Neuroprotection of Stem Cells Against Ischemic Brain injury: From Bench to Clinic

Abstract

Neurological injuries can have numerous debilitating effects on functional status including sensorimotor deficits, cognitive impairment, and behavioral symptoms. Despite the disease burden, treatment options remain limited. Current pharmacological interventions are targeted at symptom management but are ineffective in reversing ischemic brain damage. Stem-cell therapy for ischemic brain injury has shown promising preclinical and clinical results and has attracted attention as a potential therapeutic option. Various stem cell sources (embryonic, mesenchymal/bone marrow, and neural stem cells) have been identified and investigated. This review provides an overview of the advances made in our understanding of the various types of stem cells and progress made in the use of these stem cells for the treatment of ischemic brain injuries. In particular, the use of stem cell therapy in global cerebral ischemia following cardiac arrest and in focal cerebral ischemia after ischemic stroke are discussed. The proposed mechanisms of stem cells’ neuroprotective effects in animal models (rat/mice, pig/swine) and other clinical studies, different routes of administration (intravenous/intra-arterial/intracerebroventricular/intranasal/intraperitoneal/intracranial) and stem cell preconditioning are discussed. Much of the promising data on stem cell therapies after ischemic brain injury remains in the experimental stage and several limitations remain unsettled. Future investigation is needed to further assess the safety and efficacy and to overcome the remaining obstacles.

1. Introduction

Ischemic brain injury, defined as brain injury induced by oxygen deprivation, results in damage of brain tissue with loss of neurons, astroglia, and oligodendroglia. It is one of the most important central nervous system (CNS) disorders and remains a leading cause of death and disability [1]. Ischemic and reperfusion injury to the brain is thought to be irreversible [2]. Cardiac arrest (CA) is the most common cause of global ischemic brain injury, with the hippocampus, cortex, thalamus, and cerebellum being the most susceptible. Hypoxia and ischemia can lead to apoptosis and cell death in these vulnerable brain regions, leading to neurological deficits [3, 4]. Ischemic stroke is another major health problem causing severe social and economic burdens worldwide [5]. Stroke is induced by either a transient or permanent reduction of blood flow to the brain, which results in the death of neural tissue [6, 7]. Despite available treatments such as thrombolytic therapy, interventional thrombectomy, and physical rehabilitation, many patients suffer long-term disabilities and deficits.

Stem cells have self-renewal potential and are capable of differentiation into many cell types. Several types of stem cells have demonstrated therapeutic potential in ischemic stroke and brain injury induced by CA [8–13]. The therapeutic effects of stem cells are not limited to their ability to replace damaged or dead cells and tissues. They have also been shown to provide a supportive microenvironment suitable for neurogenesis [14, 15], neurotrophic factor release [16], and mitochondrial transfer [17, 18]. This review provides an overview of stem cell sources, routes of administration, and therapeutic potential for ischemic brain injury induced by CA and stroke. We aim to summarize the related studies focusing on stem cell types, transplantation methods, preconditioning, safety, efficacy, unsettled limitations, and future directions for clinical applications to enhance functional recovery after ischemic brain injury (Fig. 1).

Figure 1. Overview of stem cell application in ischemic brain injury.

Stem cells belong to four major categories based on their differentiation potential. Cell types including iPSC, ESC, NSC, and MSC were applied in the treatment of ischemic brain injury. Multiple delivery routes were selected for the administration of stem cells. NSC, neural stem cell; MSC, mesenchymal stem cell; ESC, embryonic marrow stem cell; iPSC, induced pluripotent stem cell

2. Rationale for Cell-Based Therapies for Ischemic Brain Injury

Current treatments for ischemic brain injury are limited by short treatment time windows and a lack of regenerative potential [19]. Due to its differentiation potential, cell-based therapy offers a promising alternative providing neuroprotection and promoting regeneration, and potentially extending the treatment window [20]. Cell-based therapy for ischemic brain injury can be attributed to neural regeneration, reduction of neuronal apoptosis caused by ischemia and hypoxia, replacement of dead neurons, [21–24] and enhanced neuroprotection [25]. While neural regeneration and neuroprotection are both recognized mechanisms in stem cell therapy, treatments have generally focused on harnessing one or the other. There seems to be a preference for treatments based on neural regeneration in preclinical and clinical trials [26–28]. Further, ischemic brain injuries both from stroke and from CA are associated with immune responses, including inflammatory elements involved in exacerbating the initial damage and further impairing neuronal tissue [29]. Stem cells have illustrated the capability of modulating the immune system, boosting therapeutic effects by alleviating inflammation-related injury (Fig.2) [12, 30, 31].

Figure 2. Neuroprotective function of stem cell therapy in ischemic brain injury.

Ischemia leads to activation of microglia, demyelination, and a loss of tight junction integrity in BBB. Most microglial cells transform from resting state (small, round cells with elaborate ramifications) into activated state, shown as bigger, more amoeboid cells with retracted processes. Activated microglia recruits and engulfs the debris. The BBB damage, microglia induced-neuroinflammation, neurodegeneration and necrosis can be observed post ischemic brain injury. Stem cell therapy exerts neuroprotective effects by targeting these pathophysiological mechanisms.

3. Delivery Routes of Stem Cell Therapy in Ischemic Brain Injury

Routes of stem cell transplantation include, but are not limited to, intravenous delivery (IV), intra-artery delivery (IA), intracerebroventricular (ICV) delivery, intraperitoneal delivery (IP), intranasal delivery, and intraparenchymal/intracranial delivery (IC) [13]. Intranasal administration, intravenous and intra-arterial delivery are usually less invasive when compared with ICV and intracranial administration. Intravenously administrated stem cells can cross the blood-brain barrier (BBB) and target damaged cerebral regions [32, 33]. However, stem cells administrated via IV tend to accumulate in peripheral tissues, such as the liver, spleen, kidneys, and lungs [34]. Complications of pulmonary embolism have been reported in an intravenous injection of adipose-derived mesenchymal stem cells (AD-MSCs) [35]. The IA delivery method can lower the risk of stem cells being trapped in peripheral organs [36]. However, with IA delivery, cells may accumulate in the artery of the brain, potentially causing cerebral infarction [37, 38]. ICV delivery is invasive, but it allows stem cells to reach the cerebral spinal fluid (CSF) directly. Subsequently, CSF circulation delivers the cells to multiple sites within the CNS [39]. IC delivery allows for high concentrations of stem cells to be delivered directly to the injured brain, but it carries a risk of BBB disruption and traumatic injury secondary to the highly invasive nature of the delivery method [40, 41]. Stem cells in IP delivery reach the brain in limited concentrations. Most cells are aggregated in the peritoneal cavity [42]. Intranasal delivery has the advantage of easily repeated doses [43–46]. It is non-invasive, rapid, and allows cells to migrate to the brain through the olfactory epithelium [47–49].

Thus far, ICV delivery has been demonstrated as feasible and relatively safe despite its invasive nature [8, 9, 13]. Intranasal delivery in preclinical trials has demonstrated good penetration into the CNS with little to no adverse effects [12, 13, 50, 51]. Ultimately, the ideal delivery method is yet to be determined.

4. Stem Cell Therapy in Ischemic Brain Injury

4.1. Pluripotent Cells

4.1.1. Embryonic Stem Cells

Embryonic stem cells (ESCs), derived from the inner cell mass of a preimplantation embryo, possess the potency of unlimited self-renewal and differentiation. ESCs are pluripotent cells and can differentiate into any body cell type. The unlimited expansion guarantees the efficiency of cell numbers can be expanded and the huge potential of differentiation with enhanced efficacy. ESCs were first derived and reported by Evans and Bremnes in vivo and in vitro, respectively [52, 53]. The beneficial effects of ESCs on ischemic stroke are associated with regenerative capacity, giving rise to neuronal and glial cells, which are important elements to form brain tissues [54]. ESCs do come with ethical challenges in their use.

4.1.1.1. Preclinical Evidence

Engrafted ESCs were shown to reduce brain lesion size and increased the number of micro-vessels at the border of the infarction region in a photo-chemically induced-thrombotic stroke model. ESCs restored histological and behavioral deficits by enhancing angiogenesis with endogenous endothelial cells [55]. In another study, intracranial infusion of mouse-derived ESCs 3 h after 90-min transient middle cerebral arterial occlusion (MCAO) alleviated motor dysfunction, decreased infarct volume, and restored damaged synaptic connections associated with stroke lesions in rats [56]. Pretreatment with overexpression of Bcl-2, an anti-apoptotic mitochondrial protein, increased survival and differentiation of transplanted ESCs and promoted functional benefits [57]. Intra-cortex infusion of mouse-derived ESCs 7 days after 2-h MCAO exhibited enhanced functional recovery on neurological and behavioral tests in rats with severe focal ischemia, and gene modification with Bcl-2 further enhanced this therapeutic effect. ESC-derived cells expressed cell surface markers of neurons, astrocytes, oligodendrocytes, and endothelial cells in the lesion cavity at 8 weeks after transplantation. ES cell-derived neurons exhibited dendrite outgrowth and formed neuropil [57].

