;Xiong, Liu, & Yang, 2016). Furthermore, the release of cytokines by activated microglia upregulates the expression of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and chemokine ligand 1 (CXCL1) on endothelial cells, which promotes the infiltration of peripheral monocytes/macrophages across the blood-brain barrier (BBB) which exacerbates inflammatory injury (Remus, Sayeed, Won, Lyle, & Stein, 2015). The modulation of this inflammatory cascade may be the most widely characterized NSC mode of action in preclinical animal models of stroke. The immunomodulatory mechanisms of transplanted NSCs are likely carried out through what is known as the "bystander effect," in which NSCs release neurotrophic factors such as glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), which have been shown to inhibit mechanisms induced by inflammatory actions (Lladó, Haenggeli, Maragakis, Snyder, & Rothstein, 2004;Lu, Jones, Snyder, & Tuszynski, 2003;Ourednik, Ourednik, Lynch, Schachner, & Snyder, 2002). Multiple studies in preclinical stroke models have documented decreased immune cell activation after intraparenchymal transplantation of fetal and iPSC-NSCs (Eckert et al., 2015;Kelly et al., 2004;Mine et al., 2013;Zhang et al., 2013). Ultimately, reduced inflammation mediated by NSC transplantation is neuroprotective because the secondary injury cascade is curtailed; thus, reports of reduced inflammation in the stroked brain are often correlated with reductions in final infarct volume and functional recovery (Bacigaluppi et al., 2009;Huang, Wong, Snyder, Hamblin, & Lee, 2014;Song et al., 2015).
More studies are needed to assess whether one NSC type possesses heightened immunomodulatory properties over another.
Experiments performed by the Lee group showed decreases in the expression of pro-inflammatory cytokines TNF-α, IL-6, IL-1β, cell adhesion molecules ICAM-1 and VCAM-1, and chemokines MCP-1 and MIP-1α after intraparenchymal transplantation of fetal-and iPSC-NSCs in a rodent stroke model (Eckert et al., 2015;Huang et al., 2014). This decrease in pro-inflammatory cytokines and chemokines was attributed, at least partially, to NSC-mediated increases in BDNF expression in the ipsilateral hemisphere. These data indicate that neurotrophic factor signaling by transplanted NSCs of both iPSC and fetal origin diminishes upstream inflammatory activity in the early stages of ischemic injury by reducing pro-inflammatory signaling by activated microglia, which in turn inhibits subsequent peripheral immune cell extravasation. Indeed, the documented changes in gene expression correlated to reduced expression of the microglia marker Iba1 and reduced BBB leakage. However, fetal-NSCs were more effective at reducing final infarct volume compared to iPSC-NSCs, which may be due to differences in the neurotrophic signaling capacity of the two cell types.
Although NSC immunomodulatory behavior in the stroked brain is often documented after an intraparenchymal transplantation approach, there is evidence that intravenous delivery of NSCs after stroke also reduces the expression of pro-inflammatory mediators, immune cell activation, and apoptosis in brain tissue (Bacigaluppi et al., 2009;Song et al., 2015;Watanabe et al., 2016). Whether it is necessary for these cells to accumulate in the brain parenchyma after intravenous delivery in order to carry out their anti-inflammatory effects is largely unknown. However, a study by Song et al. (2015) demonstrated that intravenously delivered, magnetically targeted fetal-NSCs accumulated in the stroked brain tissue and correlated to reduced infarct size compared to nontargeted NSCs. Taken together, these data indicate that targeting systemically infused NSCs to the brain parenchyma augments their immunomodulatory capacity after stroke. Therefore, direct intraparenchymal transplantation may be advantageous over intravenous delivery to enhance NSC-mediated tissue repair.

