Mesenchymal stem cell‐based treatments for stroke, neural trauma, and heat stroke

Abstract Background Mesenchymal stem cell (MSC) transplantation has been reported to improve neurological function following neural injury. Many physiological and molecular mechanisms involving MSC therapy‐related neuroprotection have been identified. Methods A review is presented of articles that pertain to MSC therapy and diverse brain injuries including stroke, neural trauma, and heat stroke, which were identified using an electronic search (e.g., PubMed), emphasize mechanisms of MSC therapy‐related neuroprotection. We aim to discuss neuroprotective mechanisms that underlie the beneficial effects of MSCs in treating stroke, neural trauma, and heatstroke. Results MSC therapy is promising as a means of augmenting brain repair. Cell incorporation into the injured tissue is not a prerequisite for the beneficial effects exerted by MSCs. Paracrine signaling is believed to be the most important mediator of MSC therapy in brain injury. The multiple mechanisms of action of MSCs include enhanced angiogenesis and neurogenesis, immunomodulation, and anti‐inflammatory effects. Microglia are the first source of the inflammatory cascade during brain injury. Cytokines, including tumor necrosis factor‐α, interleukin‐1β, and interleukin‐6, are significantly produced by microglia in the brain after experimental brain injury. The proinflammatory M1 phenotype of microglia is associated with tissue destruction, whereas the anti‐inflammatory M2 phenotype of microglia facilitates repair and regeneration. MSC therapy may improve outcomes of ischemic stroke, neural trauma, and heatstroke by inhibiting the activity of M1 phenotype of microglia but augmenting the activity of M2 phenotype of microglia. Conclusion This review offers a testable platform for targeting microglial‐mediated cytokines in clinical trials based upon the rational design of MSC therapy in the future. MSCs that are derived from the placenta provide a great choice for stem cell therapy. Although targeting the microglial activation is an important approach to reduce the burden of the injury, it is not the only one. This review focuses on this specific aspect.

This has promoted the hypothesis that inhibiting proinflammatory cytokine production may be a therapeutic approach in treating brain injury (Barone & Parsons, 2000). Indeed, according to an observational study that involved 629 consecutive patients with chronic neurological, neuropsychiatric, and clinical impairment after stroke and TBI, the perispinal administration of etanercept produces clinical improvement (Tobinick, Rodriguez-Romancce, Levine, Ignatowski, & Spengler, 2014). In addition, various drugs or strategies improve outcomes of experimental heatstroke by reducing the overproduction of these proinflammatory cytokines resulting from heat stress .

| Microglial activation is involved in brain injury pathology
In contrast to their well-known deleterious roles, TNF-α and IL-6 have also been shown to exhibit neuroprotective properties. In both TNF-deficient mice (Bruce et al., 1996;Gary, Bruce-Keller, Kindy, & Mattson, 1998;Lambertsen et al., 2009;Taoufik et al., 2007) and IL-6deficient mice (Herrmann et al., 2003), infarct sizes were significantly increased following cerebral injury. In addition, TNF-α and IL-6 doublereceptor knockout mice had higher mortality rates than did their wildtype controls following heatstroke collapse (Leon, Blaha, & DuBose, 2006). Adult IL-6 knockout mice have also shown to compromise neurogenesis (Bowen, Dempsey, & Vemuganti, 2011). A complete lack of TNF-α or IL-6 might be detrimental to neurogenesis in the adult brain (Monje, Toda, & Palmer, 2003;Vallières, Campbell, Gage, & Sawchenko, 2002). This can be concluded by previous studies that show that an appropriate baseline level of TNF-α or IL-6 is necessary and essential for neurogenesis or host defense, whereas higher levels of TNF-α or IL-6 are detrimental to neurogenesis or host defense.
Recent developments in magnetic resonance (MR) and positron emission tomography (PET) imaging techniques have demonstrated that increased binding of the peripheral benzodiazepine receptor (PBR) PET ligand 11 C-RK11195 is interpreted as a marker of microglial activation and hence neuroinflammation in several brain diseases (Denes et al., 2010). Increases in 11 C-PK11195 binding are found 30 days after stroke in patients, which suggests a contribution of microglial activation to ongoing processes in the ischemic brain (Price et al., 2006). In a rat stroke model, evidence supports a role for microglia as a central mediator in the ongoing processes of stroke damage (Gelosa et al., 2014). In addition, microglial activation is involved in other neurodegenerative disease models, such as Alzheimer's and Parkinson's disease (Mosher & Wyss-Coray, 2014;Walker et al., 2014), traumatic brain injury (Chio, Lin, & Chang, 2015), and heatstroke .
Tumor necrosis factor-alpha levels in both serum and cerebrospinal fluid are found to be significantly elevated in ischemic stroke, traumatic brain injury, and heatstroke Chio et al., 2015;Gelosa et al., 2014). Activation of TNF receptor 1 (TNF-R1) is believed to promote proinflammatory and proapoptotic action, astrogliosis, leukocyte extravasation, and disrupted blood-brain barrier (BBB) permeability (McCoy & Tansey, 2008). However, other results have demonstrated that TNF-R1 is required for erythropoietin receptor and vasculoendothelial growth factor expression and protective effects in primary cortical neurons after ischemic and excitotoxic injury (Taoufik et al., 2008).