Transplantation of ESC-derived differentiated cells is a promising method to avoid malignant transformation of ESCs in vivo. ESCs and ESC-derived neuron-like (ES-N) cells recovered the immunoreactivity of tyrosine hydroxylase and dopamine transporter in the striatum. Intra-striatal delivery of mouse-derived ESCs or ES-N cells 1 week after 2-h MCAO promoted dopaminergic function and subsequently enhanced neurological outcomes in MCAO-induced focal ischemia in rats [58]. Another study investigated the safety and efficacy of embryonic stem cell-derived neural stem cells (ESC-NSCs) in ischemic stroke. One week after 1.5-h MCAO, intracranial administration of ESC-NSCs exerted neuroprotective effects on rats. Transplanted ESC-NSCs differentiated into neurons, astrocytes, and oligodendrocytes through multiple passages both in vitro and in vivo. ESC-NSCs migrated to ischemia-vulnerable regions and enhanced forelimb movements. Meanwhile, no chromosomal abnormalities or tumor formations were observed after implantation [59].

Intracranial delivery of embryonic stem cell-derived neural progenitor cells (ESC-NPCs) at 1-week post-stroke had a favorable effect on behavioral recovery and reduction of infarct size in the 2-h MCAO-induced cerebral infarct model of rats. However, the delivery route induced some damage in the injected site of the basal ganglia, such as loss of neuroglial cells, reactive gliosis, and microcalcification, which was found in the Sham-operated group as well, suggesting the need for a less invasive delivery method [60]. ESC-NPCs, when transplanted into the infarct core and periphery of adult rats, survived and revealed the characteristics of electro-physiologically functional neurons. These neurons were equipped with voltage-gated sodium channels and were capable of evoking action potentials. Synaptic input with spontaneous excitatory post-synaptic currents was observed in engrafted ESC-NPCs. ESC-NPCs can also differentiate into immunohistochemically mature glial cells and neurons of various neurotransmitter subtypes [61]. Another study further confirmed the survival, differentiation, and network formation of ESC-NPCs derived from monkeys and transplanted into the ischemic mouse brain. Intra-striatum delivery of monkey-derived ESC-NPCs 24 h after 30-min MCAO resulted in favorable functional recovery in ischemic brain injury, as ESC-NPCs migrated to the peri-infarct region and differentiated into diverse types of neurons and glial cells [62].

4.1.1.2. Contradictories, Limitations, and Perspectives

In contrast, it was reported that intracranial transplantation of 8.0×10⁵ ESC-NPCs at day 7 post-MCAO failed to show therapeutic effects on cortical stroke induced by permanent occlusion of the middle cerebral artery and temporary occlusion (60 min) of both common carotid arteries in rats. ESC-NPC treatment showed low graft cells survival and no change in infarct volume and sensorimotor function [63]. These differences may be attributable to variations in stem cell dose, the timing of administration, route of administration, or pretreatment methods between studies. As these measures are not yet clearly optimized, there are not yet across-study standards for these variables.

Studies on ESC-based cell therapy for ischemic brain injuries remain limited and are primarily preclinical in nature. Treatments with ESCs suggested comorbidity of teratoma formations. One study compared the safety of transplantation of undifferentiated and pre-differentiated murine ESCs in the focal ischemic brain induced by 1-h MCAO and indicated host-dependent tumorigenesis in the mouse brain [64]. 3 weeks after transplantation of undifferentiated ESCs, microscopically visible tumors were detected in 2 out of 22 mice brains, whereas 10 out of 11 brains exhibited large tumors when using the pre-differentiated ESCs. Immunohistochemistry showed the presence of colocalization of GFP-positive cells and tumor tissue, suggesting that they were derived from the transplanted ESCs [64]. Another study analyzed the effects of the stage of ESC differentiation and the postischemic environment on the formation of teratoma [65]. The effects of the ischemic environment on the formation of teratoma by transplanted ESCs were limited to early differentiation stages [65]. These findings highlight the importance of taking cell resources into consideration to avoid potential tumorigenesis. The development of future research will continue to be hindered by ethical concerns, a lack of optimal cell sources, cell conservation, immune rejection, and the risk of teratogenic or malignant transformation, pending future studies to overcome these challenges and limitations.

4.1.2. Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs), produced by incorporating specific transcription factors into somatic cells from skin or urine, have powerful self-renewing potential and can differentiate into any tissue-specific adult body cell lineage [66, 67]. iPSCs were first identified by Takahashi and Yamanaka through a retroviral vector [68, 69]. iPSCs are a class of cloned cells with similar biological functions to ESCs [68, 69]. However, iPSCs have advantages over ESCs. iPSCs can be harvested from the somatic cells in the same body with non-invasive techniques. Therefore, iPSCs have fewer ethical challenges regarding their transplantation [70]. Although iPSCs were developed rapidly and their therapeutic effects have been studied in many CNS disorders, the use of iPSCs for ischemic brain injury has been limited due to some potential risks of tumorigenesis, poor integration, and mutagenesis [71–73]. The published literature investigating the clinical applications of iPSCs and ESCs is limited. This section reviews the application of pluripotent stem cells in rodent and large animal models of ischemic brain injury and discusses favorable outcomes, limitations, and techniques for further improving stem cell therapy.

4.1.2.1. Preclinical Evidence

CA-Induced Ischemic Brain Injury

Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) play an important role in modulating the immune system. In vitro, transplanted iPSC‑MSCs altered the polarization direction of macrophages in the oxygen and glucose deprivation (OGD) model [74]. In vivo, intravenous injection of iPSC-MSCs via the femoral vein as soon as the return of spontaneous circulation (ROSC) was shown to lower the mortality in a transcutaneous electrical epicardium stimulation-induced CA model via an immune regulatory effect in rats. Different macrophages with different functions worked together to repair the damaged tissue after CA. iPSC-MSCs increased the expression of M2 macrophages and decreased the expression of M1 macrophages after cardiopulmonary resuscitation (CPR) [75], demonstrating that iPSC‑MSCs may suppress inflammation and augment neurological recovery following CPR.

Stroke-Induced Ischemic Brain Injury

Several preclinical studies have suggested the beneficial effects of iPSCs in ischemic stroke treatment. Transplanted iPSCs enhanced motor and sensory function, narrowed infarct volume, promoted angiogenesis, neurogenesis, immunomodulation, and anti-inflammation in rodents affected by ischemic stroke [76]. Multi-site injection of iPSCs into the cerebral cortex promoted synapse formation between neurons in a 30-min MCAO stroke-injury model [76]. In another 70-min MCAO-induced stroke model in rats, a 4-site injection of iPSCs promoted sensorimotor function without comorbidity of seizure or convulsive activity. Grafted iPSCs survived and differentiated into neuronal cells [77]. Intracranial delivery of induced pluripotent stem cell-derived neural stem cells (iPSC-NSCs) 24 h after 2-h MCAO rapidly attenuated neurological deficits during the early stage of ischemic stroke and demonstrated BBB integrity protection, downregulation of inflammatory response, inhibition of microglial activation and reduction in adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1) [78].

The neuroprotective function of induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) in a translational pig ischemic stroke model was first demonstrated in 2018 [79]. Intraparenchymal injections of iPSC-NPCs 5 days after stroke promoted recovery of posture, postural reactions, mental state, and appetite in pigs that underwent permanent MCAO [79]. Similarly, another study suggested that iPSC-NSC administration intracranially 5 days after permanent MCAO surgery improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in an ischemic pig stroke model [80]. iPSC-NSC-based cell therapy protected white matter integrity, modulated brain metabolism, and improved cerebral blood perfusion in the ipsilateral hemisphere in pigs with ischemic brain injury after stroke [80].

Enhancement in iPSC Application

Extensive preclinical studies have investigated methods of improving the proliferation, migration, and function of transplanted stem cells via preconditioning methods. Pretreatment with episomal plasmids (EPs) based-reprogramming produced integration-free and non-viral iPSCs, which were advantageous over gene-integration iPSCs when transplanted in stroke models [81, 82]. Ep-iPSCs proliferated and migrated into stroke-induced brain territories and differentiated into neuronal and glial cells [83]. Intracerebrally administrated EP-iPSCs decreased the number of activated microglia, inhibited astroglia scar formation, narrowed infarct size, and enhanced sensorimotor and behavioral functional recovery in rats [83]. Another study of reprogramming iPSC therapy in the 30-min MCAO stroke model with transplantation at 1 week and 48 hours after MCAO in mice and rats respectively, both gained improved results [84]. Intracranial administration of iPSCs enhanced endogenous plasticity by upregulating the level of vascular endothelial growth factor (VEGF). Extension of axons to the globus pallidus was observed, which enhanced the recovery of forepaw movement [84]. Mixed iPSCs with fibrin glue (iPSC-FG) is another approach to enhance the therapeutic effect of iPSC in stroke. Subdural transplantation of iPSC-FG improved motor function and decreased infarct volume in rats with stroke induced by 1-h MCAO. iPSC-FG modulated inflammatory reactions and transplanted iPSCs differentiated into astroglia-like and neuron-like cells [85]. Comorbidity of teratoma formation was reported in 6 of 6 (100%) MCAO-rats at 4 weeks post-transplantation with direct intracranial injection. However, administration of iPSC-FG to subdural space minimized the chance of tumor formation, no teratomas were observed 6 weeks after subdural administration. Subdural administration was reported to be a safer route of administration by avoiding iatrogenic injury to the brain parenchyma and reducing the risk of teratoma formation. This study provided new suggestions for the clinical application of stem cell therapy [85].

