Advances in genetically modified neural stem cell therapy for central nervous system injury and neurological diseases

Abstract

Neural stem cells (NSCs) have increasingly been recognized as the most promising candidates for cell-based therapies for the central nervous system (CNS) injuries, primarily due to their pluripotent differentiation capabilities, as well as their remarkable secretory and homing properties. In recent years, extensive research efforts have been initiated to explore the therapeutic potential of NSC transplantation for CNS injuries, yielding significant advancements. Nevertheless, owing to the formation of adverse microenvironment at post-injury leading to suboptimal survival, differentiation, and integration within the host neural network of transplanted NSCs, NSC-based transplantation therapies often fall short of achieving optimal therapeutic outcomes. To address this challenge, genetic modification has been developed an attractive strategy to improve the outcomes of NSC therapies. This is mainly attributed to its potential to not only enhance the differentiation capacity of NSCs but also to boost a range of biological activities, such as the secretion of bioactive factors, anti-inflammatory effects, anti-apoptotic properties, immunomodulation, antioxidative functions, and angiogenesis. Furthermore, genetic modification empowers NSCs to play a more robust neuroprotective role in the context of nerve injury. In this review, we will provide an overview of recent advances in the roles and mechanisms of NSCs genetically modified with various therapeutic genes in the treatment of neural injuries and neural disorders. Also, an update on current technical parameters suitable for NSC transplantation and functional recovery in clinical studies are summarized.

Background

Neurological damage frequently arises from a range of traumatic injuries and disorders, including trauma [1], ischemia and hypoxia [2], and infection [3]. Within this spectrum of injuries, traumatic brain injury (TBI), spinal cord injury (SCI), and stroke are classified as CNS injuries. These diverse forms of CNS damage typically lead to varying degrees of neural tissue structural destruction, as well as either complete or partial neural functional deficits. In recent years, the incidence of nerve injury has escalated significantly, attributable to the rising occurrences of traumatic events, neurological tumors, and high prevalence of cerebrovascular diseases [4]. Consequently, nerve injuries are associated with elevated rates of disability and mortality. In general, patients constantly suffer from substantial physical and psychological distress, often resulting in permanent loss of neurological functions, thereby imposing significant economic burdens on both families and society [4]. More regrettably, the adult mammalian CNS exhibits a markedly limited capacity for neural regeneration. Damaged or degenerated neuronal cells as a result of CNS injury are typically challenging to repair and regenerate. Furthermore, they often fail to functionally integrate into the damaged nerve tracts to establish synaptic connections and reconstruct functional neuronal circuits [5]. Although endogenous immature cell types can be activated in response to injury or disease, cell replacement is generally inadequate, lacks direction, or fails to generate the appropriate lost cell types [5]. In addition, the microenvironment following CNS injury undergoes rapid deterioration, which progressively impedes nerve repair. This deterioration is characterized by a reduction in the secretion of neurotrophic factors, the formation of glial scars, and the presence of inhibitors of neuronal regeneration [6,7,8]. Nonetheless, advancements in research on NSCs have yielded valuable insights and methodologies for the repair of nerve damage.

Currently, the transplantation of NSCs has been shown to remarkably enhance neural functional recovery in both preclinical and clinical trials. This is mainly due to NSC unique potential for proliferation, secretion bioactive factors, homing and generation of distinct subtypes of neurons and glial cells [8,9,10,11,12]. Although NSCs possess the capacity to augment neuroregeneration and promote the restoration of neurological functions through a variety of mechanisms, their low long-term survival, neural differentiation and integration within the host injured nervous system diminish the functional benefits of NSC-based therapies for SCI, attributable to the hostile and progressively deteriorating microenvironment resulting from various unfavorable factors subsequent to nerve injuries [6, 7, 13]. To address this challenge, it is imperative to promptly improve, or even reverse the deteriorating microenvironment in the injured area through combinatorial strategies, such as biomaterials, genetic and pharmacological therapies aside with new emerging technologies. In comparison, an increasing body of evidence indicates that a series of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT3), among others, can effectively promote neural differentiation, survival, synaptic plasticity, and regeneration [6, 13]. Thereby the administration of these neurotrophins holds the potential to further augment neural regeneration and functional recovery through various mechanisms including trophic support, axonal induction, neural circuit reconstruction, and remyelination [14,15,16]. Despite their great potential, the therapeutic application of these bioactive factors for improving environment conducive to neural regeneration still encounters substantial challenges, such as the precise administration of the timing, dose and delivery routes, the likelihood of penetrating the BBB, and the secondary invasive insult. Intriguingly, NSCs can be strategically engineered via genetic modification to stably express specific biological molecules, including neurotrophic factors [17, 18], anti-apoptotic genes [19], and antioxidant enzymes [20], over extended durations. This capability is instrumental in alleviating damage induced by adverse microenvironments, and improves the outcome of NSC-based therapy and avoid additional injury from the transplantations themselves. Therefore, genetically modified NSCs emerge as optimal vectors for targeted molecular delivery, thereby enhancing the efficacy of cell-based therapies.

Although nearly all genetic modifications of interests dramatically increase survival, promote neurodifferentiation of the transplanted NSCs, and effectively improve microenvironment conducive to neural regeneration, the therapeutic efficacy of NSC transplantation with genetic modification may be negated when inappropriate transplantation timing and routes and dosage of transplanted cells are employed in the treatment of nerve injuries and neurological disorders [21, 22]. In addition, diverse damage models should adopt various strategies and technical parameters. This variation can be attributed to the unique dependence of transplanted cell survival and differentiation on the distinct extrinsic signals from the local microenvironment following nervous injury. This review for the first time provides an unprecedented comprehensive analysis of NSC characteristics, resources and the specialized genetic modifications conducive to neural regeneration, and the therapeutic capability of genetically modified NSCs in cell-based therapeutics for a variety of nerve injuries and neurological diseases. Additionally, we also provide a detailed summary of recent advances in the transplantation timing and routes and dosages of genetically modified NSC-based therapies for the CNS injuries and disease models. We believe this review can help better understanding of the regulatory mechanisms by which genetically modified NSCs promote nerve injury repair, and inspire future experimental research to better identify treatment options for the CNS injuries and disease.

Neural stem cells and genetically modified neural stem cells

Characteristics of NSCs

NSCs are primarily characterized by their capacity for self-renewal and clonal expansion through symmetric divisions and proliferation, as well as their ability for multipotent differentiation via asymmetric divisions to give rise to a diverse array of phenotypically distinct neurons and glial cells [8,9,10]. Besides, the ability to home is increasingly envisioned as a crucial attribute of NSCs [10]. Studies in developmental neurobiology, particularly those focusing on the rodent cortex, have identified resident stem cells that predominantly produce neurons during the early stages of brain development and glial cells at later stages [11, 13]. This observation suggests the existence of neuron-biased stem cells. The development of the CNS is initiated during early animal development through the induction of NSCs or NSC-like precursors, a process referred to as neural differentiation. The generation of the entire CNS from these initially induced NSCs underpins the rationale for employing NSCs to recapitulate CNS development in cases of CNS injury [23]. Notably, NSCs possess the capacity to home to specific regions [24], enabling their directed migration to lesioned areas following various injuries, thereby facilitating neural repair through multiple mechanisms. The homing capability of NSCs is closely linked to the expression of various chemokines and chemokine receptors, including CXCR4, as well as adhesion molecules such as integrins, immunoglobulins, and selectins [25]. These molecules facilitate the homing process towards sites of inflammatory chemokine production, such as stromal cell-derived factor-1α (SDF-1α) [25]. In fact, it is worth mentioning that in mammalian brains across different age groups, NSCs can be attracted considerable distances from neurodegeneration regions [7]. In traditional cognitive frameworks grounded in the principles of NSC therapy, cell replenishment therapy has been widely endorsed for patients with CNS injuries who exhibit poor or inadequate responses to conventional treatments. This acceptance is primarily due to the capacity of NSCs to differentiate predominantly into committed cell lineages of both the CNS and peripheral nervous system (PNS), including neurons and supportive glial cells such as oligodendrocytes and astrocytes [26]. These differentiated cells have the potential to replenish and mitigate irreversible neuronal loss resulting from neurological trauma or disease. Concurrently, numerous studies have shown that NSCs are capable of secreting a diverse array of products, including neurotrophic and other bioactive factors, which ameliorate the adverse microenvironment [27]. This secretion supports cell survival, promotes axonal regeneration, and enhances angiogenesis [27, 28], thereby significantly contributing to the efficacy of nerve repair.