| Neurogenesis
For many years, it was widely accepted that the brain possessed no regenerative potential and lost neurons could not be replaced after injury or disease. However, more recently, it has been demonstrated that endogenous NSCs are present in the subventricular zone (SVZ; Corotto, Henegar, & Maruniak, 1993;Kirschenbaum et al., 1994) and the dentate gyrus (DG; Eriksson et al., 1998;Kuhn, Dickinson-Anson, & Gage, 1996) of the adult mammalian brain, and neurogenesis in these regions is increased after stroke albeit at a level lower than what is necessary to restore tissue function (Arvidsson, Collin, Kirik, Kokaia, & Lindvall, 2002). Indeed, despite evidence of functional integration of these nascent neurons (Hou et al., 2008;Yamashita et al., 2006), about 80% of the new neuroblasts and neurons die within the first 2 weeks after formation (Arvidsson et al., 2002). The potential to enhance endogenous neurogenesis mechanisms to replace lost neuronal cells has been an exciting therapeutic target for stroke.
Previous studies have shown that stroke-induced neurogenic behavior is augmented after NSC transplantation in preclinical stroke models by increasing the proliferation of endogenous NSCs at the SVZ and DG as well as promoting the migration of endogenous neuroblasts to the damaged brain region which differentiate to mature neurons (Hassani et al., 2012;Jin et al., 2011;Mine et al., 2013;Ryu, Lee, Kim, & Yoon, 2016;Stroemer et al., 2009;Zhang et al., 2011Zhang et al., , 2013. A study by Mine et al. (2013)  with respect to duration of enhanced neurogenesis, which may be due to differences in the stroke models, transplantation location, and neurogenic potency of the cell line tested. The mechanism by which transplanted NSCs augment endogenous neurogenic behavior after stroke is not well known, but is likely mediated by the secretion of neurotrophic and regenerative growth factors that suppress immune and inflammatory responses while promoting tissue repair (Kokaia, Martino, Schwartz, & Lindvall, 2012;Lu et al., 2003;Martino & Pluchino, 2006).

| Neural reorganization
Patients show varying levels of spontaneous recovery in limb function, language, and other cognitive measures within the first month after stroke onset (Benowitz & Carmichael, 2010). This phenomenon is largely attributed to the rewiring of neuronal circuitry in which motor and sensory circuit activity is increased in other brain regions remote from the infarcted area (Cramer, 2008). VEGF, were specifically secreted by NSCs and were responsible for NSC-induced effects on dendritic and axonal plasticity (Andres et al., 2011). Other studies in preclinical stroke models have demonstrated that transplanted NSCs promote oligodendrocyte proliferation and myelination of new neuronal circuits (Daadi et al., 2010;Manley et al., 2015;Stroemer et al., 2009;Zhang et al., 2013). These results show that NSC transplantation augments key reorganization of axons, dendrites, and synapses across multiple brain regions that lead to improved recovery.
The penumbra is impacted by the ischemic event but still viable due

| Cell Replacement
In addition to the many mechanisms in which NSC-mediated trophic factor signaling protects and promotes tissue recovery after stroke, NSCs themselves can act as a cellular replacement. After transplantation into the stroke-damaged brain, grafted NSCs have been  Zhang et al., 2013), medium spiny striatal projection neuron (Chang et al., 2013;Oki et al., 2012;Polentes et al., 2012;, and cortical neuron markers (Darsalia et al., 2007;Oki et al., 2012;Tornero et al., 2013). Furthermore, some of these studies demonstrate that grafted NSC-derived neurons are electrically active, project axons to appropriate target regions, and form synapses (Chang et al., 2013;Mine et al., 2013;Oki et al., 2012;Polentes et al., 2012;Tornero et al., 2013). Glial differentiation (astrocytes and oligodendrocytes) has also been documented with a similar time course to neuronal cells (Andres et al., 2011;Darsalia et al., 2007;Kelly et al., 2004;Oki et al., 2012;Song et al., 2015;Stroemer et al., 2009;Zhang et al., 2013). However, there is much debate whether transplanted NSCs truly integrate into the host brain and contribute directly to improving functional outcome in preclinical animal models. Previous studies have shown that functional recovery often occurs earlier than the time it would take to achieve functional integration of the transplanted NSCs, so other NSC-mediated repair mechanisms such as trophic factor support may play a larger role in functional recovery than cell replacement (Oki et al., 2012). In addi- that external cues may have facilitated region-specific cortical differentiation. It is possible that effective neuronal replacement in various brain regions will require directed in vitro differentiation of NSCs to site-specific precursors prior to transplantation (Oki et al., 2012). Indeed, Tornero et al. (2013) found that cortically fated NSCs more readily differentiate to a cortical phenotype with pyramidal morphology than nonfated NSCs. Building upon this strategy, it may 1 day be possible to generate region-specific cellular composites with the appropriate combination (e.g., specific neuron subtypes, astrocytes and oligodendrocytes) and transplant this cellular milieu with exquisite regionalized specificity. However, this strategy can become quite complex. For example, the cerebral cortex is composed of six unique layers that are comprised of different numbers and ratios of specialized neural cell types and changes depending upon brain region. In addition, this approach is also plagued by other complex variables such as brain vasculature remodeling involving multiple cell types (e.g., endothelial cells, pericytes, smooth muscle cells) as well as the need to form neurovascular units, and a lymphatic system (Benarroch, 2012;Louveau et al., 2015). Through the lens of NSC transplant studies in a number of neural injury and disease models, it has become clear that there is a need to better understand the mechanisms driving differentiation of grafted NSCs before cells can be safely and reliably used as a stroke therapy.