4.1.2.2. Contradictories, Limitations, and Perspectives

Despite these promising findings, it is of note that some preclinical studies did not show the same positive results. One study demonstrated that intracerebral transplantation of iPSC-NSCs 1 week after focal cerebral ischemia resulted in survival and differentiation of neural cells in the ischemic rat brain, but without behavioral recovery improvement at 1 week, 3 weeks, and 5 weeks post-stroke. The infarct size was also similar to the vehicle group at 5 weeks post-stroke [86]. Another study showed that intracerebral delivery of iPSCs 24h after 30-min MCAO could induce abundant neuroblasts and a few mature neurons in mice brains but did not show a significant difference in behavioral recovery at 28 days post-transplantation. Further, this study demonstrated iPSC-induced tumorigenesis in the brain [87].

iPSC therapy for ischemic brain injury has shown inconsistent outcomes and even induced tumorigenesis. The discrepant results may be attributed to different cell dosages and delivery time points when administrating stem cells, and tumorigenesis may be attributed to cell resources. EPs-based reprogramming, gene integration, and biomaterials can help enhance the efficacy of iPSC therapy. More studies are needed to further verify the safety and efficacy of iPSC-based cell therapy in ischemic brain injury, as well as the optimal dose and timing.

4.2. Adult Stem Cells

4.2.1. Neural Stem Cells

Neural stem cells (NSCs) are pluripotent cells that can differentiate into major cell types of CNS in the adult body. NSCs exist in certain areas of the brain throughout life [88], with endogenous NSCs primarily distributed within the dentate gyrus (DG) and subventricular zone (SVZ) [89]. There are three main sources of therapeutic exogenous NSCs. NSCs can be derived directly from brain tissue, particularly from the region of the olfactory bulb or paraventricular nucleus. Second, pluripotent stem cells (PSCs) can differentiate into NSCs. Lastly, NSCs are able to differentiate from somatic cells, including renal tubules, skin fibroblasts, and blood cells [90]. Initially, human neural stem cells (hNSCs) had limitations of restricted donor resources and a low rate of expansion and proliferation in vitro. However, up to now, hNSCs have been successfully cultured [91], and have demonstrated the potential to differentiate into neuronal and glial cell types in brain ischemia [92]. In this section, an overview of the preclinical and clinical evidence is presented to discuss the neuroprotective effects after NSC transplantations in ischemic brain injury, as well as to introduce different mechanisms and modifications to further enhance the effectiveness of NSC transplantations therapy.

4.2.1.1. Preclinical Evidence

CA-Induced Ischemic Brain Injury

One study has demonstrated that ICV delivery of hNSCs 3 hours after ROSC in an 8-minute asphyxia model of cardiac arrest resulted in better functional recovery scores and improved electrophysiologic recovery. This study demonstrated modulation of the inflammatory Wnt/β-catenin signal pathway [8, 9]. Grafted hNSCs demonstrated the potential of proliferation and further migration from SVZ when compared to controls [8, 9]. Then, they further found that intranasal delivery of hNSCs at 3h ROSC had superior neuroprotective function over IP injection of glibenclamide by regulating neuroinflammation TLR4/NLRP3 pathway both in vitro and in vivo[12]. Another study evaluated the feasibility of NSC transplantation into the hippocampus in a 12-minute potassium-induced cardiac arrest model [93]. Intracranial delivery of NSCs 1 week after ROSC improved neurological outcomes in rats through the SDF-1/CXCR4 pathway, which was critical for the migration of NSCs to injured brain regions. Grafted NSCs migrated into the hippocampal CA1 segment and differentiated into mature neurons [93].

Stroke-Induced Ischemic Brain Injury

The idea of enhanced neurogenesis for the replacement of injured or necrotic neuronal cells has become a target for NSC therapy in ischemic stroke. Intracranial transplantation of NSCs 14 days after permanent MCAO has demonstrated the promotion of proliferation and homing of endogenous NSCs in the DG and SVZ in rats [94]. Administration of exogenous NSCs led to enhanced migration of endogenous NSCs to damaged brain regions and differentiation into mature neurons in the ischemic brain [94–97]. Intracranial delivery of NSCs 48 h after 1-h MCAO improved behavior recovery in rats. NSCs survived and differentiated into neuroblasts or mature neurons at 6 and 14 weeks from stroke onset. Administration of exogenous NSCs increased the proliferation of endogenous cells in the SVZ, enhanced the migration of neuroblasts and mature neurons, and inhibited the activation of inflammatory microglia [98]. Interestingly, neurogenesis appeared to be more active in NSC transplantation at 48 h after MCAO when compared with injection at 3 weeks, indicating that the timing of administration also influences cell transplantation effect and therapeutic efficacy [98]. Further studies are warranted to verify the optimal timing and mechanisms of NSC delivery.

Neural reorganization is attributed to the structure modification of synapses, dendrites, and axons, which can be induced by the activation of endogenous NSCs in stroke. Intraparenchymal delivery of NSCs 7 days post-permeant-MCAO enhanced dendritic plasticity and axonal transport by remodeling surviving circuits in the cortex, striatum, and thalamus, which was associated with neurological recovery in a rat stroke model [99]. Grafted NSCs upregulated the expression of vascular endothelial growth factor (VEGF), glial-derived neurotrophic factor (GDNF), insulin growth factor‐1 (IGF‐1), thrombospondins 1 (TSP-1), and thrombospondins 2 (TSP-2), among which VEGF, TPS1, and TPS2 can act as mediators for the therapeutic effects of NSCs on the reorganization of surviving circuits [99]. Intracranial transplantation of NSCs 2 days after 45-min transient MCAO augmented motor and sensory function by promoting synaptic plasticity, axonal growth, and neuronal proliferation in mice [100].

Angiogenesis contributes to cell survival, and enhanced angiogenesis improves the environment for the regeneration of exogenous transplanted stem cells and the endogenous cells involved in stroke repair and recovery. Studies have demonstrated that endogenous NSCs have the capability of migrating into the ischemia-vulnerable brain region, interchanging the growth factors and neurotrophic factors with endothelial cells to enhance angiogenesis, which led to improved functional recovery in preclinical stroke models [101]. Delivery of NSCs to the lateral ventricle 24 h after 2-h MCAO enhanced angiogenesis in the penumbra area after stroke. Increased proliferation of endothelial cells, upregulated expression of the angiogenic receptor, and higher density of microvessels were observed [96]. Another study investigated the angiogenic activity of NSCs (CTX0E03) in rats underwent 1-h MCAO. In vitro, hNSCs (CTX0E03) exerted angiogenic effects by overexpression of trophic and pro-angiogenic factors, boosting vascular regrowth. In vivo, NSCs increased the number of microvessels at the injured site and provided significant benefits for functional recovery after ischemic stroke [102]. Intracranial administration of NSCs (CTX0E03) 4 weeks after 1-h MCAO promoted behavioral recovery in a dose-dependent manner, and the surviving CTX0E03 differentiated into endothelial phenotypes in the brain. Mid-dose (4.5×10⁴ cells) and high-dose (4.5×10⁵cells) groups provided better functional recovery than that of the low-dose (4.5×10³ cells) group, indicating that the therapeutic effects of NSC therapy are related to the cell dose [97]. The optimal dose for NSC therapy in ischemic stroke remains to be further verified. Several studies have suggested that transplantation of NSCs increased the co-localization of NSC and neuron markers, indicating differentiation into neurons in the ischemic brain following ischemic stroke [103, 104]. Intracranially transplanted NSCs survived and generated several neurons in the stroke-damaged striatum of adult rats [103]. Glial differentiation in NSCs, including differentiating into oligodendrocytes and microglia, has been reported in rat stroke models, and these models demonstrated some improvement in neurological deficits after ischemic brain injury [99, 103, 105].

Immunoreaction and inflammatory processes following ischemic stroke are triggered by the activation of microglia and result in secondary brain injury. Within minutes of the onset of ischemia, activated microglia produce and upregulate the expression of pro-inflammatory factors such as tumor necrosis factor -α (TNF -α), interleukin 1β (IL-β), and interleukin 6 (IL-6), promoting the migration of macrophages across the BBB, and exacerbating ischemic injury [12, 106]. Rat stroke models have investigated the NSC regulation of this inflammatory process and reported that transplanted NSCs inhibited the inflammatory response by releasing neurotrophic factors such as brain-derived neurotrophic factor (BDNF) [107], nerve growth factor (NGF) and GDNF [108, 109]. This downregulation of the inflammatory response was associated with improved neurological function after stroke. Intraparenchymal delivery of NSCs demonstrated reduced activation of immune cells in both mouse and rat MCAO models in several preclinical studies [78, 98, 105]. Grafted NSCs played a neuroprotective role by reducing inflammation, leading to reduced infarct volume and better neurological performance [110, 111]. Most studies demonstrating the immunomodulatory effects of NSCs have utilized the intraparenchymal mode of administration. Other studies also showed that intravenous injection of NSCs following an ischemic stroke inhibited inflammatory reactions, reduced the activation of immune cells, and alleviated neuronal apoptosis [110–112].