Origin and location of NSCs

In the early 1990s, several research groups identified a specific subset of stem cells located within the CNS. The embryonic origin of NSCs is situated within the walls of the lateral ventricles, in proximity to the cortex, hippocampus, striatum, septum, and the central canal of the spinal cord (Cx, Hp, St, SP, and SC) [10, 29]. In the adult mammalian brain, the majority of NSCs predominantly reside in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus within the hippocampus [29]. In addition, an increasing body of studies demonstrated that NSCs also exist in the adult rodent and human olfactory bulb (OB) albeit several reports claiming that the majority of newly generated neurons in the granular cell layer or glomerular layer of OB migrate from the V-SVZ of the postnatal lateral ventricle along the radial migratory stream and ultimately integate into the OB [30,31,32,33,34]. However, the in vitro study of Carlos and his colleagues showed that NSCs exists in the OB core and predominantly give rise to Calr⁺ interneurons [30]. In fact, NSCs/NPCs within the olfactory system, are not confined to the OB, many protocols have been established to culture and characterize adult NSCs/NPCs derived from olfactory mucosa, which serves as an alternative source for the treatment of nervous injuries and neurological disorders [35,36,37]. Despite the intrinsic capacity of endogenous NSCs to facilitate neural repair, their reparative potential is constrained by factors such as their limited numbers, restricted distribution, and the unfavorable microenvironment following neural injury [25]. Therefore, the transplantation of exogenous NSCs emerges as a promising strategy for the treatment of neurological diseases and injuries. Given that NSCs are typically derived from various sources within the CNS. They can be directly isolated from both fetal and adult neural tissues, including the brain and spinal cord [38]. Specifically, NSCs can be harvested from regions such as the ventricular zone, striatum, and cortex of the embryonic brain [39]. During the first trimester of mammalian pregnancy, including in humans, neural tissue is predominantly composed of NSCs/NGCs, accounting for approximately 90% of the cellular composition [39].

In addition, NSCs can also be obtained through the transdifferentiation of pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) [38]. iPSCs are primarily generated from adult somatic cells by introducing specific transcription factors, which drive the reprogramming of these cells into multipotent states [40]. In addition, NSCs derived from mesenchymal stem cells (MSCs) have been proposed as an innovative cell-based therapeutic approach for various nervous system disorders [41]. It is noteworthy that NSCs can be acquired from transdifferentiated somatic cells, including monocytes in blood cells, skin fibroblasts, and urine cells, which are readily accessible in routine clinical practice [38, 42]. Besides, NSCs can be further immortalized through the introduction of genes associated with embryogenesis, allowing for the selection of NSC clones with continuous division capabilities. Furthermore, genetically homogeneous human NSC (hNSC) lines have been established using genetic perpetuation techniques [43,44,45,46]. Currently, the large SV40T-antigen, derived from viral oncogenes, is extensively utilized for the immortalization of NSCs [47]. Similarly, v-myc and adenovirus early region 1A are also commonly employed viral oncogenes for the establishment of NSC lines [47]. Until now, a large number of studies have demonstrated that immortalized NSCs, which are genetically modified in vitro, are capable of surviving, differentiating into neurons and glial cells, and integrating into host tissues following transplantation into diseased or injured brain and spinal cord [48]. More intriguingly, stable clonal lines of human NSCs derived from human fetal telencephalon using a retroviral vector encoding v-myc, demonstrate the capacity for self-renewal and differentiation into various neuronal and glial cell types, as shown in Fig. 1. These NSCs can integrate into damaged CNS regions within the brain or spinal cord, as well as in conditions such as neurodegenerative diseases and mucopolysaccharidosis [49]. This suggests that NSCs, derived from other genetically modified cell types, hold significant potential as vehicles for cell replacement and gene transfer therapies in patients with neurological disorders. In summary, the derivation or generation of NSCs is illustrated in Fig. 2.

Fig. 1

Schematic diagram of the origin and derivation of neural stem cells

Fig. 2

A detailed illustration of biological effects of gene modification to neural stem cells in neurological insults. Genetically modified NSCs serve as ideal carriers for specific molecules, maximizing the potential of cell therapy and yielding higher levels of therapeutic efficiency through a variety of mechanisms

Genetically modified NSCs

Despite extensive research demonstrating that transplanted NSCs confer therapeutic benefits not only by replenishing damaged or necrotic cells but also through bystander immunomodulatory and neurotrophic mechanisms, their clinical application remains significantly challenged. This is primarily due to the low survival rates and inefficient differentiation of grafted NSCs into neural cells. A critical factor impeding the proliferation, migration, and differentiation potential of transplanted NSCs following SCI is the deteriorating microenvironment in the damaged area, which hinders effective therapeutic outcomes [14]. Compelling studies have evidenced that several neurotrophins and bioactive factors can play a neuroprotective role by improving the microenvironment, triggering nerve repair and regeneration [13, 27, 29]. Nonetheless, the primary challenges associated with the direct delivery of the aforementioned growth factors are predominantly due to their limited permeability across the BBB, inadequate distribution within neural tissue, low bioavailability, and short half-life [14]. Fortunately, epigenetic modifications play a pivotal role in regulating gene expression related to neural development and growth, encompassing mechanisms such as DNA methylation, non-coding RNA, and histone modification [50]. MicroRNA (miR), a crucial non-coding regulatory RNA, demonstrates dynamic expression patterns throughout neurogenesis and initiates various functions, some of which serve as effective therapeutic targets for this pathological condition [51]. Accordingly, miR has also been employed to enhance NSC properties in transplantation strategies for treatment of CNS injuries and neurological disorders. Until now, several miRs, such as miR-124, miR-7b-3p, miR-29a, miR-26a, miR-20a, miR-381 etc., have been demonstrated to enhance the ability of NSCs to repair injured or diseased tissues and improve neurological functions by different mechanisms of action against CNS lesions and neurological diseases [52,53,54,55,56,57,58,59,60]. For example, transplantation of miR-124 transfected-NSCs into a rat model of contusive SCI also results in enhanced neuronal differentiation of the transplanted cells, simultaneously reduced astrocyte differentiation and decreased cavity formation [52]. Studies on miR-26a also show that transplantation of miR-26a-modified NSCs can improve brain injury in rats with cerebral palsy (CP) [58]. miR-381 was identified as a positive regulator to increase the capacity of NPCs for neurogenesis and decreasing astrocyte differentiation by modulating NSC proliferation and fate specification [54]. Noteworthy, together with miR-20a, miR-30b restores the sensory conductive function of the spinal cord [59, 60]. Consequently, the epigenetic regulation of NSCs through diverse epigenetic modifications also represents a promising strategy for NSC-based therapies for treatment of nerve injuries. Typically, nerve injuries are usually associated with inflammation, cell necrosis, apoptosis, vascular damage, and oxidative stress. In light of detrimental factors, enhancing the anti-inflammatory, anti-apoptotic, pro-angiogenic, and antioxidant stress capabilities of NSCs is essential for the treatment of CNS damage. Promisingly, the integration of combinatorial genetic engineering techniques with stem cell-based therapies offers innovative ideas to solve the intractable issues. Recent comprehensive studies have demonstrated the efficacy of specific gene transfer into NSCs for the treatment of CNS disorders [13, 60, 61]. To date, the genes commonly transduced include those encoding neurotransmitter synthases, neurotrophins, metabolic enzymes, and reporter proteins [9, 13]. Given the inherent advantages of NSCs over other cell types in addressing nerve injuries, NSCs present themselves as highly attractive candidates for the delivery of therapeutic genes. There are numerous studies highlighting the potential of genetically modified NSCs as suitable vehicles for gene delivery in the treatment of CNS injuries, showing that these modified cells not only facilitate cell survival and proliferation, but also enhance the differentiation of NSCs into neural cells [62, 63]. It is significant to note that following nerve injury, the transplantation of genetically modified NSCs can sustain the expression of specific molecules at elevated levels, thereby enhancing the microenvironment and mitigating inflammation, demyelination, and astrocyte reactivity. Taken together, the transplantation of genetically modified cells potentially offers a potentially more precise therapeutic strategy for CNS injuries through various mechanisms, including optimization of NSC survival, enhancement of neuronal differentiation, promoting axonal growth, plasticity and functional recovery.

Treatment of CNS injury using genetically modified NSCs

Transplantation of exogenously modified NSCs

Origin of genetically modified NSCs/NPCs

Genetically modified NSCs/NPCs for the treatment of CNS injuries have been prepared through following approaches: through several methodologies as follows: (i) the majority of NSCs are primarily isolated from embryonic tissues, specifically the cortex [52, 64, 65], forebrain [17, 66], diencephalon [67], spinal cord [21], and hippocampus [68] at embryonic day 14/14.5, and adult olfactory system [30, 34, 35]; (ii) immortalized neural precursor cell lines, such as the C17.2 cell line [61, 69] and the immortalized human NSC line HB1/F3 [20, 28, 70], which incorporate oncogenes to enable unlimited proliferation and thus can be produced in large quantities; (iii) NSCs/NPCs derived from ESCs [71]; (iv) NSCs/NPCs derived from bone marrow stroma cells [72]; and (v) induced pluripotent stem cell derived NPC (iPSC-NPC) [73]. iPSCs/NPCs can be generated from a patient's autologous skin fibroblasts (Fig. 1), thereby circumventing issues related to immunorejection and ethical concerns associated with stem cell transplantation. Nonetheless, one of the primary challenges that must be addressed prior to pre-clinical application is the problem of low reprogramming efficiency.

