Introduction
Spinal cord injury (SCI) is one of the most destructive neurological diseases and directly leads to axotomy and neuronal death, resulting in permanent motor and sensory dysfunction. More than 27 million people globally are reported to suffer from long-term disability after SCI (Wiles, 2022). Primary damage and traumatic damage are two causes of SCI. Spina bifida, newborn axonal dystrophy, amyotrophic lateral sclerosis, and other degenerative central nervous system (CNS) illnesses are examples of congenital and developmental conditions that cause primary spinal cord damage (Geisler et al., 2002). However, widespread spinal cord damage is more likely in stressful situations such as car accidents, assaults, and other conditions, and this usually causes traumatic spinal cord damage.
At present, the most common clinical case type is incomplete SCI. SCI lesions vary considerably in size, complexity, and functional consequences (Sofroniew, 2018), but some clinical treatments for incomplete SCI are available, such as surgical decompression, therapeutic hypothermia, and manipulation of the inflammatory response and the neuroprotective diet (Ramer et al., 2014). These pharmacological or physical rehabilitation-based therapies focus on the modulation of symptoms associated with incomplete SCI, including reducing pain (Gwak et al., 2016; Saulino and Averna, 2016), relieving muscle spasms (Koulousakis and Kuchta, 2007; McIntyre et al., 2014), and promoting motor functions (Jacobs and Nash, 2004). However, except for optimizing the treatment plan to alleviate the pain of patients as much as possible and reducing the occurrence of complications with SCI, there is no clinical method to promote the improvement of natural movements and sensory function in incomplete SCI (Rowald et al., 2022). Moreover, it is a more difficult challenge for patients with complete SCI to have a functional recovery in clinic. It has recently been reported that combined therapy with implanted NeuroRegen scaffolds and mesenchymal stem cells (MSCs) helps to improve motor function in patients with complete SCI (Xiao et al., 2018). In addition, changes in autophagy markers can be found in SCI animal models suggesting that autophagy plays a role in SCI. Moreover, although there are no published articles reporting changes in autophagy markers in clinical SCI patients, the latest literature has indicated that autophagy plays an important role in human diseases and the lack of autophagy genes can lead to motor tremor, cerebellar ataxia, etc. (Mizushima and Levine, 2020). A recent review also discussed preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer, as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders (Klionsky et al., 2021). These data illustrate the importance of autophagy in disease control. With the in-depth study of nerve regeneration strategies, stem cell treatment for SCI has progressively become a research hotspot in recent years, and potentially, regulating autophagy in stem cells may have unexpected effects on SCI treatment.
In general, this review describes the state of the art on the use of stem cells for the treatment of SCI and the synergic effect of autophagy in this therapy. Three crucial items are highlighted and extensively explored, including autophagy, the repair properties of endogenous and exogenous stem cells, and the effect of autophagy on stem cell function contributing to repair of the injured spinal cord.
Retrieval Strategy
Through a search of the PubMed database using the keywords ‘stem cells, autophagy, and spinal cord injury’, articles containing detailed modeling methods, repair outcomes, and repair mechanisms were identified for inclusion in this review, which contain research progress over the past 20 years.
Pathophysiology and Mechanism of Spinal Cord Injury and Research Models
Pathophysiology and mechanism of SCI
Oxidative stress
Traumatic SCI can be divided into primary and secondary injuries in pathophysiology, which includes three stages: acute stage (< 48 hours), subacute stage (48 hours to 14 days), and chronic stage (> 6 months) (Ahuja et al., 2017a). Different traumas lead to spinal cord compression or transection, resulting in rapid secondary injury cascade reactions, including bleeding and inflammation. Neurons and glial cells along the vascular system are destroyed, resulting in blood-spinal cord barrier damage (Choo et al., 2007; LaPlaca et al., 2007). Damage, destruction, and death of neurons are the predominant reasons for the loss of function caused by SCI. A series of rapid secondary injury reactions caused by physical injury, such as the release of inflammatory factors, misfolding of proteins, oxidative stress, etc., directly trigger the body’s endoplasmic reticulum stress response and unfolded protein response to combat this pathological environment. However, excessive stress leads to apoptosis of neurons around the injury, which significantly limits regeneration and repair after SCI. To a certain extent, reactive oxygen species (ROS) play an important role in SCI; ROS can regulate axonal regeneration through the release of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase from exosomes to the damaged site (Hervera et al., 2018). Excessive ROS leads to oxidative stress and cell death. Strategies to potentiate antioxidant pathways, based on the control of oxidative stress, are being widely researched (Jia et al., 2012). Glucocorticoid steroids have shown significant antioxidant activity in clinical experiments and have improved the rehabilitation of SCI patients. Nuclear factor E2-related factor 2 (Nrf2) is the central regulator of the antioxidant defense, and enhancement of the Nrf2 pathway can be used as protective mechanism in SCI (Liu et al., 2021).
Inflammation
Spinal cord swelling caused by overwhelming inflammatory reactions in the acute and subacute stages of SCI further leads to spinal cord compression, loss of neural circuits, and inhibition of glial scar regeneration, resulting in functional loss (Song et al., 2021). After SCI, inflammation is complicated and includes various cell types and inflammatory molecules, such as interleukin (IL)-1, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), and others (Garcia et al., 2016). Substantial immune cell infiltration contributes significantly to neurodegeneration. These immune cells are guided to the lesion site by cytokines and chemokines released by microglia, astrocytes, and macrophages inside and outside the lesion (Hellenbrand et al., 2021). Subsequently, the immune cells release many inflammatory factors to produce overwhelming inflammatory reactions, which result in the loss of neural circuits (Hellenbrand et al., 2021). MSCs have historically been preferred for the repair of SCI injury owing to their capacity to release anti-inflammatory and cell adhesion molecules and their specific differentiation potential.