| MSC therapy may improve outcomes of brain injury by modulating microglial activation
Mesenchymal stem cells (MSCs) can be derived from different sources, including bone marrow, adipose tissue, the umbilical cord, and the placenta. Currently, clinical trials are being conducted to investigate the therapeutic effects of human MSCs in many cardiovascular and neurodegenerative disorders (Kalladka & Muir, 2014;Mastri, Lin, & Lee, 2014). In addition to their multilineage differentiation potential, MSCs may exert their regenerative effect via the production of multiple paracrine factors (Kalladka & Muir, 2014;Mastri et al., 2014). Production of IL-6, vascular endothelial growth factor (VEGF), hepatocytes growth factor (HGF), brain-derived neurotrophic factors (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-3 (NT3), fibroblast growth factor (FGF), and thrombospondins can be promoted by MSCs. It is well known that neural injury results in BBB breakdown and the infiltration of tissue neutrophils and macrophages into damaged brain tissue, which causes microglial activation. In addition, microglia have been promoted as a compelling target for treating infectious and inflammatory diseases of the brain (Chio et al., 2015;Denes et al., 2010;Rock & Peterson, 2006). It is likely that MSC therapy may improve outcomes of brain injury by modulating microglial | (3 of 11) e00526 activation. Although targeting the microglial activation is an important approach to reduce the burden of the injury, it is not the only one.

| MSCs fulfill the criteria that have been established by the international society of cellular therapy
Mesenchymal stem cells are multipotent, self-renewing cells (Friedenstein, Petrakova, Kurolesova, & Frolova, 1968). They fulfill the following criteria that have been established by the International Society of Cellular Therapy (Dominici et al., 2006): (i) adherence to plastic, (ii) expression of CD105, CD73, and CD90; lack of expression of CD45, CD34, CD14, CD116, CD79a, CD19, and HLA11; and (iii) ability to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. Due to extensive selfrenewal capacity, their ease of isolation, and their presence during young and fetal life, MSCs that are derived from the placenta are an appropriate source for stem cell therapy.
In this review, we collected publications that pertain to MSC therapy and cerebral injury that is caused by stroke, neural trauma, and heatstroke. In doing so, we emphasized the mechanisms of MSC therapyrelated neuroprotection, which were identified using an electronic search (e.g., using PubMed). It reports the feasibility of MSCs to improve neurological function after injury. It focuses on adult injuries such stroke, TBI, and heatstroke. It summarizes the pathophysiology of the injury briefly and offers an overview of MSCs therapeutic approaches.

| Neonatal stroke rats or mice
Neonatal stroke occurs frequently in live birth and presents motor dysfunction, cognitive deficits, and epilepsy (Ferriero, 2004;Kirton & de Veber, 2009). However, treatment options are not currently available.
The transplantation of MSCs into neonatal animal models of ischemic stroke promotes functional recovery by stimulating neurogenesis, oligodendrogenesis, and axonal remodeling (van Velthoven, Kavelaars, van Bel, & Feijene, 2010a,b;Yasuhara et al., 2008). The beneficial effect of MSC transplantation might involve the augmentation of the secretion of growth and differentiation factors and the fostering of an environment that stimulates both angiogenesis and neurogenesis (van Velthoven et al., 2010b(van Velthoven et al., , 2012(van Velthoven et al., , 2013 (Table 1). The secretome that has been obtained from MSCs contains several neurotrophic factors, including insulin-like growth factor-1 and brain-derived neurotrophic factor, which are responsible for the protective effects of MSCs that were observed in studies with in vitro and in vivo neuronal injury models (Wei et al., 2009). When compared with adults, it is believed that newborns benefit more from cell therapy because newborns have an increased brain plasticity as well as a different pathophysiology of the injury. In addition, in newborns the microglial activation is more pronounced as microglial activation is present during physiological brain development as well.