The principal approaches for transplantation of genetically modified NSCs/NPCs

Although there is no consensus on the most effective NSC transplantation strategy for SCI treatment, employing various transplantation approaches can yield different therapeutic outcomes in the host. To achieve optimal therapeutic effects, the selection of the transplantation method should be adaptable and primarily based on the type and severity of the injury. Currently, the principal methods for nerve injury transplantation include local intracerebral lesion region transplantation, intraventricular CNS injection, and percutaneous venous transplantation [12, 13, 53, 70, 74]. The transplantation strategy for TBI primarily involves intracerebral transplantation, such as the injection of cells into the ipsilateral injured hemisphere [53, 66]. In the context of SCI, various transplantation routes are employed, including intralesional (IL) injection, which involves the direct administration of cells into the center of the lesion [12], or direct injection into the perilesional area. Additionally, intrathecal (IT) administration of NSCs, intravenous injection [52], and intraventricular injection, such as the delivery of cells into the spinal cord [19], are utilized. For hemorrhagic stroke, cellular transplantation is predominantly performed by injecting cells into the overlying cortex [75] or the ipsilateral striatum. For ischemic stroke, transplantation involves the direct administration of NSCs into the ipsilateral lateral ventricle, cortex, and striatum, as documented in various studies [22, 74, 76, 77]. Similarly, in models of cerebral palsy, NSCs suspended in saline are stereotactically injected into the left sensorimotor cortex of rats, demonstrating efficacy [58, 78]. Overall, the optimal strategy for the transplantation of genetically modified NSCs/NPCs remains contingent upon the specific situation of nerve injury.

Dosages for transplantation of genetically modified NSCs/NPCs

Although there is currently no consensus on the exact number of cells successfully transplanted to the lesion site by each in vivo procedure, the quantity of cells for initial transplants is closely linked to the therapeutic efficacy of cell-based therapies for nerve injuries. From the perspective of clinical trials, it is essential to establish an optimal cell number for transplantation in cases of nerve injury. While an inadequate number of transplanted cells may fail to achieve a satisfactory therapeutic outcome in nerve repair, or may even be less effective, an excessive number of transplanted cells is likely to induce adverse effects, such as the formation of cell clots. Unfortunately, there is presently no established cell dosage for stem cell-based therapy. Therefore, the current determination of cell quantity for transplantation using genetically modified NSCs/NPCs often rely on pre-clinical trials. Furthermore, the variations in animal species, disease modeling, the duration and extent of injury, and delivery routes also influence the option of number of transplanted cells and the outcomes. Generally, the quantity of genetically modified NSCs/NPCs used for transplantation typically ranges from 2.5 × 10⁴ to 2 × 10⁶ cells [12, 71]. In a particular study, 1 × 10⁵ and 3 × 10⁵ cells were transplanted into a mouse model of ischemic stroke, representing the low-dose and high-dose groups, respectively [79]. The results indicated that, at 7 and 14 days post-transplantation, mice receiving iPSCs/NPCs with overexpression of SDF-1α exhibited a significant improvement in locomotor activity. However, no significant improvement was observed in the low-dose group [73]. The transplant dosages of genetically modified NSCs are summarized in Table 1. Consequently, it is essential to investigate and optimize the quantity of transplanted cells for nerve regeneration based on the specific circumstances.

Table 1 Illustration of transplantation of a variety of gene modified neural stem cells to different animal models, transplantation time, cell number, and the resultant outcomes

Timing of transplantation of genetically modified NSCs/NPCs

Nerve damage encompasses a series of intricate pathophysiological changes, making the determination of the optimal timing for NSC transplantation crucial for the effective treatment of SCI. Previous animal studies have predominantly conducted the transplantation of genetically modified NSCs/NPCs either immediately following the injury or at 24 or 72 h post-traumatic brain injury (TBI) [80]. In the context of SCI, cell transplantation is performed either immediately following the injury or at intervals of 7 or 9 days post-injury. In ischemic stroke models, cell transplantation typically occurs immediately, at 6 h, or at 3 days following ischemia–reperfusion [81]. For middle cerebral artery occlusion (MCAO), transplantation is performed at 2 h, 1 day, 2 days, and 7 days post-occlusion. The majority of studies select 3 and 7 days post-cerebral hemorrhage as the transplantation time points. In hypoxic-ischemic encephalopathy models, cells are transplanted 3 days after the injury. Even though no study has systematically evaluated the effects of NSCs/NPCs based on the timing of transplantation, the timing of cell transplantation is a crucial factor influencing cell engraftment and subsequent functional improvements [82]. Consequently, the timing of NSC delivery for CNS injuries should be adaptable. The timing of transplantation of genetically modified NSCs in pre-clinical studies is summarized in Table 1.

Co-transplantation of NSCs with other types of cells for neural regeneration

Although transplantation of NSCs has emerged as a promising neurorestorative approach for the neural damage and compensate for the lost neural structures for recovery of interrupted neural communications caused by various injuries, the NSC-based therapy approach often suffers from numerous challenges primarily owing to NSC own biological features such as low survival rates, migration, and inefficient neurodifferentiation in the deteriorating microenvironment following nervous injuries. Therefore, it is relatively difficult to more effectively achieve neural regeneration and function repair in CNS injury, relying solely on the transplantation of NSC alone. With recent advances in regenerative medicine, compelling studies in the experimental animal models and preclinical studies in human patients of CNS injury have shown that co-transplantation strategies and combination therapies for neural regeneration further compensate or enhance the capability of NSCs to promote neural tissue remodelling and function recovery through various mechanisms.

To date, apart from co-transplantation of NSCs with OECs, Schwann cells and astrocytes, respectively, in augmenting neural repair [83,84,85,86], co-transplantation of NSCs with other types of cells including mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), endothelial progenitor cells (EPCs), brain microvascular endothelial cells (BMECs) and their derivative cells, can significantly enhance the treatment efficacy of grafted NSCs by promoting survival and efficient neurodifferentiation of grafted NSCs, improving the deteriorating microenvironment of lesioned CNS and remodelling host damaged neural network [13, 86,87,88,89,90,91,92,93,94,95,96]. The synergistic effect of co-transplantation of NSCs with other cells is superior to the treatment with either of NSCs or other cells alone. For instance, Meng et al. [97], found that co-transplantation of NSCs with AECs-expressing bFGF effectively improves the NSCs survival and neurodifferentiation microenvironment repair of the injured spinal cord, and consequently have benefits for SCI. Another interesting report demonstrated that co-transplantation with NSCs and astrocyte and BMECs can improve the memory ability in ischemic stroke rats by improving microenvironment via the support and modulation of astrocyte and BMECs [86]. Currently, most studies on the use of co-transplantation of NSCs with other types of cells, mainly including MSCs and OECs, for the treatment of neural injury have demonstrated that the co-transplanted MSCs or OECs with NSCs can aid NSCs in damaged neural tissue replacement, enhance the therapeutic potential of the grafted NSCs, and resultantly reverse neurological damages in various damage animal models [13, 84, 88, 92,93,94,95]. Regarding this point, OECs co-transplants with NSCs has been demonstrated that OECs exert supporting and trophic roles for NSCs survival and for enhancing neuronal differentiation of NSCs for lasting functional recovery in a traumatic milieu in the adult mammalian CNS [84, 85]. As for co-transplantation with MSCs, the MSCs as a potentially better source for therapeutic solution to repair the damaged nervous system usually include bone marrow-derived MSCs, amniotic epithelial cells (AECs), umbilical cord derived mesenchymal stem cells (UMSCs), placenta-derived mesenchymal stem cells (PMSCs) and adipose mesenchymal stem cells (AMSCs) [13, 83, 89, 92,93,94,95]. The underlying mechanism of therapy for use of co-transplantation of NSCs with other types of cells mainly includes the followings: (i) the other types of grafted cells can create a permissive microenvironment following nervous injury by suppressing pro-inflammatory responses, oxidative stress and neurotoxic insults [84, 85, 87,88,89, 93, 94]. (ii) The co-grafted cells with or without genetic modification produce high amounts of neurotrophic substances, which are capable of improving the NSCs survival and neuronal differentiation, and aiding in lost cellular population replacement [84, 85, 87, 88, 92,93,94]. (iii) The co-grafted cells secret NCAM to enhance migration and neurite extension of the differentiated cells[13, 84, 85, 90, 91, 93, 94]. (iv) Both of them collaborate or crosstalk to enhance integration into the host nervous tissue and remodel the damaged neural network and neural functional recovery [13, 83, 94, 95]. Despite the enhanced therapeutic efficacy using co-transplantation of NSCs with the other types of cells compared to treatment with either of the cell type alone in various neurological disease models, such as SCI or brain injury, and neurodegenerative diseases, many of the technical and biological issues including combining proportion of NSCs with other cell types, secondary injury of transplantation, and the risk of tumorigenesis and immunogenicity need to be seriously considered and further investigated.