| NEUR AL S TEM CELL-COND ITI ONED MED IA
Recent studies have demonstrated that NSC culture-conditioned media and purified media products inhibit apoptosis, reduce le- . The let-7 family of microRNAs, which are also expressed by NSCs, have been shown to protect against neuroinflammation by regulating the expression of caspase 3, inducible nitric oxide synthase (iNOS), TNF-α, and IL-12, which improve stroke-induced neurological deficits in mice (Akerblom et al., 2012;Banerjee et al., 2013;Ni et al., 2015). Furthermore, miR-210 expression is upregulated in NSCs exposed to a hypoxic environment, and overexpression of this microRNA led to enhanced neurogenesis and angiogenesis in mice (Wang et al., 2013). Together, this indicates that NSC-conditioned media is enriched with bioactive, restorative factors that are packaged into EVs or in their free form. These released factors leverage NSC therapies to a point in which whole cells may no longer be required in the future.

| TISSUE ENG INEERING APPROACHE S
While many reports outline the robust therapeutic effects of NSCs transplanted alone, recent evidence suggests that engineering approaches with biomaterials limits stroke-induced tissue architecture disruptions and enhances NSC therapeutic function and engraftment (Bible, Qutachi et al., 2012;Jin, Mao et al., 2010;Lam, Lowry, Carmichael, & Segura, 2014;Lee, Yun, Park, & Jang, 2016;Yu et al., 2010). When cotransplanted with NSCs, the main role of biomaterials is often to cultivate an adequate structural microenvironment to foster the survival, cross talk, and integration of transplanted cells into host tissue. Furthermore, biomaterials can be enriched with neurotrophic growth factors to augment transplantation success (Bible, Qutachi et al., 2012). To this end, naturally occurring and synthetic biomaterials have been developed and co-administered with NSCs in preclinical stroke models. A number of hydrogel materials seeded with NSCs have been designed and evaluated in preclinical stroke models including hyaluronic acid, collagen, Matrigel, and other xenogenic sources (Bible, Dell'Acqua et al., 2012;Jin, Mao et al., 2010;Jin et al., 2011;Lam et al., 2014;Lee et al., 2016;Moshayedi et al., 2016;Yu et al., 2010). These studies show that transplanted cells survive quite well in hydrogels due to excellent nutrient and oxygen permeability, and co-administration of NSCs with hydrogel reduces infarct size, increases host neurogenesis, and promotes functional recovery.
However, cell migration in hydrogels is often poor due to weak mechanical structure, and neurons do not extend their neurites through these three-dimensional matrices efficiently (Skop, Calderon, Cho, Gandhi, & Levison, 2014).
Opposed to hydrogels which are soft and become gelatinous upon brain injection, synthetic microparticles have a rigid structure on which neuronal growth cones can be sustained more efficiently (Park, Teng, & Snyder, 2002;Skop et al., 2014). Bible et al. (2009) transplanted a scaffold consisting of plasma polymerized allylamine (ppAAm)-treated poly(D,L-lactic acid-co-glycolic acid; PLGA) particles along with NSCs into the lesion cavity of stroked rats. Utilizing MRI to monitor integration of the NSC-scaffold matrices, they demonstrate primitive de novo tissue formation within 7 days posttransplantation. Subsequent histological analysis showed that the graft was fibrous in appearance along the periphery and consisted of neurons and astrocytes. However, the newly formed tissue was completely void of vasculature that could sustain long-term viability.
In a follow-up study, the group demonstrated that encapsulating the PLGA microparticles with VEGF promotes endogenous endothelial cell migration to the graft site which contributes to neovascularization (Bible, Qutachi et al., 2012). These findings were corroborated by Yamashita et al. who reported an increased number of endothelial cells and astrocytes after the administration of VEGF-enriched scaffold into the stroke cavity of their animal model, which led to increased tissue volume in the graft (Yamashita, Deguchi, Nagotani, & Abe, 2011). Future studies evaluating the benefit of enriching biomaterials with other potent neurotrophic factors, such as BDNF, GDNF, and NGF, would be useful to elucidate what signaling cascades will maximize NSC transplantation success.