| MSC therapy improves outcomes of stroke mainly by secreting paracrine factors
Mesenchymal stem cells have the potential to differentiate into osteoblasts, chondrocytes, adipocytes, hepatocytes, and neurons (Sanchez-Ramos et al., 2000). Although, MSCs are able to pass through the BBB (Kopen, Prockop, & Phinney, 1999), MSCs that are transplanted by intracerebral or intravenous routes minimally and selectively migrate to the ischemic boundary sites Zhao et al., 2002).
Considering the small number of MSCs in injured brain tissue, the presence of therapeutic neurotrophic factors that are secreted by MSCs

Treatment regimens
Main results References no.

| MSC therapy improves outcomes of stroke by stimulating angiogenesis, neurogenesis, and synapse formation
The mechanisms that underlie the beneficial effects of transplanted MSCs include transdifferentiation into the neural lineage as well as the induction of neurogenesis, angiogenesis, and synapse formation in rodents (Kurozumi et al., 2005;Li et al., 2002;Shen et al., 2006;Wislet-Gendebien et al., 2005). Transplantation of MSCs protects against cerebral injury and upregulates IL10 expression in Macaca fascicularis , thereby suggesting the activation of endogenous neurotrophins. Angiogenesis that is induced by MSC transplantation promotes endogenous neurogenesis, which may produce functional recovery after cerebral injury in rats with ischemic stroke (Bao et al., 2011). Both histology and MRI reveal that human umbilical MSCs promote recovery after ischemic stroke in rats . The beneficial effects of MSC therapy are associated with improved revascularization in ischemic injured tissues.

| MSC therapy attenuates neuronal death by suppressing activated microglia
During the acute phase of cerebral injury, the expression of neuronal and microglial IL-6 is elevated in the injured penumbra (Berti et al., 2002;Block, Peters, & Nolden-Koch, 2000). Direct intrathecal implantation of MSCs results in enhanced neuroprotection. The implantation of MSCs into the injured brain activates resident stem cells niches via an NF kappa B-mediated increase in IL-6 production (Walker et al., 2010). Microglia have also been implicated in the pathogenesis of a number of neurodegenerative diseases, such as stroke, Alzheimer's disease, dementia, and multiple sclerosis (Danton & Dietrich, 2003).
Microglia can defend against brain damage, but excessive or sustained microglia activation can contribute to apoptotic cell death (Ohmi et al., 2003). Bone marrow MSCs result in the suppression of activated microglia and to a delay of neuronal death (Ohmi et al., 2003;Wei, Fraser, Lu, Hu, & Yu, 2012). Human MSCs also stimulate angiogenesis in focal cerebral injury by increasing expression of α-tubulin and angiopoietin 1 and 2 . MSC treatment reduces the expression of inflammatory cytokines in lipopolysaccharide-activated microglia and subsequently reduces aquaporin-4 expression and apoptosis of astrocytes after cerebral injury (Tang, Cai, et al. 2014, Tang, Liu, et al., 2014. In addition, the survival and function of transplanted MSCs after focal cerebral injury can be enhanced by melatonin pretreatment (Tang et al., 2014b). Both neurological deficit and brain edema and infarct volume are significantly decreased postischemic stroke with MSC treatment via the tail vein (Tang et al., 2014a).
MSCs also protect against brain injury in the mouse by stimulating the production of TGF-β (transforming growth factor), but reduce proinflammatory cytokines (e.g., IL-1, TNF-α) (Cheng et al., 2015). Thus, it appears that MSCs improve outcomes of ischemic stroke in animal models by stimulating neurotrophic factors production and endogenous neurogenesis and modulating neuroinflammation.