Genetically engineered NSCs for the treatment of traumatic brain injury

Traumatic brain injury (TBI) is a devastating neurological disorder resulting from an external mechanical force [79, 98]. It encompasses both primary and secondary injuries and represents the foremost cause of mortality and morbidity among individuals under the age of 45 globally [80]. The primary injury is typically a consequence of the immediate mechanical disruption of brain compartments due to the initial physical impact, such as a hit, blow, or jolt to the brain [99]. This disruption may manifest as diffuse axonal injury, vascular injury (hemorrhage), and other forms of tissue structure damage [100]. Secondary injury occurs within minutes to days following the primary insult, culminating in the compromise and leakage of the blood–brain barrier (BBB). This breach facilitates the extravasation of immune cells, thereby exacerbating neuroinflammation and cerebral edema [81]. Concurrently, excitotoxicity, apoptosis, necrosis, oxidative stress, and mitochondrial dysfunction are observed [99]. These pathological processes ultimately result in neuronal death, tissue damage, and atrophy [85]. Recent research has shown that the enhancement of neurogenesis and synaptogenesis, axonal remodeling, and the induction of angiogenesis can significantly improve functional brain recovery in experimental traumatic brain injury (TBI) [101]. More importantly, NSCs have been observed to facilitate functional recovery in TBI by secreting neurotrophic factors [27], increasing the expression of synaptic proteins [102], and differentiating into functional neurons. Consequently, NSC transplantation is emerging as a promising therapeutic strategy for TBI. Encouragingly, through the targeted modification of specific genes, genetically modified NSCs have demonstrated superior therapeutic outcomes compared to naïve cells in the treatment of traumatic brain injury (TBI).

Genetically modified NSCs possess the capability to differentiate into both neurons and glial cells. Notably, genetic modifications have been shown to significantly enhance the survival of NSCs [17, 69]. Furthermore, these modifications increase the proportion of NSCs or NPCs that differentiate into neurons [17, 63,64,65,66, 69]. This enhancement is critical for achieving improved behavioral outcomes and facilitating the recovery of motor function following TBI. In a recent study, Makri et al. demonstrated that the transplantation of Cend1-overexpressing NPCs into the cortex of injured mice resulted in a higher expression of the neuronal marker NeuN compared to non-genetically modified NPCs [63]. Furthermore, Cend1-overexpressing NPCs exhibited an enhanced capacity to differentiate into neurons within the damaged cortex 1 month post-transplantation [62]. Notably, the majority of these graft-derived neurons were characterized as interneurons expressing the inhibitory neurotransmitter γ-aminobutyric acid (GABA) [63]. The overexpression of Cend1 has been shown to enhance the proportion of GABAergic neurons by approximately 5.5-fold in the injured cortex following NPC transplantation [63]. This finding further suggests that Cend1 genetically modified NSCs possess an enhanced capacity to differentiate into functional neurons, thereby replacing damaged cells more effectively. Similarly, another study also demonstrated that rats receiving transplants of NSCs overexpressing BDNF exhibited an increased number of NF200-positive cells, and surviving NF200-positive cells have larger cell bodies and longer axons as compared with NSCs-transplanted rats, indicating improvement of neuronal survival and differentiation [82]. It is well-known that TBI is widely recognized to be frequently associated with neurite injury of varying degrees, and the sprouting of damaged axons play a crucial role in neural recovery. The microtubule-associated protein 2 (MAP2) family constitutes a significant group of cytoskeletal components predominantly expressed in neurons. These proteins may perform a diverse array of independent and specialized functions, including the regulation of organelle transport within axons and dendrites, the nucleation and stabilization of microtubules (and potentially microfilaments), and the anchorage of regulatory proteins such as protein kinases responsible for transduction [103]. A noteworthy study demonstrated that the overexpression of BDNF can enhance the expression of MAP2 [82]. Importantly, BDNF significantly augments MAP2 expression not only in neuron-like cells derived from transplanted NSCs but also in host cells following transplantation [82]. Moreover, the expression levels of β-catenin [17], actin, and calmodulin (CaM) were also increased in BDNF-overexpressing NSCs after transplantation into the adult traumatic brain [17, 82]. These findings suggest that NSCs genetically modified to overexpress BDNF may play a crucial role in promoting neural differentiation, neuronal survival, and axonal regeneration.

The formation of a glial scar by proliferating reactive astrocytes is widely viewed as a significant impediment to CNS regeneration [7, 104, 105]. Interestingly, genetically modified NSCs have been shown to markedly diminish the proliferative capacity of astrocytes [63, 65]. The study demonstrated that host astrocytes contributing to the glial scar exhibited increased cell density and appeared more hypertrophied in the vicinity of the lesion site compared to those in which Cend1-overexpressing NSCs were transplanted [63]. These findings suggest that Cend1 inhibits the transformation of host astrocytes into reactive astrocytes, thereby restricting glial scar formation at the site of SCI. The impaired restoration of synaptic function and the establishment of synaptic contacts are critical prerequisites for the reconstruction of neuronal circuits. Genetic modifications seem to augment the role of NSCs in restoring synaptic function. For instance, several studies have demonstrated that the expression of presynaptic and postsynaptic proteins, such as synaptophysin (SYP) and Shank2, along with regeneration-related genes, is significantly upregulated in rats transplanted with BDNF-genetically modified NSCs compared to those transplanted with naïve NSCs, particularly during the early post-transplantation period [17]. The findings suggest that genetically modified NSCs significantly enhance the recovery of neural functions through the restoration of synaptic function and the reconstruction of synaptic contacts. Importantly, GAP-43, a growth-associated protein, is closely associated with presynaptic vesicle function, axonal growth, and the regulation of plasticity in response to injury-related behavioral deficits [106]. The study indicated that following TBI, the expression levels of GAP-43 mRNA and protein in BDNF-overexpressing NSCs, as well as in NSC-transplanted rats, showed a continuous increase for up to eight weeks post-transplantation. In addition, the BDNF-overexpressing NSCs-transplanted rats exhibited a higher content of GAP-43 protein compared to the naïve NSCs-transplanted group [17], underscoring the significant impact of genetically modified NSCs on neural plasticity. A series of studies have documented the presence of pro-regenerative genes in genetically modified NSCs (Table 1).

Apoptosis, a principal mode of cell death, frequently occurs in conjunction with various forms of cellular injury and is initially characterized by a series of stereotypical morphological alterations [107, 108]. The significance of the overexpression of anti-apoptotic proteins such as Bcl-2, Bax, and Bcl-x in effectively mitigating apoptosis is further underscored by the involvement of adapter molecules Ca²⁺ and p⁵³ [108, 109]. Moreover, the transplantation of genetically modified NSCs has demonstrated considerable neurological improvement. For instance, NSCs overexpressing miR-17-92, when transplanted into mice with brain injuries, demonstrated a marked enhancement in motor coordination abilities [53]. Similarly, another study indicated that brain-injured animals receiving transplants of NSCs overexpressing GDNF exhibited significant improvements in learning abilities compared to those receiving transplants of naïve NSCs [69]. Interestingly, the overexpression of Cend1 leads to a reduction in the protein levels of Notch1 and cyclin D1, while concurrently elevating the level of p²¹ in NSCs transfected with Cend1, thereby facilitating their exit from the cell cycle [65]. Furthermore, both in vivo and in vitro studies have demonstrated that Cend1 overexpression inhibits the proliferation of NSCs [65]. This finding holds substantial significance in mitigating the risk of tumor formation subsequent to NSC transplantation.

Gene-modified NSCs for spinal cord injury repair

SCI is a devastating neurological and pathological insult to the spinal cord, usually resulting in temporary or permanent significant deficits in autonomic function, sensory perception, and motor control [110]. Pathophysiologically, SCI is characterized by an initial primary injury phase, which compromises the integrity of neurons and glial cells, followed by a secondary injury phase that induces progressive cellular apoptosis and further spinal cord damage over the ensuing weeks [111]. The secondary injury phase is particularly critical in the pathophysiological progression of SCI, as it often accompanies with an uncontrolled and deleterious cascade of reactions, including inflammatory responses, secondary cellular dysfunction and death, vascular alterations, and aberrant molecular signaling [112]. During the transition stage from acute to chronic SCI, the progressive deleterious changes exacerbate the imbalance of the microenvironment. This is attributed to an increase in growth-inhibitory factors and a decrease in growth-promoting factors within nervous tissues [17, 112]. Consequently, the occurrence of multiple pathological events, such as hemorrhage, ischemic imbalance, glial scarring, demyelination, and the dysregulation of microglial and macrophage phenotypes, as well as imbalances in neurotrophins, their propeptides, cytokines, and chemokines, seriously hinders neurogenesis, neuroplasticity, and neurite regeneration [113]. As the time following an injury extends, neural regeneration is significantly impeded by the formation of an astroglial-fibrous scar encircling coalesced cystic cavities [114]. Despite advancements in therapeutic strategies for SCI, including pharmacological interventions, surgical procedures, and rehabilitative efforts, these approaches have only led to limited improvements in clinical outcomes, and no definitive cure for SCI currently exists. Reassuringly, cell-based therapy represents a promising approach for the treatment of SCI. Furthermore, the implantation of biomaterials can improve the microenvironment, bridge the spinal cord stumps, and provide physical and directional support for axonal regeneration [115]. Currently, a combinatorial therapy involving biomaterial implantation and cell transplantation significantly promotes neural regeneration and functional recovery by enhancing cell survival, regulating cell differentiation, and providing directional support for neural circuit remodeling and axon regeneration [116]. In comparison, genetically modified NSCs have served as a more efficacious approach to the treatment of SCI. Through the alteration of specific molecular expressions, these modified NSCs have demonstrated enhanced efficacy in promoting cell survival and differentiation, facilitating axonal regeneration and myelin formation, mitigating inflammation, counteracting cell apoptosis, and reducing the formation of glial scars [11, 13].