Enhancement in NSC Application

Modification of hNSCs in a glycoengineering-manipulation manner with new thiol-modified N-acetylmannosamine (ManNAc) analogs, a type of sugar analog, leads to greater potency of hNSCs while still allowing differentiation to a glial lineage [10]. In the hNSCs, the biochemical endpoint was upregulated, which was consistent with Wnt signaling in the absence of a thiol-reactive scaffold. The study provided novel tools for manipulating human stem cells to enhance the therapeutic effects during cell-based therapy [10]. Recently, they applied these metabolic glycoengineering (MGE) -treated hNSCs to their asphyxia-induced CA model, which enhanced the therapeutic effects of stem cells and improved neurological recovery after CA [11].

NSCs pretreated with overexpression of MicroRNA-26a, a microRNA that modulates apoptosis, showed improved survival of grafted NSCs in a drug-induced hypoxia injury model in vitro. In vivo, MiR-26a-modified NSCs protected NSCs from apoptosis and enhanced neurological function via the β-catenin signaling pathway in a 6-min CA-induced ischemic brain injury model [113]. Bone marrow mesenchymal stem cells (BM-MSCs) interplay with NSCs through extracellular vesicles (EVs) [66]. ICV administration of co-cultured NSCs and BM-MSCs 20min after resuscitation enhanced neuronal cell survival and alleviated CA-induced brain injury via microRNA-133b preconditioned EVs. BM-MSCs promoted NSC differentiation into neuronal cells, while NSCs stimulated BMMSC-EVs to release miR133b, which activated AKT-GSK-3β-WNT-3 pathway by targeting JAK1, ultimately promoted neuronal cell survival in the brain after CA [66].

4.2.1.2. Clinical Evidence

An open-label, single-site, and dose-escalation study of NSCs showed neuroprotective benefits in 11 stroke patients with a stable disability (National Institutes of Health Stroke Scale, NIHSS ≥ 6 and modified Rankin Scale, mRS 2–4) [114]. Single doses of 2 ×10⁶, 5×10⁶, 10×10⁶, or 20×10⁶ NSCs were applied through ipsilateral putamen injection 6–60 months post-stroke. Intracranial infusion of NSC improved neurological functions and no serious adverse event was observed with a 2-year follow-up [114]. It is of note that these studies were very small pilot studies, with only 8–11 patients recruited in the treatment group, and larger controlled studies are needed to further clarify the safety and efficacy.

Combination Therapy

A small pilot study investigated the effects of transplantation of combined autologous NSCs and MSCs in 8 patients with acute ischemic stroke. These autologous NSCs were derived from the subependymal zone of the fetal brain and the autologous MSCs were extracted from the umbilical cord. This study demonstrated lower NIHSS scores and mRS scores and higher Barthel index (BI) scores after stem cell treatment [115]. This study administered two types of treatment: either MSCs at 0.5 ×10⁶/kg body weight intravenously followed by three ICV injections of MSCs at 5 ×10⁶/patient + NSCs at 6 ×10⁶ /patient, or MSCs at 0.5 ×10⁶/kg body weight intravenously for four times if the patients could not endure the invasive operation. All cells were transplanted in the acute setting after stroke and the interval between each injection was one week. A 2-year follow-up reported improved outcomes in patients’ daily living with only mild side effects, including mild fever in 6 patients and minor dizziness in 1 patient, which resolved within 24 hours without intervention [115]. Another study investigated the therapeutic effects of transplanting multiple types of stem cells through different administrative routes at least 6 months after stroke. In this study, different types of cells were administrated: intracranial delivery of olfactory ensheathing cells (OECs) alone (1 ×10⁶, n=2); intracranial delivery of OECs (1–2 ×10⁶) + Neural progenitor cells (NPCs, 2–4 ×10⁶, n=8); ICV delivery of NPCs alone (2–5 ×10⁶, n=4); ICV delivery of NPCs (2–5 ×10⁶) + Schwann cells (SCs, 2 ×10⁶, n=1) and intravenous delivery of umbilical cord mesenchymal stromal cells (UCMSCs, 1–2.3 ×10⁷, n=2). NPCs treatment in the chronic phase (6 months to 20 years) indicated improved muscle strength, muscular tension, balance, and speech in a 2-year follow-up [116]. Combination therapy showed favorable outcomes in clinical patients with ischemic brain injury, which indicated the possible synergistic effects among different cell types, pending further verification with larger clinical trials.

Overall, NSC therapy improves neurological recovery by promoting neurogenesis, enhancing vascular reconstruction, improving synaptic plasticity, and modulating neuroinflammation after ischemic brain injury in rodents, pending further verification with preclinical studies in big animals (pig/monkey) and larger clinical trials. MGE modification, EVs, and microRNAs may further enhance transplantation efficacy towards future clinical applications.

4.2.2. Mesenchymal Stem Cells

MSCs are characterized by the capability of unlimited differentiation and can be harvested from various sources, primarily from bone marrow (BM-MSCs) and adipose tissues (AD-MSCs). MSCs have become the most studied stem cell type in ischemic brain injury due to their features of multipotent differentiation potential, paracrine ability, easy availability, and fewer ethical concerns [13, 117]. BM-MSCs are common but require an invasive method for harvesting. Alternatively, AD-MSCs can be harvested using less-invasive techniques. Both BM-MSC and AD-MSC are easy to culture and expand [118, 119]. This section discusses the neuroprotective functions of MSC treatment in ischemic brain injury after CA and stroke. MSC therapy after CA is summarized according to different delivery methods, and MSC therapy after stroke is reviewed based on the preclinical and clinical evidence in different conditions after a stroke.

4.2.2.1. Preclinical Evidence

CA-Induced Ischemic Brain Injury

The application of MSCs in the treatment of ischemia in CA has been recently reported in preclinical animal studies and has demonstrated several different neuroprotective mechanisms. Wistar rats were mostly used to establish the asphyxia or ventricular fibrillation (VF) CA model. Intravenously transplanted BM-MSCs through jugular vein 2 hours after ROSC improved 35-day survival rates, sensory function, and motor behavior in a 6min-VF rat model [120]. This study demonstrated that labeled MSCs were distributed in the hippocampus, cortex, pons, medulla, and cerebellum and that these MSCs expressed protein markers of neuronal nuclei and glial fibrillary acidic protein (GFAP) [120]. Similarly, intravenous injection (the right femoral vein) of BM-MSCs increased post-resuscitation survival in a 6min-VF rat model [121, 122]. Intravenous administration of BM-MSCs 2 hours after ROSC also demonstrated improved neurological outcome and inhibited inflammatory response by increasing the level of tumor necrosis factor-α-induced protein 6 (TSG6) and downregulating the expression of serum S-100B in a 6min-asphyxial CA model in SD rats. These grafted BM-MSCs primarily migrated into the cerebral cortex, which is one brain region vulnerable to ischemia [123]. Another study showed reduced neuronal necroptosis after transplantation of BM-MSCs in a rat CA model in which BM-MSCs migrated to the cortex and lowered the expression of cell necroptosis proteins – receptor interacting protein kinase 1 (RIP1) and RIP3, reducing the percentage of dead neurons [124]. The Lactate/creatine (Lac/Cr) ratio in the ischemic brain can be used as an indicator of lactate change and possible ischemic damage degree within the brain [125]. IV injection of human MSCs 3h after resuscitation from CA resulted in a decreased Lac/Cr ratio and improved neurological function in rats [126]. Transplanted MSCs alleviated nuclear DNA fragmentation, reduced neuronal apoptosis, and increased the level of BDNF in the hippocampus in a global cerebral ischemia CA model [126].

One study explored the effect and mechanism of different methods of BM-MSC administration in rats after CPR [127]. BM-MSC therapy (including IV delivery, IA delivery, and ICV delivery) all demonstrated marked improvement in neurological function in a 6min-asphyxia CA model, with ICV administration appearing to be the optimal method of delivery when comparing efficacy. BM-MSC treatments improved functional recovery, increased the level of VEGF and BDNF, and decreased the expression of serum S100 (an inflammatory marker). BM-MSC aggregation and migration to the hippocampus and the temporal cortex were observed [127]. In another study investigating the various routes of BM-MSC administration, treated animals, regardless of the site of administration, demonstrated better neurological outcomes after CPR for CA [128]. ICV delivery allowed for the highest cell volume localized within the temporal cortex and hippocampus, leading to the best neurological performance [128].