| Stroke patients
Systemic delivery of MSCs has also been shown to be a feasible and safe therapy for treating ischemic stroke patients (Tang et al., 2014b).
Long-term follow-up data further indicate a contribution of trans-

| Microglial activation as a biomarker for neural trauma
Mechanical injury to the brain or spinal cord results in glutamate excitotoxicity, BBB disruption, ischemia, mitochondria dysfunction, apoptotic and necrotic cell death, and inflammation (Mannix & Whalen, 2012). Secondary injury following TBI (traumatic brain injury) includes microglial activation (Davalos et al., 2005 neurotrophic factors, including nerve growth factor and transforming growth factor β (TGF-β) (Chio et al., 2015). Evidence has accumulated that indicates microglial activation as a biomarker for traumatic brain injury (Hernandez-Ontiveros et al., 2013).

| THERAPY WITH CONDITIONED MEDIUM FROM CULTURED MSCS IMPROVES OUTCOMES OF NEURAL TRAUMA
MSCs transplantations via different routes of administration have been widely studied in different species of SCI (spinal cord injury), and have been proven to produce beneficial effects following SCI ( Table 2) MSCs is shown to improve recovery after SCI in rats (Cantinieaux et al., 2013). In addition, in TBI mice, TBI rats, or TBI patients, intravenous or intrathecal administration of MSCs (Arien-Zakay et al., 2014;Chen et al., 2014;Lu et al., 2002;Nichols et al., 2013;Wang et al., 2013;Zanier et al., 2011;Zhang et al., 2013Zhang et al., , 2015 significantly improves the outcomes of TBI. The systemic injection of the secretome of cultured MSCs also improves the outcomes of TBI in rats . The transplantation of hypoxia-preconditioned MSCs improves infracted heart function via the enhanced survival of implanted cells and angiogenesis (Hu et al., 2008). It seems that while MSCs exhibit a prominent multilineage differentiation potential, the MSCs-derived mediators contribute to cytoprotection, angiogenesis, tissue repair, and alleviation of inflammation during neural injury (Mastri et al., 2014). Cell incorporation into the vessels or neurons is not a prerequisite for the beneficial effects that are exerted by MSCs. MSCs may improve the outcomes of neural injury by modulating multiple mechanisms, such as the secretion of trophic factor and immune function (Kalladka & Muir, 2014).

| THERAPEUTIC EFFECTS OF HUMAN MSCS IN EXPERIMENTAL HEATSTROKE
Heatstroke can be induced by severe heat exposure (i.e., classic heatstroke) or strenuous exercise (i.e., exertional heatstroke). Heatstroke syndrome is characterized by critical hyperthermia, which is associated with systemic inflammatory responses that result in multiorgan dysfunction, including delirium, convulsion, or coma . After the onset of heatstroke, the reduction in blood flow to the brain (or cerebral ischemia) results in hypothalamic neuronal damage, which induces multiple-organ dysfunction or failure.

Treatment regimens Main results
References no.

SCI rats received intravenous injection of MSCs-derived secretome
In vitro, secretome obtained from MSCs protects neurons from apoptosis, activates macrophages, and is proangiogenic. In vivo, MSCs secretome improves motor recovery. Cantinieaux et al. (2013) 3. SCI dogs, rabbits, or monkeys received intraspinal cord injection of MSCs Reducing spinal cord damage or neurological deficits by stimulating both de novo neurogenesis and production of BDNF Deng et al., 2006;Penha et al., 2014;Moon et al., 2014 4

. SCI patients received intrathecal or intraspinal cord injection of MSCs
Reducing spinal cord damage or neurological deficits Jiang et al., 2013;Mendonça et al., 2014;Cheng et al., 2014 5. TBI rats or mice received intravenous, intra-arterial, or intracerebroventricular injection of MSCs Reducing cerebral damage or neurological deficits by stimulating production of BDNF, NGF, VEGF, and IL-10, angiogenesis, and neurogenesis. Lu et al. 2002;Zanier et al., 2011;Zhang et al., 2013Zhang et al., , 2015Nichols et al., 2013;Chen et al., 2014 Heat-tolerant rats exhibit low levels of both IL-1β and TNF-α mRNA in the hypothalamus as well as high corticosterone levels in serum (Hu et al., 2008). In contrast, heat-intolerant rats present higher hypothalamic levels of both IL-1β and TNF-α mRNAs, but lower serum corticosterone level (Michel et al., 2007). Hypothalamic levels of IL-6, TNF-α, IL-1β, and nitrite in the hypothalamus were upregulated by heatstroke (Hsu, Chen, Lin, & Yung, 2014). It has been suggested that the inflammatory response in the acute phase of tissue injury may be related to aggravating tissue injury; however, in the later phase, these inflammatory mediators may contribute to tissue repair (Kadhim, Duchateau, & Sébire, 2008). Cytokines, such as IL-6 and TNF-α, may be essential at physiological levels for the maintenance of the endogenous neurogenesis in the brain (Bowen et al., 2011). Neither lack nor excess of IL-6 or TNF-α is beneficial for homeostasis of the inflammatory mechanisms.
It has been estimated that approximately 2% of HUCBC are positive for CD34 expression (Bender et al., 1991). CD34+ cells transplantation also attenuates the outcomes of heatstroke by reducing TNF-α production in serum, stimulating IL-10 production in serum, and stimulating production of GDNF in brain (Chen et al., 2007;Hwang et al., 2008).