The proliferation and survival of NSCs at the transplantation site are crucial prerequisites for nerve regeneration and repair. It has been reported that the transplantation of a human NSC line (HB1.F3) overexpressing Bcl-XL can extend the survival of grafted human NSCs and enhance functional recovery in a rat model of contusive SCI [19]. The number of Bcl-XL-modified HB1.F3 cells was observed to be 1.5 and 10 times greater than that of unmodified HB1.F3 cells at 2 and 7 weeks post-transplantation, respectively [19]. Notably, the quantity of Bcl-XL-modified HB1.F3 cells did not decrease between the 2 and 7-week time points, indicating that the overexpression of Bcl-XL effectively prevented cell death during this period. Similarly, other studies have demonstrated that the overexpression of E-cadherin and Olig2 in neural stem cells (NSCs) enhances their proliferative capacity [67, 117]. Despite the pro-proliferative effects of Olig2, no tumor formation was observed [118]. These findings suggest that genetically modified NSCs with increased proliferative potential may serve as an alternative, unlimited cell source for potential therapeutic applications in promoting nerve function recovery, facilitating cellular replenishment and the reconstruction of neuronal circuits.

The extensive cell death observed following SCI, particularly affecting neurons and oligodendrocytes, is regarded as especially detrimental, as neuronal death leads to axonal loss, and the subsequent demyelination of axons compromises conductivity, particularly in cases of incomplete injury [71]. Furthermore, the relatively low differentiation rates of neurons and oligodendrocytes present a challenge for NSCs to effectively exert neuroprotective effects. Nonetheless, genetically modified NSCs exhibit enhanced differentiation capacity to give rise to both neurons and glial cells. For example, the overexpression of mammalian achaete-scute homologue-1 (Mash-1) [68] and Nogo extracellular peptide residues 1-40 (NEP1-40) [118], as well as the knockout of Neurofibromatosis-1 (NF-1) [119], have been shown to facilitate the differentiation of NSCs into neurons, thereby enhancing functional recovery in SCI rat models. Additionally, NSCs overexpressing NEP1-40 exhibited increased protein levels of axon regeneration indicator GAP-43 and mature neuronal marker MAP-2, suggesting an enhancement in neural regeneration. Furthermore, the converted neurons were demonstrated positivity for NeuN and exhibited characteristics of Nissl staining. More importantly, the transplantation of NF-1 knockout NSCs significantly enhanced the survival of neurons located 5 mm both rostral and caudal to the lesion epicenter [76], as evidenced by markedly improved Basso, Beattie, and Bresnahan (BBB) scores and spinal cord evoked potential (SCEP) amplitudes. Critically, the introduction of NF-1 knockout NSCs led to progressive improvements in neurological function, indicating that NF-1 knockout is crucial for neural differentiation, neuronal relay formation, and axonal extension and conduction [119].

Oligodendrocytes are the myelin-producing cells within the CNS, characterized by their ability to tightly envelop neuronal axons through extensions of their plasma membranes [120, 121]. Their main function is to insulate axons and partition the axonal surface into distinct functional domains [120]. The transcription factor Olig2 plays a crucial role in the development of oligodendrocytes. Previous research has demonstrated that the transplantation of NSCs overexpressing Olig2 can facilitate the remyelination of axons in the context of SCI [21]. Myelin basic protein (MBP) serves as the primary myelin protein responsible for the adhesion of the cytosolic surfaces within multilayered compact myelin, thereby contributing to the functional maintenance and stability of the myelin sheath. Interestingly, another study demonstrated that the introduction of Olig2 gene into NSCs significantly upregulated MBP expression levels [117]. This genetic modification not only augmented the capacity of NSCs to differentiate into oligodendrocytes but also facilitated their maturation into myelinated mature oligodendrocytes. Significantly, NSCs overexpressing Olig2 demonstrated a pronounced tendency to migrate towards white matter, and their transplantation resulted in a substantial increase in the volume of myelinated white matter [117]. Furthermore, the introduction of Olig2 gene is likely to facilitate the differentiation of NSCs into functional oligodendrocytes, thereby contributing to the remyelination of denuded axons in the residual ventrolateral white matter [117]. In addition, a growing number of studies have shown that NSCs overexpressing NGF can counteract the downregulation of the cellular marker CNPase in oligodendrocytes within the injured spinal cord [12]. More intriguingly, the transplantation of brain-derived NPCs that overexpress human arginine decarboxylase (hADC) has been found to facilitate the differentiation of endogenous mNPCs into oligodendrocytes following SCI [122]. In summary, genetically modified NSCs/NPCs exhibit superior benefits compared to unmodified NSCs/NPCs, particularly in enhancing the formation of more robust and thicker myelin and in minimizing cavity formation post-SCI [117, 119, 122].

Following SCI, reactive astrocytes initially migrate centripetally towards the injury epicenter and contribute to tissue repair. However, they subsequently became scar-forming astrocytes, participating in the formation of glial scars at the injury site. These glial scars serve as both biochemical and physical barriers to axonal regeneration, and produce inhibitory factors that impede nerve regeneration. Given the unidirectional and irreversible nature of this continuous phenotypic change [123], it is imperative to mitigate or eliminate glial scars to facilitate neural regeneration. Studies have demonstrated that genetic modification can inhibit the differentiation of NSCs into astrocytes both in vitro and in vivo [119]. In addition, it can reduce GFAP expression in aberrant astrocytes surrounding the lesion areas [12], as well as decrease astrocyte aggregation and the volume of glial scars at the injury site [122]. Furthermore, transplantation with BDNF-overexpressing NSCs has been shown to decrease the number of Iba1 and iNOS-positive inflammatory cells, as well as GFAP-positive astrocytes in rats with SCI [124]. Therefore, this intervention further reduces inflammatory and gliosis responses, which are associated with demyelination, axonal regeneration failure, and cavitation following SCI.

Neuropathic pain, a debilitating outcome of SCI, presents significant treatment challenges due to the incomplete understanding of its pathogenesis and the intricate underlying pathophysiological mechanisms [125]. In regard to the treatment of neuropathic pain, recent research indicates that the overexpression of BDNF relieves hyperalgesia in response to mechanical stimuli in rat models of SCI, with this therapeutic effect likely resulting from reduced astrocyte reactivity and diminished activation of local immune cells [124]. Furthermore, NSCs overexpressing NGF appear to engage in crosstalk with resident NSCs, thereby triggering endogenous neurogenesis in the perilesional area [12]. On the other hand, the NGF-activated TrkA signal transduction pathway enhances the levels of cAMP response element-binding protein (CREB) and microRNA-132 in the vicinity of the lesion center, while concurrently upregulating the expression of BDNF, GDNF, and VEGF [12]. Cumulatively, genetically modified cells also exert anti-apoptotic effects through the targeted knockdown of NF-1, which leads to a reduction in cleaved caspase-3 expression and an increase in Bcl-2 expression [119]. Overall, in comparison to non-modified NSCs, the transplantation of genetically modified NSCs significantly enhanced functional recovery and concurrently abrogated behavioral deficits in animal models of SCI [52, 71, 122]. Regarding the view, another interesting study demonstrated that marmosets receiving transplants of NSC expressing Galectin-1 exhibited markedly improved functional recovery in spontaneous locomotor activity and treadmill tests at an early stage, at an early stage of approximately five weeks post-SCI [82]. This functional improvement may be intimately linked to Galectin-1-expressing NSCs which give rise to more 5HT-positive serotonergic fibers, LFB-positive myelin, and CaMKIIa-positive corticospinal tracts [126]. Collectively, the neurological recovery following SCI through the transplantation of gene-modified NSCs is largely attributed to their facilitative effects on NSC proliferation and survival, neuronal and oligodendrocyte differentiation, axonal regeneration, apoptosis resistance, and the reduction of astroglial scar formation, in addition to the activation of endogenous NSCs (see Table 1).