Stroke-Induced Ischemic Brain Injury

Similar to the findings in global cerebral ischemia, the demonstrated therapeutic mechanisms of MSCs in focal ischemia caused by stroke focus on angiogenesis, neurogenesis, and immunomodulation leading to reduced inflammation and apoptosis [129, 130]. Transplantation of MSCs following 90-min MCAO ischemic stroke in healthy animals protected neurons from apoptosis, reduced infarct volumes, and led to improved neurological function in rats [131]. MSCs were shown to exert anti-inflammatory effects and regulate immune responses during tissue repair by influencing macrophage polarization in OGD in vitro cell models, which may be related to the inflammatory Notch-1 signaling pathway [74].

Therapeutic effects of MSCs on stroke models in animals with aging, hypertension, or diabetes were reported. Intra-arterial injection (ipsilateral internal carotid artery) of BM-MSCs in middle-aged female rats 24 h post-stroke (90-min MCAO) markedly reduced axonal loss and enhanced the synaptic connections. BM-MSCs survived in the injured brain and differentiated into neurons and astrocytes. The study showed that only a few of BM-MSCs aggregated in peripheral tissues such as the lungs, heart, liver, spleen, and kidneys. The therapeutic benefits on aging rats lasted for more than 1 year from the ischemic injury [132]. Grafted MSCs were also shown to enhance neuronal homeostasis by modulating the expression of calcineurin in middle-aged ovariectomized female rats exposed to 90-min MCAO. Intra-arterial infusion of MSCs exerted antioxidant effects to normalize oxidative indicators and improved neurological outcomes [133].

The lineage of stroke-prone spontaneously hypertensive (SHRSP) rats, which usually suffer from severe hypertension and the common comorbidity of stroke, are a suitable animal model for studying primary hypertension and its complications [134]. Intracranially transplanted MSCs showed antioxidant and neuroprotective potentials in SHRSP rats. Grafted MSC led to reduced neuronal apoptosis, normalized oxidant parameters, and restored damaged tissues in the hippocampus [135]. In another study, intravenous infusion of placenta-derived MSCs reduced sensorimotor deficits and infarct volume in a permanent MCAO model with spontaneous hypertension. The therapeutic effects were better with a double dose of MSCs (at 8h and 24h post-stroke) when compared with a single dose (only at 24h post-stroke) [136], suggesting the need for further study and understanding of the optimal dose and timing of stem cell therapies.

Intravenous delivery of AD-MSCs 48h after permanent MCAO enhanced angiogenesis, protected neurons, and improved post-stroke neurological outcomes in hyperglycemic rats [137]. Other studies have also demonstrated the neuroprotective function of MSCs in ischemic stroke model with diabetes in rats [138, 139]. In vitro, BM-MSCs promoted the capillary formation and axon growth in cultured primary cortical neurons. In vivo, administration of BM-MSCs via tail vein promoted angiogenesis and white matter restoration in a rat stroke model with type 1 diabetes (T1DM) via the metabolic miR-145/ABCA1/IGFR1 signal pathway [138]. Intravenous delivery of MSC-exosomes attenuated weight loss, promoted BBB integrity, decreased activated microglia, and inhibited inflammation in a 2-h MCAO stroke model with type 2 diabetes (T2DM) [139].

Enhancement in MSC Application

Growth, differentiation, and homing remain particular challenges in stem cell therapies, and various preconditioning and modification practices have been studied to address these obstacles [13]. Intravenous administration of hypoxia-pretreated BM-MSCs enhanced the proliferation and migration of cells to the ischemic injury area through the inflammatory PI3K/AKT signal pathway, reduced the inflammatory response, and alleviated neuronal apoptosis within the cortex, thereby improving the neurological function in rats [140]. Upregulation of the expression of specific trophic factors is another form of stem cell modification. Pretreatment with co-overexpression of BDNF and VEGF resulted in enhanced neuroprotective functions of BM-MSCs in rats with ischemic brain injury after CA [141]. Biomaterials provide another option to enhance stem cell therapy. MSC therapy reduced brain edema and infarct volume, inhibited microglia activation, promoted vascular growth and attenuated neurological deficits in rats after 1-h of MACO. Loading MSCs into hydrogel/nanofiber composite scaffolds further ameliorated ischemic brain injury, providing stronger therapeutic effects than directly administering MSCs [142].

4.2.2.2. Clinical Evidence

Bone Marrow Derived-Mesenchymal Stem Cells

A small study of 5 ischemic stroke patients demonstrated improved brain function after intravenous delivery of autologous MSCs. This study was the first to explore the feasibility, efficacy, and safety of autologous MSC transplantation in clinical application, and no adverse events were reported, indicating autologous MSCs therapy as an effective and safe way for ischemic brain injury following stroke [143]. Another unblinded study of intravenous administration of autologous MSCs 36–133 days post-stroke showed that mean lesion volumes were reduced by >20% at 1-week post-cell infusion in 12 stroke patients with ischemic grey matter, white matter, and mixed lesions. When analyzing adverse events, the study confirmed no venous thromboembolism, abnormal cell growth, tumors, or infection [144]. Autologous MSC transplantation enhanced neuronal plasticity in 6 patients with chronic stroke compared with the control group [145]. Recently, another single-center, open-label, randomized controlled trial evaluated the feasibility, efficacy, and safety of autologous BM-MSC in subacute stroke patients (less than 2 weeks after moderate-severe ischemic carotid stroke). Patients who received BM-MSCs intravenously performed better on neurological assessments and functional magnetic resonance imaging activity compared to controls, with no serious adverse effects in a 2-year follow-up [146]. Besides, an open-label, observer-blinded clinical trial evaluated the long-term efficacy and safety of intravenous infusion of MSCs in 16 patients with stroke and displayed similar beneficial effects. Although comorbidities including seizures or recurrent vascular events were reported over the 5-year follow-up, the incidence did not differ from the control group [147]. Another 2-year, open-label, and single-arm study demonstrated that modified BM-MSCs (termed SB623) were safe in 18 patients with stable and chronic stroke (6–60 months). The 3 cohorts of 6 patients who received a single dose of 2.5×10⁶, 5.0×10⁶, or 10×10⁶ SB623 cells intracranially showed improvement from baseline in clinical outcomes at all time points starting from 1 month to 12 months [148].

Adipose-Derived Mesenchymal Stem Cells

An acute stroke study reported the beneficial effects of AD-MSC administered in acute stroke patients [149]. This phase II randomized, double-blind, placebo-controlled, single-center, pilot trial investigated the safety of AD-MSCs, without reporting any side effects, neurological complications, and systemic complications in primary outcomes. Secondary outcomes, including the assessment of mRS, NIHSS, infarct volume, and biochemical markers of brain repair, showed the efficacy of intravenous administration of AD-MSCs. The study affirmed safety and augmented rehabilitation in patients treated with AD-MSCs after stroke [149].

Other Sources of Mesenchymal Stem Cells

Limited studies have included other sources of MSCs, such as umbilical cord MSCs (UC-MSC), an enriched population of aldehyde dehydrogenase-bright stem cells. Laskowitz et al. conducted a phase I open-label trial to assess the safety and feasibility of a single intravenous injection of 5 × 10⁶ to 5 × 10⁷ human UC-MSCs 3–9 days post-stroke in 10 adult patients. The study concluded that UC-MSC therapy was safe and provided support for a phase II, randomized, placebo-controlled trial [150]..

4.2.2.3. Contradictories, Limitations, and Perspectives

In a phase II randomized, sham-controlled study, BM-ALD-401 cells were delivered via the internal carotid artery to 60 patients with recently stable ischemic stroke with no clinical adverse effects. However, BM-ALD-401 did not demonstrate efficacy when comparing the 90-day mRS, BI index, the NIHSS, or the 1-year disability with the control group [151]. Several other small randomized controlled trials of MSC therapies in ischemic stroke have also failed to demonstrate efficacy [152–154]. In a recent phase 2, randomized, open-label, standard-of-care controlled and multicenter trial, intra-arterial delivery of BM-MSCs were safe in patients with acute ischemic stroke, but no significant improvement was found at 180 days on the mRS [155]. The discrepancy between preclinical versus clinical investigations may be attributed to unclear optimal doses, delivery, and timing of therapies. Different criteria for neurological assessments might also account for the different outcomes.

In summary, MSC therapy has shown beneficial effects in ischemic brain injury. Hypoxic pretreatment and bioactive factor overexpression may further enhance the efficacy of stem cell application. MSC therapy shows neuroprotective effects on stroke in otherwise healthy and aging animals, as well as stroke in animals with hypertension and diabetes. However, there is a lack of consideration for common risk factors among older adults in current preclinical studies of stem cell therapy for stroke. The inclusion of such comorbidities in future preclinical studies may improve future translational potential from bench to clinic.

4.2.3. Other Stem Cell Types

4.2.3.1. Human Umbilical Cord Blood Cells

Human umbilical cord blood (UCB) contains great numbers of mesenchymal progenitor cells, endothelial cell precursors, and immature stem cells [156]. In contrast to invasive methods of bone marrow extraction, cord blood can be gained in a non-invasive manner. Human umbilical cord blood cells (HUCBCs) possess the capacity for proliferation and differentiation, including the ability to differentiate into neurons, and a minority of HUCBCs possess the ability to differentiate into astrocytes under certain conditions [157, 158]. This part reviews the beneficial effects and limitations of HUCBC transplantation in ischemic brain injuries.