| The potential use of granulocyte-colony stimulating factor (G-CSF) as a prophylactic agent for heatstroke
G-CSF is a polypeptide that promotes the mobilization of stem cells into peripheral blood (Lu & Xiao, 2006) and results in a reduction in mortality, infarct volume, and neurological deficits after cerebral ischemia in heatstroke rats (Lu & Xiao, 2006). Preconditioning with G-CSF attenuates heatstroke-induced hypothalamic apoptosis and neuronal damage by stimulating GDNF and VEGF overproduction in hypothalamus, thereby reducing levels of TNF-α, increasing levels of IL-10, and stimulating the expression of endothelial progenitor cells in the serum of rats (Yung et al., 2011).

| Transplantation of human dental pulp-derived stem cells protects against heatstroke
Human dental pulp-derived stem cells are self-renewing stem cells that reside within the perivascular niche of the dental pulp (Gronythos et al., 2002). They enhance recovery of focal cerebral injury in rats (Inoue et al., 2013). Human dental pulp-derived stem cells are also shown to attenuate ischemia and oxidative damage to the hypothalamus and the overproduction of systemic response syndrome molecules, including TNF-α and ICAM-1, in the peripheral blood stream in heatstroke mice (Tseng, Chen, Lin, & Lin, 2015). When considering the data presented herein, it appears that human MSCs may improve outcomes of heatstroke by reducing the overproduction of systemic response syndrome molecules as well as multiple-organ dysfunction or failure (Table 3).
Therefore, it can be concluded that some spontaneous but not extensive recovery (or increased endogenous neurogenesis) is typical following brain injury caused by stroke, neural trauma, and heatstroke. Exogenous cell therapy is promising as a means of augmenting brain repair by modulating microglial activation as depicted in Fig. 1.
As mentioned in the foremost section, the proinflammatory M1

Treatment regimens Main results
References no.

Heatstroke rats received intravenous or intracerebroventricular injection of HUCBCs
MSCs attenuate cerebral ischemic damage by reducing overproduction of TNF-α, IL-1β, and IL-6, but stimulating production of IL-10. 3. Heatstroke rats received subcutaneous injection of granulocyte-colony stimulating factor The factor attenuates cerebral ischemic damage by reducing overproduction of both TNF-α and ICAM-1, but stimulating production of IL-10, EPSs, GDNF, and VEGF. Yung et al. (2011) 4. Heatstroke mice received intravenous injection of human dental pulp-derived stem cells or HUCBCs MSCs attenuate cerebral ischemic damage by reducing overproduction of TNF-α, intercellular adhesion molecule 1, and oxidative damage markers, but promoting both hypothalamo-pituitary-adrenocortical axis activity and IL-10 production. Tseng et al. (2014) Tseng et al. (2015 HUCBCs, human umbilical cord blood cells.
Please see the legends of Table 1 for the explanation of abbreviations.
Hsuan et al.

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phenotype of microglia is associated with tissue destruction, whereas the anti-inflammatory M2 phenotype of microglia facilitates repair and regeneration. Therefore, MSC therapy may improve outcomes of ischemic stroke, neural trauma, and heatstroke by inhibiting the activity of M1 phenotype of microglia but augmenting the activity of M2 phenotype of microglia.