Genetically modified NSCs in the therapeutic intervention for cerebral ischemia

Stroke is the second most prevalent cause of mortality globally, accounting for 5.2% of deaths worldwide [127]. It is also a leading cause of disability and cognitive impairment, with increasing prevalence in developing nations [128]. The majority of strokes are attributable to the transient or permanent obstruction of cerebral blood vessels [128]. Ischemic stroke can lead to cerebral infarction, brain tissue necrosis, and localized neuronal injury [98], culminating in long-term disabilities and potentially fatal outcomes, thereby imposing a substantial economic burden on a global scale annually. Following a stroke, diminished oxygen levels and an inadequate glucose supply in the infarcted region initiate a cascade of harmful signaling pathways, collectively referred to as the "ischemic cascade" [129]. This process is exacerbated by the simultaneous release of neurotransmitters, reactive oxygen species (ROS), chemokines, and inflammatory cytokines, which further aggravates the ischemic damage [130]. Therefore, prompt reperfusion is essential to restore blood flow to the ischemic brain tissue as soon as possible, which is likely to mitigate further injury. While rapid reperfusion via intravenous thrombolysis and endovascular thrombectomy has been shown to reduce disability [128], the clinical utility of these techniques is constrained due to a narrow therapeutic window, the risk of hemorrhagic transformation, and limited availability [131]. Consequently, there is an urgent need to seek novel alternatives with greater efficacy and fewer restrictions in the treatment of ischemic stroke. Such alternatives should have many advantages in protecting the brain from damage prior to recanalization, extending the therapeutic time window of interventions, and further improving functional outcomes. Following exposure to hypoxia–ischemia, intrinsic response mechanisms involve the stabilization of neuronal transcription factors, specifically hypoxia-inducible factors (HIF)-1 and HIF-2, as well as the upregulation of various downstream cytokines and growth factors [132]. These upregulated expressions, in turn, activate multiple signaling pathways that mediate some alterations in angiogenesis, inflammation, apoptosis, cell proliferation, and differentiation [132]. Consequently, growth factor therapy has been proposed as a potential therapeutic strategy for ischemic stroke. Nonetheless, the short half-lives of these factors constrain their direct clinical applications. In recent years, the transplantation of NSCs has emerged as a promising neurorestorative approach for ischemic stroke, and confer beneficial effects not only by providing structural replacement but also through mechanisms such as neurotrophic support, immunomodulation, and vascular repair [129]. However, genetically engineered NSCs selectively modify their secretome through the overexpression of specific factors, thereby exerting neurorestorative effects in the context of ischemic stroke. These effects are manifested through enhanced survival and differentiation potential of transplanted cells, promotion of angiogenesis, reduction of infarct volume, inhibition of apoptosis, and attenuation of inflammation (see Table 1). Currently, the genetic modification of NSCs extends beyond the upregulation of specific growth factors and pathways [72, 133, 134] to include the involvement of non-coding RNAs [76, 135] and global SUMOylation, which play crucial roles in maintaining cellular function and homeostasis under ischemic stress [136].

The present study reveals that the transplantation of genetically modified NSCs markedly reduces lesion volume in ischemic stroke [74, 137], enhances the recovery of sensorimotor functions [74], and ameliorates cognitive dysfunction and behavioral deficits [137], exhibiting a more pronounced effect compared to non-genetically modified NSCs. For instance, the transplantation of NSCs overexpressing Galectin-1 (Gal-1) was found to reduce infarct volume and attenuate white matter damage in the corpus callosum and striatum for a duration of at least 28 days post-stroke [77]. In addition, Noggin-transfected NSCs have been shown to significantly curtails pathological damage in the ischemic cortex and dentate gyrus, as well as improve neurological scores [22]. Genetically engineered NSCs offer potential neuroprotection against ischemic stroke. The enhancement of proliferation and survival of transplanted cells is essential for the effective exertion of their functions. It has been reported that the overexpression of Breast Cancer Susceptibility Protein 1 (BRCA1) stimulates cellular proliferation in oxygen–glucose deprivation/reoxygenation (OGD/R) NSCs and substantially increases the survival of grafted cells [138]. Following an ischemic stroke, the cognitive and memory deficits, along with motor dysfunction observed in patients, are intimately associated with the disruption of neuronal function. This includes alterations in synaptic activity, interhemispheric connectivity, the secretion of neurotrophic factors, and the consequent disturbance of normal neural circuits [139, 140]. The investigation of neuronal damage post-ischemic stroke has been a central topic in recent research endeavors [117]. A separate study demonstrated that the overexpression of miR-145 facilitated the upregulation of Cyclin D1, Nestin, neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP), thereby enhancing NSC activity, which in turn promoted cellular proliferation and differentiation [76]. In addition, genetic modifications to NSCs were shown to improve the survival rates of transplanted cells, as well as to enhance beneficial immune modulation, cell migration, and the differentiation of neuronal and glial subtypes in the context of stroke. Chang and colleagues conducted a study in which they transplanted rat NSCs modified to overexpress BDNF into the ipsilateral lateral ventricle. They found that the BDNF-overexpressing NSCs expressed CXCR4, a marker for chemokine receptor 4 that responds to stroke-induced inflammatory signals [133], and subsequently migrated to the ipsilateral stroke-injured region. While a portion of the transplanted cells retained their identity as nestin-positive NSCs, others differentiated into DCX-positive neuroblasts or MAP2-positive neurons [133]. Importantly, the presence of TH-positive neurons and GAD65/67-positive neurons within the infarct area indicates the synthesis of dopamine and GABA, respectively, which are crucial neurotransmitters involved in the regulation of striatal function [133]. Additionally, transplanted BDNF-overexpressing NSCs exhibited expression of DAPPRP-32, a marker indicative of striatal projection neurons. These findings robustly support the efficacy of genetically modified NSCs in the treatment of stroke in animal models.

Gene modification not only reduced apoptosis in NPCs [136], but also alleviated morphological damage in differentiated cells [22]. In a study conducted by Xu and colleagues, the overexpression of miR-145 in NSCs was found to inhibit the expression of Cleaved-caspase 3 [138]. Furthermore, the overexpression of BRCA1 in NSCs was shown to prevent apoptosis by suppressing OGD/R-induced upregulation of pro-apoptotic proteins p53 and Bax, while also preventing the downregulation of Bcl-2 [138, 139]. The survival of transplanted cells can be delineated into two distinct phases: the initial phase, referred to as short-term survival or immediate death, concludes approximately one week post-transplantation; the subsequent phase, known as long-term survival or serial cell death, extends over several months. In another experimental study, the overexpression of four pro-survival factors namely Bcl-xl, Bcl-2, Akt1, and Hif1a, significantly suppressed apoptosis mediated through the caspase-3-dependent pathway [141]. Among these factors, the overexpression of Bcl-2, Bcl-xl, and Akt1 not only significantly protected transplanted cells from immediate apoptosis but also effectively prevented ongoing cell death. In contrast, Hif1a expression merely postponed immediate cell death [141]. Notably, the survival rate of Hif1a-expressing cells decreased from 20 to 6.3% within the first month post-transplantation; however, beyond this period, cell death was markedly inhibited [141]. This finding suggests that while Hif1a expression delays immediate cell death, it ultimately prevents continuous cell death [141].

Adenosine triphosphate (ATP) is essential for maintaining neuronal membrane potential via the Na+/K+-ATPase pump [98]. During ischemic conditions, the Na+/K+-ATPase pump fails to regulate the intracellular and extracellular concentrations of sodium and potassium ions [142]. This dysfunction leads to the rupture of the cell membrane and the release of intracellular contents into the extracellular space, and ultimately culminates in nuclear degradation [142]. The degradation of the nucleus and subsequent release of cellular components into the extracellular space elicit an inflammatory response in the vicinity of dying cells [142]. Studies have demonstrated that NSCs/NPCs overexpressing SDF-1α can differentiate into cells exhibiting mature neuronal characteristics, including the expression of synaptic markers and functional sodium/potassium channels, as well as the ability to fire action potentials [73]. Given that neuroinflammation following ischemic brain injury exacerbates tissue damage, genetically modified NSCs have shown greater efficacy in mitigating inflammation compared to unmodified NSCs [137, 143]. This modification to NSCs has the potential to reduce the expression of pro-inflammatory cytokines and mediators, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and cyclooxygenase 2 (COX-2), as well as inhibit NF-κB-mediated inflammation [137, 143]. Furthermore, another study demonstrated that NSCs hypersecreting Gal-1 modulated the polarization of microglia and/or macrophages towards the M2 anti-inflammatory phenotype, thereby decreasing the population of CD16⁺ cells and increasing the prevalence of CD206⁺ cells in the ischemic striatum [77].