4.2.3.1.1. Preclinical Evidence

Intraperitoneal transplantation of HUCBCs following hypoxic-ischemic brain injury has been shown to induce angiogenesis and neurogenesis, as well as enhance neurobehavioral recovery [159]. Grafted HUCBCs upregulated the expression of angiogenesis-related proteins Tie-2 and occludin, leading to increased levels of BDNF and VEGF, which support blood vessel growth [159]. Stem cell therapy with HUCBCs has also shown protective effects by suppressing pro-inflammation [160] and inhibiting lectin binding to microglia/macrophages [161] to attenuate neuroinflammation. HUCBC therapy can also reduce the proliferation of T cells by decreasing the level of proinflammatory factors interferon‐γ (IFN‐γ) and IL‐10 [162]. Intravenous administration of HUCBC 24 hours after hypoxic-ischemic brain injury showed a dose-dependent effect to enhance tissue recovery and cognitive recovery in a rat model [163]. High-dose (1×10⁸ cells) and medium-dose (1×10⁷ cells) showed better outcomes than low-dose (1×10⁸ cells) HUCBC treatments [163]. In another rat model of focal ischemia, HUCBC therapy was investigated in a dose-dependent study (3×10⁵, 1×10⁶, 3×10⁶, 1×10⁷ cells) followed by a therapeutic time window study (Day 1, Day 7, Day 30, Day 90 post-stroke at a dose of 5×10⁶ cells). Treatment with intravenous infusion of HUCBCs after 2-h MCAO showed neuroprotective effects at a dose ≥ 3×10⁶ cells up to 30 days post-stroke, which enhanced synaptogenesis, neurogenesis, angiogenesis, and reduced cellular apoptosis [164].

Hematopoietic stem cells (HSCs), produced in bone marrow, have the potential for differentiation into red blood cells and lymphoid cells [165]. Intravenous administration of lineage-negative bone marrow-derived hematopoietic stem cells (Lin^--HSCs) 24 hours post-stroke reduced cerebral inflammation, inhibited apoptotic neuronal cell death, and induced neuroprotective effects in mice stroke model induced by 45-min MCAO [166]. In another study, intravenous transplantation of HSCs reported timing- and dose-dependent effects in a 2-h MCAO stroke model, suggesting the optimal timing in two doses at Day 1 and 2 after ischemic stroke. Administered HSCs narrowed infarct volume in the cerebral cortex and reduced neurological morbidity (paralysis or weakness) in surviving mice [167]. Combining therapy of HSCs with stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) resulted in a reduction in atrophy in the ipsilateral brain and a reduction of neurological impairments after hypoxic-ischemic brain injury in neonatal rats [168].

4.2.3.1.2. Contradictories, Limitations, and Perspectives

In contrast, in a rat model of neonatal hypoxic-ischemic brain injury, a single intraperitoneal injection of HUCBCs 6 hours post-ischemia reduced cellular apoptosis and oxidative stress but failed to show long-term morphological or functional protections [169]. Another study failed to demonstrate the benefits after intravenous administration of HUCBCs 3 weeks after neonatal hypoxic ischemia [170].

Overall, HUCBC therapy promoted synaptogenesis, neurogenesis, and angiogenesis while suppressing neuroinflammation in a dose-dependent manner after ischemic brain injury. However, some studies showed inconsistent results when stem cells were delivered in different therapeutic time windows or delivery routes. Further studies are required to optimize timing, routes of delivery, or combination therapies to ensure sustained protective functions.

4.2.3.2. Endothelial Progenitor Cells

4.2.3.2.1. Preclinical Evidence

Endothelial progenitor cells (EPCs), another type of stem cell derived from bone marrow, may contribute to blood vessel growth and paracrine cytokine release and have demonstrated the capability of proliferation, migration, angiogenesis, and neurogenesis in ischemic stroke [171, 172]. Jugular vein injection of EPCs 1h post-MCAO (occlusion for 1 hour) enhanced cerebral blood flow, blood vessel reconstruction, and neurogenesis and decreased ischemic infarct volume and neuronal apoptosis, ameliorating neurological deficits in mice [171]. Intravenous delivery of EPCs 1.5 h after 90-min MCAO resulted in enhanced neovascularization, decreased cerebral atrophy, and improved neurobehavioral outcomes in MCAO mice [172]. C3 is the core component in complement activation pathways after ischemia. EPCs have the capability of decreasing the inflammatory response via the inflammatory C3/C3aR pathway. In a mouse model of 90-min MCAO ischemic stroke, intracranial delivery of EPCs immediately after brain ischemia reduced the expression of astrocyte-derived C3 and showed neuroprotective effects [173].

Enhancement in EPC Application

The chemokine CXCL12, also known as stromal-derived factor-1, played an important role in EPC homing and vascularization in ischemic regions. Modified EPCs that were treated with CXCL12 (CXCL12-EPC), outperformed a single treatment of EPCs by enhancing EPC proliferation and increasing levels of VEGF in vitro and increasing blood vessel density, myelination, neurogenesis, and angiogenesis in vivo [174]. Taken together, these findings imply that EPCs are a potential candidate for cell-based treatments in ischemic stroke and bioactive factors preconditioning may further enhance the efficacy.

5. Immune Responses in Stem Cell Transplantation

One major challenge that highly promising stem cell-based therapies faces in clinical translation is immune rejection. Different cell sources may require varying generation methodologies and are likely to express distinct antigens, leading to different immunogenicity. Due to the partial retention of epigenetic memory from the parental induced cells, pluripotent stem cells are more prone to generating aberrant antigen presentation compared with adult stem cells, which could trigger an immune response even in autologous transplantation [175].

Immunosuppressive therapy may directly affect the immune responses process, or indirectly by altering the immunological microenvironment. However, all preclinical studies in Table 1 showed that stem cell transplantation after CA-induced ischemic brain injury didn’t cause immune rejection in the absence of immunosuppressive drugs. In Table 2, except for a few studies using cyclosporine A to suppress graft rejection in ESC therapy [56, 59, 61–63] and iPSC therapy [83, 84, 86] in ischemic stroke treatment, most of the preclinical evidence was conducted without using immunosuppressants after stem cell administration. All clinical studies in Table 3 didn’t use immunosuppressants, and one trial stated that there were no reports of subjects experiencing graft-versus-host disease during a 1-year follow-up [150].

Table 1.

Stem cell therapy application in cardiac arrest-induced brain injury

Cell type	Dosage	Pretreatment	Delivery Route	Timing	Outcome Measured	Proposed Mechanisms	References
NSC	2.0×105	-	ICV	3h after ROSC	Improved neurological outcome	β-catenin signaling	[8, 9]
NSC	2.0×105	MEG	ICV	3h after ROSC	Better neurological recovery and less anxiety- and depression-related behaviors	-	[11]
NSC	4.0×105	-	Intranasal	3h after ROSC	Improved neurological function and superior over GBC	TLR4/NLRP3 pathway	[12]
NSC, BM-MSC	1.0×106	miRNA-133b, exosome	ICV	20min after ROSC	Alleviated brain damage	MicroRNA-133b incorporated in extracellular vesicles	[66]
iPSC-MSC	1.0×106	-	-	OGD model	Altered the polarization direction of macrophages	-	[74]
iPSC-MSC	2.5×106	-	IV	ROSC	Neuroprotection	Modulating neuroinflammation	[75]
NSC	5.0×107	-	Intra-hippocampus	7d after ROSC	Supported brain regeneration	SDF-1/CXCR4 pathway	[93]
NSC	2.0×105	miR-26a	ICV	1h after ROSC	Improved neurological function	β-catenin signaling	[113]
MSC	5 × 106	-	IV	2h after ROSC	Improved brain function	-	[120]
MSC	5 × 106	-	IV	2h after ROSC	Improved functional recovery	Upregulation of TSG-6 expression	[123]
MSC	1.0×106	-	IV	1h after ROSC	Improved neurological function	Inhibiting necroptosis	[124]
MSC	1.0×106	-	IV	3h after ROSC	Elicited functional improvement	-	[126]
MSC	1.0×106	-	ICV, IA, IV	1h after ROSC	Improved nerve function; ICV was an optimal method	-	[127]
MSC	1.0×106	-	ICV, IA, IV	1h after ROSC	ICV produced the best neurological outcome.	-	[128]
MSC	4.0×107	Hypoxia	IV	1h after ROSC	Suppressed brain injury	PI3K/AKT signaling	[140]
MSC	3.0×106	BDNF and VEGF	IV	2h after ROSC	Neuroprotection	-	[141]
MSC	1.0×106	-	IV	2h after ROSC	Improved neurological function	Induction of TSG-6	[178]

NSC, human neural stem cell; ROSC, return of spontaneous circulation; ICV, intracerebroventricular; NSC, neural stem cell; BM-MSC, bone marrow derived mesenchymal stem cell; MSC, mesenchymal stem cell; MSC, mesenchymal cell; IV, intravenous; IA, intravenous; iPSC-MSC, induced pluripotent stem cell induced-mesenchymal stem cell; GBC, Glibenclamide; TSG-6, tumor necrosis factor-α-induced protein 6

Table 2.