| The targeting of microglial activation in clinical trials as a rational design of NSC therapy in the future
During ischemic stroke, after brain trauma, and during heatstroke, the intrinsic inflammatory mechanisms of the brain as well as those of the peripheral blood stream are mediated by the release of proand anti-inflammatory cytokines and chemokines. According to a more recent review (Chio et al., 2015;Tuttolomondo, Pecoraro, & Pinto, 2014), microglia are the first source of the inflammatory cascade during brain ischemia and after brain trauma. Additionally, an important mediator of this inflammatory event is TNF-α. Etanercept, a TNF-α antagonist, which has been used therapeutically in animal models of ischemic stroke and neural damage (Kinnaird et al., 2004).
In addition, various drugs or strategies have improved the outcomes of experimental heatstroke by reducing the overproduction of these proinflammatory cytokines in both the brain and the peripheral blood stream . Although some spontaneous recovery (due to endogenous neurogenesis) in humans is thought to contribute to repair, exogenous MSC therapy is promising as a means of augmenting brain repair. MSCs, when administered systemically, are observed in the injured brain areas but not in noninjured brain areas and are never seen in corresponding brain areas of noninjured animals (Nishio et al., 2006). Nevertheless, paracrine signaling, rather than cell incorporation into vessels or neurons, is a prerequisite for the beneficial effects that are exerted by MSCs (Kinnaird et al., 2004). The multiple mechanisms of action of MSCs include enhanced angiogenesis and neurogenesis (by the secretion of trophic factors), immunomodulation, and antiinflammatory effects (Kalladka & Muir, 2014). Cytokines, including TNF-α, IL-1, and IL-6, are greatly produced by microglia in the brain after experimental stroke (Clausen et al., 2005(Clausen et al., , 2008Hill et al., 1999;Lambertsen et al., 2005). Appropriate baseline levels of TNF-α or IL-6 are necessary and essential for neurogenesis or host defense, whereas higher levels of TNF-α or IL-6 are detrimental to neurogenesis or host defense (please see the Introduction). The appropriate level of microglial activation is necessary and crucial for normal neurogenesis and host defense, whereas microglial overactivation results in deleterious and neurotoxic consequences (please see the Introduction section).
The exogenous administration of MSCs may promote tissue repair by stimulating trophic factor release and endogenous neurogenesis (Chamberlain, Fox, Ashton, & Middleton, 2007;Chen, Tredget, Wu, & Wu, 2008;Phinney & Prockop, 2007). The expression of prosurvival and proangiogenic markers in MSCs can be enhanced by hypoxic preconditioning (Chacko et al., 2010). This review offers a testable platform for the targeting of microglial activation in clinical trials that are based upon rational design of MSC therapy in the future.
It is most probable that the central concern that is considered in this review is that MSCs may exert their neuroprotective effects mainly by modulating the production of both cytokines and neurotrophic factors. In addition, immunosuppression of allogenic MSC transplantation after neural injury improves graft survival and beneficial outcomes in rats (Torres-Espín, Redondo-Castro, Hernandez, & Navarro, 2015).
Conversely, the intravenous, intranasal, or intracerebral administration of MSCs is found to be beneficial in treating neurological damage.
F I G U R E 1 The mechanisms of MSC therapy-related neuroprotection. Microglia are the first source of the inflammatory cascade in brain injury. Microglia are activated rapidly in response to central nervous system injury and produce proinflammatory cytokines, growth factors, reactive oxygen species, nitric oxide, and glutamate (Block & Hong, 2005;Jin et al., 2010;Stolp & Dziegielewska, 2009). The proinflammatory M1 phenotype of microglia is associated with tissue destruction, whereas the anti-inflammatory M2 phenotype of microglia facilitates repair and regeneration. During brain injury, the activity of M1 phenotype microglia and M2 phenotype microglia is augmented and inhibited, respectively. In contrast, MSCs might improve outcomes of brain injury by inhibiting the activity of M1 phenotype microglia and augmenting the activity of M2 phenotype microglia To produce a similar beneficial effect, the intravenous route injection needs a lesser dosage than does the intracerebral route injection, suggesting the central action of MSCs. Additionally, compared to MSCs therapy adopted postinjury, pretreatment regimens of MSCs has significantly better beneficial effects. However, expansion of MSCs without fetal bovine serum is a big problem during different pretreatment regimens and preparation of MSCs. Nevertheless, because substances that are administered via the peripheral blood stream are able to reach multiple organs (including brain tissues) during diseased conditions, intravenous route injection is the most practical approach for cell therapy in general.
6.2 | MSCs that are derived from the placenta may be the most practical for use in the treatment of brain ischemic injury

CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.