Current therapeutic approaches for the recanalization of acute ischemic stroke primarily encompass intravenous administration of tissue plasminogen activator (t-PA) and endovascular interventions [144]. However, these treatments have some significant drawbacks, as reperfusion can lead to the excessive production of reactive oxygen species (ROS), thereby exacerbating cerebral injury post-stroke [144]. Of note, compelling evidence suggests that the swift surge in ROS production following an acute ischemic stroke can quickly surpass the antioxidant defenses, leading to additional tissue damage [144]. Furthermore, the rapid restoration of the blood flow results in a surge of ROS production, subsequently leading to reperfusion injury [144]. The excessive presence of ROS causes irreversible oxidative damage to macromolecules such as DNA, lipids, and proteins, resulting in significant cellular damage [145]. This damage ultimately leads to cell necrosis, apoptosis, and autophagy [145]. The hostile brain environment created by excessive ROS following cerebral ischemia and reperfusion is likely to accelerate the death of transplanted stem cells, thereby reducing the efficacy of stem cell therapy [145]. Antioxidant enzymes constitute a primary mechanism for counteracting the harmful effects of ROS. Currently, Copper/zinc-superoxide dismutase 1 (SOD1) functions as a dimeric cytosolic enzyme that catalyzes the conversion of superoxide anions into hydrogen peroxide (H2O2) [145]. It has been reported that the overexpression of SOD1 effectively reduces ROS levels and enhances the survival of NSCs following ischemia–reperfusion injury [145]. Strikingly, in another study, the transplantation of Noggin-transfected NSCs was found to significantly augment SOD activity and reduce malondialdehyde (MDA) levels [22]. Furthermore, Nuclear factor-erythroid 2-related factor 2 (NRF2) has been identified as a key regulator of cellular defense mechanisms against toxic and oxidative challenges by modulating the expression of genes involved in oxidative stress response and drug detoxification [146]. Notably, the overexpression of BRCA1 has been shown to enhance the upregulation of NRF2, Heme oxygenase 1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO1) [138], thereby significantly contributing to the deoxidation of ROS. Moreover, NSCs with overexpression of choline acetyltransferase (ChAT) have been observed to inhibit lipid peroxidation (LPO) in brain tissue [137]. Ischemic stroke typically causes extensive neurovascular damage, and vascular repair is regarded as a crucial strategy for enhancing long-term recovery outcomes following such events. Although NSCs demonstrate various potential therapeutic benefits against neurovascular injury, their efficacy is often limited. Interestingly, genetically modified NSCs demonstrate superior efficacy. For instance, NSCs modified by specific factors that trigger endogenous repair mechanisms in the early stages of ischemic stroke have been shown to effectively facilitate neurovascular repair [138]. Therefore, the transplantation of gene-modified NSCs that promote vasculogenesis presents a promising therapeutic approach for neurovascular injury. The transplanted cells facilitated neurogenic differentiation and the formation of vascular-like structures [133], while promoting angiogenesis in the ischemic border zone via the upregulation of vascular endothelial growth factor [145]. In fact, the implantation of ChAT-overexpressing NSCs effectively preserved microvessels within the penumbra of brain injury and significantly augmented the number of vWF-positive vessels in the brain [137]. In addition, accumulating evidence suggests that NSCs overexpressing HIF-1 lead to an increased presence of factor VIII-positive cells in regions affected by ischemic injury [134]. More significantly, there is also increasing studies demonstrating that mice receiving transplants of NPCs overexpressing SDF-1α exhibit a higher number of endothelial cells co-labeled with Glut-1 and BrdU in the peri-infarct area, alongside improvements in locomotor function [73].

Neurotrophic and growth-promoting factors are crucial for neuroprotection and neural regeneration. Numerous studies have demonstrated that the transplantation of NSCs genetically engineered to overexpress BDNF and NGF into animal models of ischemic stroke can effectively improve neurological function [138]. Notably, NSCs overexpressing ChAT exhibited more amounts of VEGF, GDNF, NGF, and VEGF compared to standard NSCs [137]. In addition, an interaction among the expression of these growth factors in gene-modified NSCs has been observed. For instance, NSCs modified with the GDNF gene exhibit a significant upregulation of BDNF and NT-3 protein expression [148]. Inspiringly, genetic modification also enhances the expression of MAP2, GAP-43, postsynaptic density protein 95 (PSD-95), and synaptophysin (Syp) [74, 136, 148]. These elements are essential for dendritic formation, synaptic integration, and synaptic plasticity, suggesting the potential therapeutic efficacy of genetically NSCs in treating neurological disorders, including ischemia.

Gene-modified neural stem cells for hemorrhagic stroke

Stroke is one of the most devastating medical conditions, characterized by significant consequences including mortality and long-term disability. It can be broadly categorized into hemorrhagic and non-hemorrhagic types. Hemorrhagic stroke accounts for approximately 20% of all stroke cases and can be further classified into intracerebral hemorrhage (ICH), intraventricular hemorrhage (IVH), and subarachnoid hemorrhage (SAH) [149, 150]. Among these, ICH is the most prevalent subtype of hemorrhagic stroke [149] and poses considerable treatment challenges, impacting millions of individuals globally [151]. Epidemiological studies suggest that ICH is linked to increased mortality rates and substantial disability [152]. The onset of ICH triggers a cascade of events leading to both primary and secondary brain injuries, ultimately resulting in persistent neurological deficits [151]. The brain injury following ICH involves the activation of thrombin and inflammatory processes [153]. Thrombin activation has the potential to alter the morphology and functions of brain endothelial cells, usually resulting in the disruption of the BBB and the subsequent development of cerebral edema [154]. Furthermore, ICH is plagued by neuroinflammation during both the acute and chronic phases [155]. Cerebral hemorrhage leads to oxygen and glucose deprivation in perilesional tissue and triggers a secondary inflammatory response that exacerbates lesion expansion [156]. Following ICH, microglia become activated and secrete cytokines and chemokines [157]. In addition, leukocyte infiltration exacerbates cerebral injury by producing chemokines, pro-inflammatory cytokines, matrix metalloproteinases [158, 159], and ROS [153]. Unfortunately, neuroinflammation induces both primary and secondary cell death, which is likely to contribute to the pathophysiology of stroke. Although current therapeutic strategies for hemorrhagic stroke, such as invasive surgical interventions to evacuate intraventricular blood or intracranial thrombi and manage intracranial pressure, have demonstrated efficacy in reducing mortality rates [150, 151], the overall clinical outcomes remain suboptimal. This is largely attributable to the limited time window available for effective intervention. Accordingly, there is an urgent need to explore novel treatment approaches. Recent research indicates that stem cell transplantation has become a valuable tool in the field of regenerative medicine for the treatment in hemorrhagic stroke [153, 155, 160, 161]. Comparatively, genetically modified NSCs exhibit enhanced therapeutic potential over unmodified NSCs. Based on this, these genetically engineered NSCs are highly promising candidates for the treatment of hemorrhagic stroke.

To address the adverse microenvironment following hemorrhagic stroke, the overexpression of neurotrophic factors such as BDNF, VEGF, GDNF, and various growth factors, along with survival signaling molecules in transplanted NSCs, has been shown to effectively ameliorate the hostile conditions, thereby promoting cell survival and neural regeneration [18, 28, 70, 75]. For example, NSCs overexpressing Akt1, when transplanted into the striatal epithelium, demonstrated extensive migration to the hippocampus [75], and this genetic modification significantly promoted the survival of transplanted cells following ICH [18, 28, 70, 75]. Similarly, the transplantation of GDNF-overexpressing NSCs led to a significant enhancement in cell survival between the second and fifth weeks post-transplantation in time-dependent manner, when compared to non-genetically modified cells [18]. Importantly, the majority of the transplanted genetically modified NSCs differentiate into neurons or astrocytes in response to cues from the local microenvironment [18, 70]. Furthermore, genetic modification has been shown to augment the neuronal differentiation of NSCs, thereby exerting neuroprotective effects in the context of hemorrhagic stroke. At two weeks and eight weeks following transplantation, the Akt1-overexpressing NSCs resulted in a significantly higher proportion of hNuMA-positive cells in the border area of the hemorrhagic core compared to the non-genetically modified NSCs, as reported in study [75].

It is significant to highlight that genetically modified NSCs confer neuroprotective effects via various mechanisms, particularly through anti-apoptotic pathways. In this context, studies have demonstrated a markedly reduced number of TUNEL-positive cells in ICH models transplanted with NSCs overexpressing GDNF, VEGF and BDNF, compared to those receiving phosphate-buffered saline (PBS) or non-genetically modified NSCs [18, 28, 70, 162]. This phenomenon is primarily attributed to the down-regulation of pro-apoptotic proteins, including Bax, caspase-9, p53, and caspase-3, alongside the up-regulation of anti-apoptotic proteins such as Bcl-2, as well as survival signaling pathways like Akt1 and ERK-MAPK. These changes, coupled with cell survival-promoting molecules, are observed in the ICH brain as a result of the overexpression of neurotrophic factors in transplanted NSCs [18, 28]. Furthermore, functional angiogenesis plays a crucial role in orchestrating these underlying mechanisms [132]. Lee and colleagues demonstrated that the transplantation of VEGF-overexpressing NSCs into brains affected by ICH led to a significant increase in the number of von Willebrand factor (vWF)-positive microvessels [70]. Similarly, another noteworthy study also revealed that transplantation with BDNF-overexpressing NSCs resulted in a three-fold increase in vWF-positive microvessels in ICH-affected brains [28]. Compared to the administration of PBS, the extent of neovascularization increased by six to eight times [28]. Therefore, the transplantation of NSCs genetically modified with various angiogenic factors at the site of ICH injury demonstrates substantial potential for enhancing neural regeneration.

