Neural stem cells (NSCs) are a class of pluripotent cells with division potential, self-renewal ability, and capability of differentiating into neurons, astrocytes and oligodendrocytes. NSCs are ubiquitously present in a wide array of tissues, including the cerebral cortex, olfactory bulb, hippocampus, striatum, lateral ventricle ependymal/subventricular zones, and the spinal marrow of embryos, fetuses, and adults (Tuazon et al., 2019).

Initially, the notion of using cell transplantation for the treatment of NP was premised on the fact that stem cells act as a kind of totipotent cells that can replace damaged nerve cells and deliver trophic factors to the site of the lesion. NSCs make a superior candidate cell type for cell therapy since they are highly capable of differentiating into neurons and glial (Franchi et al., 2014).

Previous studies have shown that NSCs can be induced to differentiate into stem cells and promote axonal regeneration. Wang et al. (2017) retrospectively reviewed the role of NSCs in the repair of peripheral nerve injury, and found that, NCSs not only possessed nerve regenerative and neuroprotective effects, but also secreted a variety of factors to enhance the survival of motor and sensory neurons and to promote angiogenesis. Further experiments confirmed that neural stem cells could interact bidirectionally with resident cells in the damaged microenvironment. Du et al. (2019) locally injected NSCs into the injury site of the spinal cord injury (SCI) rat model, and the transplanted NSCs could regulate the expression of P2X receptor and improve the microenvironment after SCI, thereby enhancing neural regeneration and functional movement. A study treated patients with chronic SCI damage by multiple intramedullary injections of human-derived CNS stem cells and found that, after transplantation, an interaction took place between stem cells and the microenvironment (Levi et al., 2018).

In addition to relieving central NP, NSCs transplantation could also effectively ease NP caused by peripheral nerve injury. In a rat sciatic nerve transection (SNT) model, neural crest stem cells attenuated NP and improved motor function (Zhang et al., 2021). Lee et al. (2016) found that NSCs could relieve NP and promote nerve regeneration in a rat model, and a small amount of persistently expressed vascular endothelial growth factor (VEGF) could effectively modulate nerve function. However, uncontrolled overexpression of VEGF might pose the risk of causing tumor formation.

Currently, a great many animal studies confirmed that NSCs provide effective relief of NP. Prior studies showed that NSCs appeared to be more effective for the peripheral NP than for the central one, but the relevant literature is scanty and further comparative studies are needed.

A small number of clinical trials employed human-derived neural stem cells for treating NP due to chronic SCI (Curtis et al., 2018), and their efficacy and safety were experimentally supported, but the sample size was small, and more trials are warranted to support their clinical application.

Glial cells that surround and enclose the olfactory nerve bundle and the outer layers of the olfactory bulb, are collectively known as olfactory ensheathing cells (OECs). They possess the unique ability to transgress the peripheral nervous system (PNS) and central nervous system (CNS) environments (Andrews et al., 2016). And OECs have also been shown to have neuro-regenerative functions (Zhang et al., 2020a). Because of this property, olfactory ensheathing cells are also seen as a good choice for treating nerve injury and NP.

Zhang et al. (2019a,b, 2020a,e) in a series of studies, used OECs transplantation for treating NP in chronic constrictive injury (CCI) model rats, and confirmed that OECs transplantation could promote motor recovery and mitigate pain. Their studies found that OECs secreted various neurotrophic factors (e.g., neurotrophic Y, neurotrophic factor 3, enkephalin, and endorphin), inhibited the formation of colloid scar and cavities, and facilitated regeneration and myelination of new axons, and they were effective for the treatment of NP. At the same time, they also found that the inhibition of the P2 purinoceptor family was an important part of the process. Further studies have revealed that OECs transplantation inhibit P2X2 receptor (Zhao et al., 2015; Zhang et al., 2020c), P2X4 receptor (Zheng et al., 2017; Zhang et al., 2019b, 2020b), and P2X7 receptor (Zhang et al., 2019a, 2020a) overexpression mediated NP. Wu et al. (2011) found that delayed OECs transplantation also can effectively inhibit hyperalgesia and allodynia after dorsal root injury.