Stem cell therapy application in ischemic stroke-induced brain injury

Cell type	Dosage	Pretreatment	Delivery route	Timing	Outcome Measured	Proposed Mechanisms	References
ESC	7.5×106	-	IC	3h after MCAO in rats	Restored damaged synaptic connections	-	[56]
ESC	1.0×105	Bcl-2	IC	1w after MCAO in rats	Promoted functional benefits	-	[57]
ESC	1.2×105	-	IC	1w after MCAO in rats	Enhanced neurological outcomes	-	[58]
ESC-NSC	1.0×105	SD56	IC	1w after MCAO in rats	Enhanced forelimb movements	-	[59]
ESC-NPC	5.0×105	-	IC	1w after MCAO in rats	Behavioral recovery and reduction of infarct size, but induced damage in the injected site of the basal ganglia	-	[60]
ESC-NPC	1.0×105	-	IC	1w after MCAO in rats	Better brain function	-	[61]
ESC-NPC	1.0–4.0×105	-	IC	24h after MCAO in mice	Favorable functional recovery	-	[62]
ESC-NPC	8.0×105	-	IC	1w after MCAO in rats	No change in infarct volume and sensorimotor function	-	[63]
iPSC	2.4×106	-	IC	-	Promoted sensorimotor function	-	[77]
iPSC-NSC	1.0×105	-	Intra-hippocampus	24h after MCAO in mice	Attenuate early-stage neurological deficits	-	[78]
iPSC-NPC	1.0×107	-	IC	5d after MCAO in pigs	Promoted recovery of posture, postural reactions, mental state, and appetite	-	[79]
iPSC-NSC	-	-	IC	5d after MCAO in pig	Robust recovery response	-	[80]
iPSC-NPC	2.0×105	Episomal plasmid-based reprogramming	IC	7d after MCAO in rats	Treating functional deficits	-	[83]
iPSC	1.0×105 in mice 4×105 in rats	-	IC	1w after MCAO in mice 48h after MCAO in rats	Improved results both in rats and mice	-	[84]
iPSC	1.0×106	Fibrin glue	IC, Subdual	1h after MCAO in rats	Functional improvement and subdual transplantation was safer	-	[85]
iPSC-NSC	2.5×105	-	IC	1w after MCAO in rats	No improvement in behavioral recovery and infarct size	-	[86]
iPSC	5.0×105	-	IC	24h after MCAO in rats	No better behavioral recovery and tumor formation	-	[87]
NSC	1.0×105	-	Intra-hippocampus	24h after MCAO in mice	Ameliorated behavior dysfunction	Ameliorated inflammation	[31]
NSC	6.0×105	-	ICV	24h after MCAO in rats	Improved behavior performance	Neurogenesis	[96]
NSC	4.5×103 4.5×104 4.5×105	CTX0E03	IC	4w after MCAO in rats	Promoted behavioral recovery in a dose-dependent manner	Neurogenesis	[97]
NSC	1.0×105	-	Intra-striatum	48h/ 3w after MCAO in rats	The therapeutic efficacy of transplantation at 48h was superior to 3w	Neurogenesis	[98]
NSC	3.0×105	-	IC	7d after MCAO in rats	Better neurological recovery	Enhance structural plasticity and axonal transport	[99]
NSC	1.0×105	-	IC	48h after MCAO in mice	Promoted functional recovery	Enhance brain plasticity	[100]
NSC	3.0×105	-	IC	7d after MCAO in rats	Improved functional recovery	-	[101]
NSC	2.25×105 in mice 4.5×105 in rats	CTX0E03	IC	4w after MCAO	Provided significant benefits for functional recovery	Angiogenesis	[102]
NSC	3.0×105	-	IC	1–2w after MCAO in rats	Improved functional recovery	-	[103]
iPSC-NSC	1.5×105	-	Intracortical	48h after MCAO in rats	Improved functional recovery	-	[104]
NSC	3.0×105	-	IC	7d after MCAO in rats	Improvement of neurological deficits	-	[105]
NSC	0.8–1.0×105	-	IC	-	-	Rescued dysfunctional neurons	[109]
NSC	1.0×106	-	IV	72h after MCAO in mice	Better neurological performance	-	[110]
NSC	4.0×106	-	IV	24h after MCAO in rats	Long-term effects	-	[111]
NSC	3.0×106		IV	6h after MCAO in rats	Improved neurological performance	Interfered of neuroinflammatory system	[112]
NSC	1.0×106	Hypoxia	Intranasal	3d after hypoxic-ischemia induction in rats	Migrated toward injured areas along the brain blood vessels	-	[179]
MSC	1.0×106	-	IA	1h, 6h, 24h, 48h after MCAO in rats	Improved neurological function	-	[131]
MSC	1.0×105	Hypoxia	IA	-	Improved neurological function	-	[180]
MSC	2.0×106	-	IA	1d after MCAO in aged rats	Long-term improvements in functional outcome	-	[132]
MSC	1.0×105	-	IA	6h after MCAO in aged rats	Normalize oxidative indicators and improved neurological outcomes	-	[133]
MSC	1.0×106	-	ICV	SHRSP model	Restored damage in the hippocampus	-	[135]
MSC	1.0–2.0×105	-	IV	8h and 24h after MCAO 24h after MCAO in rats	A repeated dose at 8h and 24h was superior to the single dose only at 24h post-stroke	-	[136]
MSC	1.0×106	-	IV	48h after MCAO in hyperglycemic rats	Improved neurological outcomes	-	[137]
MSC	5.0×106	-	IV	24h after MCAO in rats with diabetes	Neuroprotective function	miR-145/ABCA1/IGFR1 signaling	[138]
MSC-Exo	3.0×1011	-	IV	72h after MCAO in rats with diabetes	Neuroprotective function	-	[139]
MSC	5.0×105	-	Intranasal	10d after hypoxic-ischemia induction in rats	Long-lasting improvement of sensorimotor and cognitive function	-	[50]
MSC	2.0×105	-	Intranasal	24h after hypoxic-ischemia induction in rats	Better behavior performance	-	[181]
MSC	5.0×105	Scaffold	IC	24h after MCAO	Obvious neuroprotective effects	PI3K/AKT signaling	[142]
HUCBC	1.0×107	-	IP	24h after insult	Ameliorated lesion-impaired neurological and motor functions	-	[159]
HUCBC	2.0×105	-	IP	24h post-injury	Effective neuroprotective therapy	-	[160]
HUCBC	1.0×106		IV	Subsequent after MCAO	Neuroprotective effects	Neuroinflammation	[161]
HUCBC	1.0×106 1.0×107 1.0×108	-	IV	24h after insult	Promoted robust tissue repair and stable cognitive improvement in a dose-dependent manner	-	[163]
HUCBC	3.0×105 1.0×106 3.0×106 1.0×107	-	IV	24h after MCAO in rats	Showed neuroprotective effects at a dose ≥ 3×106 cells	-	[164]
HUCBC	5.0×106	-	IV	1d after MCAO in rats 7d after MCAO in rats 30d after MCAO in rats 90d after MCAO in rats	Showed neuroprotective effects up to 30 days post-stroke	-	[164]
HUBCB	1.0×107	-	IP	6h after insult in rats	Failed to show long-term morphological or functional protections	-	[169]
HUCBC	1.0×107	-	IV	6h after insult in rats	Behavior deficits were not attenuated	-	[170]
HSC	5.0×106	-	IV	24h after MCAO in mice	Ameliorated brain injury	-	[166]
HSC	-	-	IV	2h, 1d, 2d, 3d and 4d after MCAO in mice	Alleviated neurological morbidity	-	[167]
lt-NES	1.5×105	-	IC	48h after MCAO in rats	Promoted motor and sensory function	-	[76]
EPC	1.0×106		IV	1h after MCAO	Ameliorated neurological deficits	-	[171]
EPC	1.0×106	HMGB1	IV	1.5h after MCAO in mice	Promoted functional recovery	-	[172]
EPC	3.0×105	CXCL12	IC	1w after MCAO in mice	Promoted functional recovery	-	[174]

NSC, neural stem cell; ICV, intracerebroventricular; MCAO, middle cerebral arterial occlusion; C, intracranial; IV, intravenous; iPSC-NSC, induced pluripotent stem cell-derived neural stem cells; iPSC, induced pluripotent stem cell; MSC, mesenchymal stem cell; IA, intra-arterial; AD-MSC, adipose induced-mesenchymal stem cell; MSC-Exo, mesenchymal stem cell derived-exosome; ESC, embryonic stem cell; ESC-NSC, embryonic stem cell-derived neural stem cell; lt-NES cell, human iPSC-derived long-term neuroepithelial like cells; UCB, umbilical cord blood; MPC, multipotent progenitor cells; BM-MNC, Bone marrow mononuclear cell; HSC, hematopoietic stem cell

Table 3.