Post-ICH, hemoglobin (HB) serves as a critical blood component and a significant mediator of oxidative stress [163]. HB released from extravasated erythrocytes has been associated with the development of brain edema following ICH. Furthermore, HB and its breakdown products are primary sources of ROS, which play a crucial role in the pathological processes post-ICH [163, 164]. The oxidative damage mediated by ROS adversely affects host intracellular components and the survival of transplanted NSCs in the context of ICH [20]. Therefore, the removal of ROS is essential for enhancing the microenvironment and promoting neural regeneration. Based upon this premise, certain researchers have transplanted NSCs overexpressing the antioxidant enzyme SOD1 into mice models of ICH, and observed a significant reduction in ROS expression [20]. Additionally, the overexpression of SOD1 in NSCs reduced levels of carbonyl proteins and increased secretion of paracrine factors such as GDNF and VEGF, thereby enhancing the survival of these cells within the hemorrhagic stroke milieu. Nonetheless, the precise mechanisms underlying the protective effects of gene-modified NSCs in the context of ICH hitherto warrant further investigation.

Gene-modified NSCs for the treatment of hypoxic-ischemic encephalopathy

Hypoxic-ischemic encephalopathy (HIE) constitutes a form of cerebral injury resulting from insufficient oxygen supply to the brain [165]. The prevalence of HIE is notably elevated during the neonatal period, with 15–20% of affected neonates succumbing in the early neonatal phase, while the survivors had severe neurological deficits, encompassing cerebral palsy (CP), cognitive impairments, epilepsy, visual and auditory impairments, behavioral issues, intellectual disabilities, and social dysfunctions [166]. Among these deficits, CP represents one of the most severe outcomes of prenatal hypoxic-ischemia (HI) [167, 168]. The mechanisms underlying HI encompass the activation and/or stimulation of numerous pathways, such as oxidative stress, enhanced glutamatergic excitotoxicity, compromised mitochondrial energy production, induction of inflammation, overstimulation of N-methyl-D-aspartate (NMDA) receptors, cellular edema, impaired maturation, and the depletion of trophic support [169, 170]. Although hypothermia remains the predominant standard treatment for neonates with HIE, as it curtails brain damage and enhances neurological outcomes associated with neonatal HIE [171], its efficacy is limited, with less than 50% of infants experiencing improved outcomes [135]. Therefore, while hypothermia is a suitable therapeutic strategy for treatment of HIE, its partial effectiveness underscores the need for alternative intervention. Recently, NSC-based therapy has demonstrated promising therapeutic effects for HIE, garnering significant attention for its potential to augment endogenous brain repair mechanisms [51]. Increasing studies utilizing animal models has demonstrated that the transplantation of NSCs suppresses neuroinflammatory damage, contributes to neuronal remodeling, and significantly enhances behavioral functions following hypoxic-ischemic injury [51]. Notably, the application of genetic engineering techniques, the expression of special protein molecules or epigenetic regulators can be effectively altered, thereby amplifying these therapeutic effects [58, 78, 166].

Hypoxic-ischemic injury (HI) frequently leads to the disruption of myelin gene expression and the death of oligodendroglial precursors in certain infants, potentially resulting in myelin loss and subsequent cerebral palsy [165]. Therefore, the regulation of NSC differentiation into oligodendrocytes and the mitigation of myelin sheath loss are critical areas of research aimed at ameliorating the effects of such injuries. Notably, bone morphogenetic protein 4 (BMP4) inhibits oligodendrogliogenesis from NSCs within the SVZ as well as the differentiation of adult OPCs. Consequently, BMP4 may influence cell-mediated remyelination following demyelination at various levels within the CNS. A study has shown that the targeted knockout of the BMP signaling pathway in neural progenitor cells effectively prevents the loss of oligodendrocytes [172]. Similarly, the deletion of the BMP subtype 2 receptor in NPCs effectively prevented the loss of CNPase, a marker indicative of immature oligodendrocyte (OL) expression, and MBP, a marker of mature myelin OL expression, following hypoxia–ischemia (HI) in the corpus callosum and internal capsule [172]. These findings suggest that the transplantation of gene-modified NSCs may alleviate the neurological and behavioral deficits caused by disruption of myelination due to hypoxia–ischemia, and further reduce the extent of pathological brain damage [58, 78]. In addition to affecting oligodendrogliogenesis and myelination, genetic modifications in NSCs have been found to predominantly reduce apoptosis in hypoxic-ischemic encephalopathy (HIE). Empirical evidence indicates that the transplantation of NSCs modified with lentivirus-mediated microRNA26a results in a altered expression of apoptosis-related proteins, including caspase-3, Bax, and BCL-2, within the cerebral cortex and hippocampus [58]. This intervention appears to suppress the complex pathological processes associated with these proteins. Alternatively, the transplantation of VEGF-transfected NSCs has been shown to alleviate a majority of the neurological deficits observed in the cerebral palsy (CP) model, notably by reducing neuronal apoptosis [78]. Furthermore, this intervention led to an enhancement in microvessel and neuronal density within the cortex and hippocampus [78], as evidenced by a significant increase in CD34-positive cells and the presence of sprouting or spindle-shaped microvessels in CP animals. Importantly, the transplantation of transgenic NSCs markedly mitigated the substantial loss of pyramidal cells in the CA1 region of the hippocampus in a rat model of unilateral carotid artery occlusion combined with hypoxia, which serves as a model for cerebral palsy [78]. Furthermore, this approach represents an effective strategy for restoring the lost functions of damaged or necrotic neurons post-injury by facilitating neuronal differentiation. A study demonstrated that the implantation of NT-3-overexpressing NSCs into subacute cortical infarcts resulted in an increase in donor-derived neurons, ranging from 5% to over 80% in the penumbra region [61]. This suggests that NT-3 may enhance both the neuronal differentiation of donor cells and the survival of host cells through autocrine and paracrine mechanisms [61]. Eventually, these neuronal precursors differentiated into specific subtypes of neurons, such as cortical-appropriate cholinergic, GABAergic, and glutamatergic neurons, through various ways, thereby supporting the recovery of neurological function [61].

Conclusion and prospects

Genetic modification of NSCs/NPCs can intentionally enhance their proliferation and survival, and also effectively modulate cell differentiation pathways and the secretome. This is propitious to survival of both donor and host cells in adverse conditions caused by oxidative stress and inflammation. Furthermore, it contributes to the creation of an endogenous nerve regeneration microenvironment subsequent to diverse injuries, including TBI, SCI, and stroke (see Fig. 2 and Table 1). The integration of gene and stem cell therapies has the potential to address the limitations of individual therapeutic approaches, thereby offering enhanced benefits for nerve injury treatment and functional recovery. Notwithstanding genetically modified NSCs have demonstrated numerous advantages and hold significant promise for the treatment of various neurological insults, such as TBI, SCI, AD, and strokes, numerous challenges remain in translating these therapies into clinical practice as follows. (i) Prior to the utilization of NSCs/NPCs for transformation and transplantation, there have not been comprehensive and detailed comparative studies involving NSPCs derived from various stem cell sources. (ii) Contemporary research on NSCs predominantly concentrates on embryonic neural tissue sources. However, the transplantation of cells derived from human embryonic tissues is unable to address several critical challenges, including ethical and moral constraints. (iii) The genetic modification of NSCs poses a potential risk of tumorigenesis due to the increased proliferative capacity that may result from specific gene integrations into the genome. (iv) There remains a lack of comprehensive research and consistent guidelines regarding the optimal timing, route, method of cell administration, and appropriate cell dosage for transplantation. (v) The present research predominantly emphasizes animal experimentation and is deficient in clinical trials, rendering it uncertain whether genetically modified NSCs can produce comparable therapeutic outcomes in humans as observed in post-transplantation animal models in vivo. Aside from the above, it remains unclear whether the transfected target gene can maintain stable expression over an extended period or if the overexpression of a specific gene might impact other related pathways, potentially leading to unintended side effects. Therefore, the safety, efficacy, and potential adverse effects of this approach still need to be further investigated. Despite the inherent challenges associated with gene transduction therapy in NSCs, its unique advantages and promising potential have garnered significant attention from researchers in the field of neuroregeneration. Given the rapid progress in research on NSC-based therapies for clinical application, there is optimism that transgenic cell therapy will be successfully translated into clinical practice. Moreover, specialized NSC treatment strategies could be developed based on the underlying causes of injury, the characteristics of the injury, and individual patient profiles.