A meta-analysis published in 2018 (Nakhjavan-Shahraki et al., 2018) reviewed 40 studies related to the effect of OEC transplantation on NP and functional recovery following spinal cord damage, and concluded that OEC transplantation significantly improved post-injury motor function, but had no effect on allodynia and might even lead to a relative exacerbation of nociceptive hyperalgesia, especially 8 weeks after OECs transplantation. Animal experiments by Lang et al. (2013) found that transplantation of olfactory bulb OECs into the rat hemispheric spinal cord caused hyperalgesia, and they observed the activation of ERK and the up-regulation of the brain derived neurotrophic factor (BDNF) following OECs transplantation, which may be related to hyperalgesia. BDNF is an important regulator of pain, and studies have shown that it exerts both anti-nociceptive and anti-inflammatory actions and pro-nociceptive effects (Cao et al., 2020). The BDNF/tyrosine receptor kinase B (TrkB) signaling pathway is closely related to NP. BDNF polymorphisms can interfere with BDNF role in pain perception, resulting in pain relief or exacerbation, and existing researches suggested that genetic factors such as Val66Met are involved (Cappoli et al., 2020). The authors consider that this may be related to the controversial result of relieving NP after OECs transplantation, but no specific studies on this pathway and BDNF genotypes after transplantation have been reported.

However, two studies (Feron et al., 2005; Tabakow et al., 2013) on autologous OECs in the treatment of human SCI failed to observe any hyperalgesia or serious adverse effects. A study by Mackay-Sim et al. (2008) performed OECs transplantation in patients with traumatic injuries to the thoracic spine and observed no adverse effects during 3-year follow-up. These experiments have confirmed the feasibility of olfactory sheath cell transplantation to some extent, but further validation is needed. In order to further address the risk of exacerbated hyperalgesia upon OECs transplantation, some studies used combined transplantation to treat NP. Co-transplantation of NSCs and OECs not only promoted the survival of NSCs, but also reversed the hyperalgesia caused by OECs (Luo et al., 2013).

Due to their source abundance, easy availability, and high viability in ex vivo culture, OECs have good prospect of being used for treating NP in clinical practice. However, experiments with longer observation time are still needed to verify their long-term effects and safety.

As a kind of adult stem cells, mesenchymal stem cells (MSCs) can be obtained from various sources, such as bone marrow, umbilical cord, adipose tissue and placenta, and can be induced to differentiate into endoderm, mesoderm, and ectoderm cell lines. Mesenchymal stem cells are abundant in source and easy to expand, do not express the major histocompatibility complex class II cell surface receptor HLA-DR, and are characterized by low immunogenicity, high immunomodulation and good phenotypic stability. As a result, they can be used for the treatment of diseases involving neuroinflammatory components, such as immune diseases, NP, etc (Franchi et al., 2014; Jwa et al., 2020) A great number of animal experiments have demonstrated that MSCs have great potential to be used for alleviating NP symptoms, such as allodynia and hyperalgesia. Moreover, most of the trials did not observe MSCs-related adverse reactions (Wang et al., 2020). MSCs can be administered intravenously, intrathecally, and topically at the site of injury, and can be transported and collected at the site of injury regardless of the route of administration, a phenomenon known as post-transplantation homing (Chakravarthy et al., 2017; Han et al., 2019; Xie et al., 2022).

Bone marrow mononuclear cells (BMMCs), a mixed population of cells, include monocytes, lymphocytes, hematopoietic stem cells, and endothelial cell precursors. BMMCs can be isolated from bone marrow by density gradient centrifugation and have been shown to be able to relieve experimental NP. Takamura et al. (2020) found that intrathecal injection of BMMCs attenuated NP in a mice model of spinal nerve transection (SNT). Animal experiments by Klass et al. (2007) and Usach et al. (2017) exhibited that intravenous injection of BMMCs could relieve NP caused by sciatic nerve crush or constriction. In addition to suppressing microglial migration and inflammation associated with nerve injury, a range of angiogenic ligands (basic fibroblast growth factor, angiopoietin-1, VEGF) and cytokines (IL-1b, TNF-a) were secreted by BMMCs to promote local neovascularization and tissue repair (Naruse et al., 2011). BMMCs have unique advantages over bone marrow-derived mesenchymal stem cells in that they are readily available, require no in vitro expansion, facilitate transplantation, and minimize the risk of contamination (Alvarez-Viejo et al., 2013).