Clinical evidence of stem cells in patients with stroke

Cell type	Dosage	Precondition	Delivery Route	Timing	Enrollment	Outcomes	Adverse Effects	References
MSC	5.0×107	-	IV	first boosting at 4–5w second boosting at 7–9w	30 patients (5 received MSCs and 25 patients as control)	Safe and feasible	-	[143]
MSC	0.6–1.6×108	-	IV	36–133d post-stroke	12 patients (age between 20 and 75 years old, stroke onset within the past 6 months)	Reduced lesion volumes by >20% at 1week post-cell infusion	No adverse effects	[144]
MSC	5.0–6.0×107	-	IV	Chronic stroke	12 patients (6 patients were administrated MSCs and 6 patients served as control)	Restored function	No mortality or cell-related adverse reaction	[145]
MSC	5.0×106	-	IV	Chronic stroke	31 enrolled patients (16 patients received MSC treatment; 15 patients as control)	Better neurological assessments and functional MRI activity	Safe	[146]
MSC	5.0×107	-	IV	first boosting at 5–7w second boosting at 7–9w	52 patients (16 patients in MSC group and 36 patients in the control group)	Clinical improvement	Seizures or recurrent vascular events, but did not differ from the control group	[147]
BM-MSC	2.5×106 5.0×106 10×106	SB623	IC	Chronic stroke	18 patients (3 cohorts of 6 patients)	showed improvement from baseline in clinical outcomes	Safe	[148]
AD-MSC	1.0×106/kg	-	IV	Within the first 2w of stroke	20 patients (10 patients receive MSCs; 10 patients receive placebo)	Safe and effective	No allergic reactions, immunologic rejection, or tumor was observed	[149]
UCB	5.0×106/kg	-	IV	3–10d post-stroke	10 patients (6 Caucasians, 3 African Americans, and 1 American Indian)	Feasible and safe	No	[150]
Stem cell	3.08×106	ALD-401	IA	13–19d post-stroke	100 patients (60 patients received ALD-401 and 40 patients received sham infusion)	Not showed efficacy when comparing 90-day mRS, BI index or the NIHSS, or 1-year disability with the control group	-	[151]
MPC	4–12×108	-	IV	24–48h post-stroke	129 patients (67 receive MPCs; 62 receive placebo)	Failed to demonstrate efficacy	-	[152]
BM-MSC	1.59×108	-	IA	5–9d post-stroke	20 patients (10 cases and 10 control subjects)	No significant differences in neurological function at 180 days	2 cases had an isolate partial seizure	[153]
BM-MSC	2.80×108	-	IV	18.5d post-stroke	120 patients (60 patients receive MSCs; 60 patients receive placebo)	Failed to demonstrate efficacy	-	[154]
BM-MNC	2×106/kg 5×106/kg	-	IA	4–7 days after stroke onset	77 patients (38 in the control group, 20 in the low-dose group, and 19 in the high-dose group)	No significant improvement at 180 days on the mRS	Safe	[155]
NSC MSC	Group 1: 0.5×106/kg MSC (IV, four times) Group 2: 0.5×106/kg MSC (IV, one time) + 5×106 MSC + 6×106 NSC, (ICV, three times)	-	IV, ICV	Acute stage in 1 patient Subacute stage in 3 patients Stroke sequelae in 2 patients	6 patients (2 patients in group 1 and 4 patients in group 2)	Improved outcomes in patient’s daily living	Mild side effects (fever, minor dizziness) and resolved within 24 hours	[115]
OEC NPC SC UCMSC	1.0–2.0×106 (OEC, IC) 2.0–4.0×106 (NPC, IC) 2.0–5.0×106 (NPC, ICV) 2.0×106 (SC, ICV) 1.0–2.3×107 (UCMSC, IV)	-	IC, IV, ICV	Chronic phase stroke	17 patients: IC delivery of OECs (n=2); IC delivery of OECs + NPCs (n=8); ICV delivery of NPCs (n=4); ICV delivery of NPCs + SCs (n=1); IV delivery of UCMSC (n=2)	Improved neurological performance	-	[116]

NSC, neural stem cell; ICV, intracerebroventricular; IC, intracranial; IV, intravenous; hCNS-SC, human central nervous system stem cell; iPSC-NSC, induced pluripotent stem cell-derived neural stem cells; iPSC, induced pluripotent stem cell; MSC, mesenchymal stem cell; IS, intra-arterial; AD-MSC, adipose induced-mesenchymal stem cell; MSC-Exo, mesenchymal stem cell derived-exosome; ESC, embryonic stem cell; ESC-NSC, embryonic stem cell-derived neural stem cell; UCB, umbilical cord blood; MPC, multipotent progenitor cells; BM-MNC, Bone marrow mononuclear cell; OEC, olfactory ensheathing cell; NPC, neural progenitor cell; SC, Schwann cell; UCMSC, umbilical cord mesenchymal stem cell; Acute stage indicates treatment within 1 week from onset; Subacute stage indicates 1 week to 1 month from onset; Chronic stage indicates treatment after 6 months from onset; During stroke sequelae indicates 0.5–2 years from onset

Persistent systemic immune suppression with suppressants may lead to serious side effects. New strategies have been reported to induce immune tolerance. The pursuit of low-immunogenic cellular therapies has been made possible due to the development of genetic engineering techniques and the CRISPR/Cas9 system, which has been proven to be at least partially effective in enabling allogeneic cells to evade immunological attack in xenotransplantation experiments [176]. Shielding stem cells with encapsulated biomaterials is another approach to overcome the host immune responses. Microencapsulated MSCs showed higher level of cell–cell contacts and less host immune responses [177].

6. Conclusion and Prospects

Investigations of stem cell therapy in ischemic brain injury have emerged over several decades. As technology has matured, a substantial body of literature has accumulated demonstrating strong potential for the application of stem cell therapies in the preclinical arena. Different cell types, including iPSCs, ESCs, NSCs, and MSCs, have exerted various neuroprotective effects after ischemic brain injury. Despite the favorable outcomes, there are still some limitations and challenges that need to be addressed. Clinical translation is largely concerned with adverse effects, and assessing the safety of the treatment requires more well-designed preclinical studies in large animals (monkeys/pigs) and larger randomized clinical trials to determine the risk of immune rejection and teratoma formation. How to further optimize stem cell therapy for ischemic brain injury is another critical area. Along with a better understanding of underlying therapeutic mechanisms, effective clinical translation of these promising findings requires further investigations to determine the optimal dose, routes of administration, and timing of treatment. Meanwhile, several techniques can further improve the efficacy of stem cell therapy, including EVs, biominerals, MGE, hypoxia-preconditioning, microRNAs, and overexpression of bioactive factors. Once these topics can be satisfactorily addressed, stem cell therapy will be an alternative option for patients suffering from an ischemic brain injury after CA and stroke, with a goal of improving neurological function recovery and quality of life.

List of abbreviations

AD-MSCs

Adipose-Derived Mesenchymal Stem Cells

BBB

Blood-Brain Barrier

BDNF

Brain Derived Neurotrophic Factor

BM-MNC

Bone Marrow Mononuclear Cell

BM-MSCs

Bone Marrow Mesenchymal Stem Cells

CA

Cardiac Arrest

CNS

Central Nervous System

CPR

Cardiopulmonary Resuscitation

CSF

Cerebral Spinal Liquid

DG

Dentate Gyrus

EEG-IQ

Electroencephalogram Information Quantity

ESCs

Embryonic Stem Cells

ESC-NSCs

Embryonic Stem Cell-derived Neural Stem Cells

ESC-NPCs

Embryonic Stem Cell-derived Neural Progenitor Cells

Eps

Episomal Plasmids

EVs

Extracellular Vesicles

GDNF

Glial-derived Neurotrophic Factor

hASCs

Human Adipose Stem Cells

hCNS-SCs

Human Central Nervous System Stem Cells

hNSCs

Human Neural Stem Cells

HUCBCs

Human Umbilical Cord Blood Cells

IA

Intra-artery

IC

Intracranial

ICV

Intracerebroventricular

IP

Intraperitoneal

IV

Intravenous

iPSCs

Induced Pluripotent stem cells

iPSC-MSCs

Induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells

iPSC-NSCs

Induced Pluripotent Stem Cell-derived - Neural stem cell

IGF-1

Insulin Growth Factor-1

MCAO

Middle Cerebral Arterial Occlusion

MGE

Metabolic Glycoengineering

MPC

Multipotent Progenitor Cell

MSCs

Mesenchymal Stem Cells

NDS

Neurological Deficit Score

NGF

Nerve Growth Factor

NPCs

Neural Progenitor Cells

NSCs

Neural Stem Cells

OEC

Olfactory Ensheathing Cell

OGD

Oxygen and Glucose Deprivation

PBS

Phosphate Buffer Solution

RCT

Randomized Controlled Trials

ROSC

Return Of Spontaneous Circulation

RIP1

Receptor-Interacting Protein Kinase 1

SCs

Schwann Cells

SVZ

Subventricular Zone

TSP-1

Thrombospondins 1

UCB

Umbilical Cord Blood

UCMSC

Umbilical Cord Mesenchymal Stem Cell

VEGF

Vascular Endothelial Growth Factor

VF

Ventricular Fibrillation

Data Availability

N/A