| CLINI C AL CON S IDER ATI ON S FOR TR AN S L ATI ON
There are a number of challenges to address before NSC therapies can be widely adopted for clinical stroke treatment-many of which have yet to be assessed. The stroke brain environment is truly unique with respect to immune system challenges relative to studying cell replacement therapies in other organs. The brain is typically immunoprivileged, yet in the stroke environment the BBB is compromised and undergoes dynamic changes in permeability allowing for infiltration of systemic immune cells. This has raised questions pertaining to the immune system response to transplantation of allogeneic NSC lines in stroke and the potential need for autologous iPSC-NSCs to overcome this response (Manley et al., 2015  in the brain due to the propensity of cells to accumulate systemically in nontarget organs such as the lungs and liver (Fischer et al., 2009;Lappalainen et al., 2008). Previous studies have shown that IV NSC delivery results in neuroprotection and improved neurological performance without evidence of cell engraftment in the stroked brain, indicating that anti-inflammatory and regenerative trophic factor release is the main mechanism behind this treatment strategy (Watanabe et al., 2016). Taken together, safety considerations along with intended NSC therapeutic action should be considered when developing effective, translatable NSC therapies for stroke.

| Treatment window and dosing
A wide range of NSC treatment windows have been assessed in rodent models, ranging from immediately after reperfusion to 4 weeks post-stroke (Table 1). In these preclinical studies, the treatment window is often dictated by the predicted mode of therapeutic action. If the main objective is to maximize the neuroprotective and immunomodulatory roles of transplanted NSCs, then transplantation often occurred within the acute stage of stroke before infarction is complete and tissue can be rescued (Kokaia, Andsberg, Martinez-Serrano, & Lindvall, 1998 furthermore, the delayed transplantation did not augment NSC migration, proliferation, or neuronal differentiation (Darsalia et al., 2011). Another technical consideration that warrants further study for clinical translation is the optimal NSC dose (i.e., number of cells and number of transplantations). In rodent studies, cell dosing ranges from 1.0 × 10 5 to 4.5 × 10 5 cells in one to three transplantation sites for IP and a single dose of 3.0 × 10 6 to 4.0 × 10 6 cells for IV with no evidence of outcome discrepancies between transplantation protocols. Indeed, Darsalia et al. (2011) demonstrated that transplanting a greater number of NSCs does not result in a higher number of grafted cells or increased neuronal differentiation. Taken together, this suggests that an early treatment time window, before the inflammatory response is established, may be a more important factor determining engraftment success compared to dosing.

| CON CLUS ION
NSCs provide the unique opportunity to mitigate stroke pathology through multimodal therapeutic action. A number of preclinical studies in rodent stroke models have demonstrated promising evidence that NSCs are able to act as a neuroprotectant by limiting secondary injury through anti-inflammatory mechanisms, promoting endogenous neurogenesis and synaptic remodeling, and even act as a cell replacement thereby promoting tissue and functional recovery.
These preclinical findings have led to human clinical trials assessing NSC safety and efficacy in stroke patients with promising results.
However, additional studies designed to better understand important factors determining NSC engraftment success such as cotransplantation of multiple cell types, treatment time window, dosing number, and the effect of age and comorbidities will ultimately augment therapeutic efficacy and hopefully improve stroke prognosis and future treatment paradigms.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.