γ-aminobutyric acid (GABA) is an important neurotransmitter that inhibits neurotransmission. The nerve cells in the dorsal horn of the spinal cord that can synthesize GABA are referred to as GABAergic neurons. Upon nerve damage, peripheral or central, GABAergic inhibition at the spinal cord level is decreased, and the excitability of neurons is increased in the dorsal horn of the spinal cord, resulting in the development of persistent post-injury NP (Dugan et al., 2020). Therefore, early intervention aimed to restore GABA levels is an efficacious way to preempt NP development. However, the drugs targeting the GABAergic system (e.g., benzodiazepines, gabapentin, etc.) are of limited value and have side effects (Mason et al., 2018).

Transplantation of GABAergic precursor/progenitor cells to enhance central inhibition and reduce pain is a new approach to managing chronic NP. Hwang et al. (2016) intrathecally transplanted embryonic stem cell-derived spinal GABAergic neural precursor cells into the injury area of the spinal cord. They found that the NP was significantly relieved and the cells survived for more than 7 weeks after transplantation. Manion et al. (2020) using pluripotent stem cell-derived GABAergic interneurons transplantation, also confirmed that the GABAergic cells are involved in spinal cord injury-induced NPs, and synaptic integration occurred after the transplantation. A new analysis reviewed 13 studies on the transplantation of GABAergic precursor cells (Askarian-Amiri et al., 2022), and concluded that GABAergic cells can ameliorate allodynia and hyperalgesia in the rat model, but the extrapolation of the conclusion to mice and other large animals and human subjects still requires a higher-grade evidence. In years to come, multi-specie modeling and multi-model studies on the effect of GABAergic cells on NP are warranted to verify their therapeutic effects.

While cell transplantation can provide better pain relief than drug therapy, researchers have begun to integrate genetic engineering into stem cell transplantation to improve its efficacy. The neurotrophic factor VEGF introduced into gene-modified neural stem cells can enhance the analgesic effect of NSCs by increasing the expression of VEGF, inducing high-speed migration of NSCs and promoting cell viability (Lee et al., 2016). By transfecting mesenchymal stem cells with fibroblast growth factor (FGF-1; Forouzanfar et al., 2018), glial neurotrophic factor (GDNF; Yu et al., 2015) and sirtuin 1 (SIRT1; Tian et al., 2021) genes, researchers found that the analgesic effect induced by transfected MSCs was better than that of simple mesenchymal stem cells alone. To our knowledge about the mechanism of NP, analgesic peptides, such as inhibitory neurotransmitters (e.g., beta-endorphins), anti-inflammatory peptides (e.g., IL-10), neurotrophic factors (e.g., NT-3, VEGF) and soluble receptors (such as soluble tumor necrosis factor receptor) have potential to serve as targets for genetic engineering (Wang et al., 2020). It is worth mentioning that attention should be paid to the biosafety and the regulation of the gene engineering to avoid the occurrence of occasional tumor-like aggregation caused by overexpression or proliferation of transplanted cells and other possible risks.

Nerve injury can cause morphological, functional, and behavioral changes at the site of nerve damage, resulting in intractable NP. Hwang et al. (2016) found that intrathecally administered GABAergic cells successfully integrated into or around the injured tissue, with analgesic effect persisting up to 7 weeks post-modeling. Wu et al. (2020) marked the transplanted BM-MSCs with GFP and showed that the cells successfully migrated to the injured segment of spinal cord. In addition to intrathecal injection, intravenously transplanted cells (Siniscalco et al., 2011; Li et al., 2017; Usach et al., 2017) also showed directional migration to the damaged site. Chen et al. (2015) found that BM-MSCs that migrated to the border of the dorsal root ganglia survived for more than 2 months, providing sustained pain relief in mice model induced by sciatic nerve contraction. The process by which transplanted cells migrate to the site of injury is dubbed homing of cells. This phenomenon is beneficial to the survival of transplanted cells, and the migrated cells provide timely and long-lasting analgesia for pain relief by interacting bidirectionally with the residing cells in a damaged microenvironment via paracrine secretion (Naruse et al., 2011; Franchi et al., 2014; Du et al., 2019; Kotb et al., 2021).

The homing behavior of transplanted cells has been well described in many studies, but the definitive mechanisms and regulatory processes of their migration process remain unclear (Deak et al., 2010; Liesveld et al., 2020). According to the different transplantation pathways, the homing phenomenon can be divided into systemic homing and non-systemic homing. The directional migration behavior of transplanted cells in the damaged tissue locally or in the adjacent area (e.g., within the sheath) is non-systemic homing. Systemic homing involves multiple steps, including entry of graft cells into circulation, extravasation around the lesion, and interstitial migration toward the injury site (Nitzsche et al., 2017). Although the two processes are different, new advances revealed that the homing of transplanted cells both require chemokine guidance (Siniscalco et al., 2011; Nitzsche et al., 2017; Asgharzade et al., 2020). However, how chemokine receptors such as CCR2 and CXCR4 promote the process of transendothelial extravasation of transplanted cells is not clear, and further clarification of this process will greatly promote the efficiency of homing and overcome one of the existing impediments of cell therapy.

Activation of microglia and astrocytes leads to neuroinflammation, which plays a pivotal role in the development and persistence of NP (Zhao et al., 2017; Li et al., 2019). Activated glial cells release inflammatory cytokines, leading to upregulation of glutamate receptors. Glutamate released from A-delta and C-fibers is the main nociceptive excitatory neurotransmitter, and is highly associated with the central sensitization of dorsal horn neurons and the maintenance of hyperexcitability (Siniscalco et al., 2007). Transplanted bone marrow-derived mesenchymal stem cells can modulate microglial activation (Forouzanfar et al., 2018; Joshi et al., 2021). Zhong et al. (2020) found that bone marrow MSCs suppressed the M1 phenotype of microglia by secreting GDNF while promoting their conversion to the M2 phenotype, and that the PI3K/AKT signaling pathway is activated during this process.

Although the mechanism that promotes polarization of glial cells towards an anti-inflammatory phenotype is not fully understood, the researchers found that it is related to the inhibition of MAPK-related pathways (Figure 2). The mammalian mitogen-activated protein kinase (MAPKs) family includes three related pathways, that is, p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N2-terminal kinase (JNK) pathways, and the MAPK cascade is a key signaling pathway governing a wide array of cellular processes, including proliferation, migration, differentiation, apoptosis and responses of cells (Kim and Choi, 2010, 2015). Existing studies have shown that the p38 MAPK pathway and ERK 1/2 channels play a central role in neuroinflammation and pain regulation. Intraspinal injection of BM-MSCs after SCI reportedly inhibited the NF-κ B and p38 MAPK pathways (Zhang et al., 2021). Watanabe et al. (2015) documented that transplantation of MSCs derived from bone marrow inhibited MAPK signaling cascade and inflammatory cell recruitment after spinal cord injury, leading to pain relief. Xie et al. (2019) also found that intrathecally injected BM-MSCs inhibited ERK1/2 up-regulation in the dorsal root ganglia of CCI model rats. However, the exact mechanism by which transplanted stem cells, glial cells and MAPK signaling pathway interact with each other needs further studies at the molecular level.

Initially, cell transplantation for NP treatment was meant to substitute injured nerve cells, but several studies have shown that the transplanted cells support and enhance the regeneration and remyelination by secreting various trophic regulators of nerve regeneration, such as VEGF, GDNF and NGF, rather than directly replace the lost or damaged cells. Andrews et al. (2016) and Zhang et al. (2019a, 2020a) found that transplanted OECs not only inhibited formation of colloidal scars and cavities after nerve injury, but also promoted regeneration of new axons and remyelination. Lee et al. (2016) found that VEGF-expressing neural stem cells could enhance cell viability under hypoxic conditions and facilitate remyelination of injured sciatic nerves. Transplanted bone marrow mononuclear cells could migrate to the damaged site and promote neovascularization and tissue repair, electromyogram compound muscle action potential (CMPA) verified the presence of regenerated axons (Usach et al., 2017).