Autophagy
When traumatic SCI occurs, extensive secondary injury occurs in the acute stage, including ischemia and hypoxia, severe oxidative stress, and inflammatory reactions, which result in the accumulation of many cytotoxic fragments. In the regeneration and repair process of SCI—whether through stimulating repair or exogenous transplantation repair of endogenous stem cells—the cell survival and functioning of the damaged part of the spinal cord are largely related to the microenvironment of SCI, and the poor pathological microenvironment of the damaged part often eclipses the therapeutic effect of treatment. For example, MSC-derived factors (e.g., IL-6, IL-10, and TGF-β) create an immunosuppressive microenvironment in the process of repair (Harrell et al., 2021), which improves the survival rate of stem cells by reducing immunological rejection. Autophagy is the core molecular pathway used to maintain functional homeostasis of the cellular environment. During this process, double-membrane vesicles (autophagosomes) isolate cytoplasmic components—including damaged cytoplasmic organelles and toxic protein aggregates—and then fuse with lysosomes to allow lysosomal proteases to degrade cargo, which usually has protective functions. Within the cell, autophagy can help decrease oxidative stress and increase the elimination of waste to maintain stability. Outside the cell, autophagy may help to decrease inflammation, improve the balance of the neuroendocrine system, and remove aging cells. In addition to inflammation and oxidative stress at the lesion site, autophagy is often an effective way to prevent the accumulation of misfolded proteins after SCI. Mutations in autophagy-related processes can lead to serious human diseases, including cardiovascular, neurological, metabolic, and musculoskeletal diseases (Klionsky et al., 2021). In an early study of autophagy, mice with CNS-specific knockout of the autophagy gene Atg7 had serious neurodegeneration (Komatsu et al., 2006), resulting in abnormal motor function and reflexes, highlighting the importance of autophagy in the nervous system (Figure 1).
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Figure 1: Under the regulation of ATGs, autophagic cargo is sequestered by double-membrane phagocytosis into double-membrane vesicles (autophagosomes), which subsequently fuse with acidic lysosomes to form autolysosomes, and then the cargo is degraded.Autophagy is a complex self-degradative process that involves the following key steps: (A) control of phagophore formation by VPS34 at the endoplasmic reticulum and other membranes in response to stress signaling pathways. For example, TOR is the main pathway for autophagy in response to nutrient sensing. Upstream of TOR, AMPK activates autophagy in response to low ATP levels. (B) Atg5-Atg12 conjugation, interaction with Atg16L, and multimerization at the phagophore, with simultaneous LC3 processing and insertion into the extending phagophore membrane. In this process, the E1-like ubiquitin-activating enzyme Atg7 activates Atg12 in an ATP-dependent manner, and then transfers Atg12 to Atg10, enhancing the conjugation of Atg12 to Atg5. (C) LC3-II binds to the autophagy substrate p62 with ubiquitination, and acidic lysosomes fuse with autophagosomes to form autolysosomes. (D) Fusion of the autophagosomes and lysosomes results in proteolytic degradation of engulfed molecules by lysosomal proteases. AMPK: Adenosine 5′-monophosphate-activated protein kinase; Atg: autophagy-related gene; LC3: microtubule-associated protein light chain 3; mTORC1: mammalian target of rapamycin complex 1; PE: phosphatidylethanolamine; TOR: target of rapamycin; Ub: ubiquitin; ULK1: mammalian autophagy-initiating kinase; VPS34: vesicular protein sorting 34.
Recent studies detected changes in autophagy markers in SCI animal models (Wu et al., 2021; Rong et al., 2022), but the mechanism and function of those changes are still disputed; rather autophagy has a double-edged effect on SCI repair. It was initially believed that Beclin1 was abnormally increased in the injured nerve tissue, and autophagic cell death was induced in response to SCI (Kanno et al., 2009), thus inhibition of autophagy was more conducive to functional recovery after SCI (Bisicchia et al., 2017). However, as further research has been conducted, the function of autophagy in various cells in SCI has been gradually elucidated. For example, blocking autophagy in oligodendrocytes limits the recovery of hindlimb motor function after injury (Saraswat Ohri et al., 2018); transcription factor E3 reduces neuronal cell death after SCI by increasing autophagic flux and decreasing endoplasmic reticulum stress (Zhou et al., 2020a); and transplanted neural stem cell (NSC)-derived small extracellular vesicles regulate apoptosis and inflammatory processes after SCI by inducing autophagy (Rong et al., 2019a), indicating that autophagy may be indispensable in the repair of SCI. Furthermore, rapamycin-activated autophagy combined with surgical decompression was effective in treating SCI in rabbit models (Zhang et al., 2020). In general, regulating autophagy after SCI contributes to neuroprotection and functional recovery of traumatic SCI (Ray, 2020), and activation of the autophagy lysosome pathway reduces neuronal cell death and inflammation (Lipinski et al., 2015), which can both improve secondary injury after SCI. However, this regulation of autophagy may need to be altered according to the different stages of SCI. The massive accumulation of damaged autophagosomes in the early stage of SCI may lead to the death of neuronal cells, while autophagy may have a neuroprotective role in the later stage of SCI with the recovery of autophagic flux. It remains to be determined how autophagy homeostasis can be maintained in the pathological state and how this double-edged sword can be utilized to beneficial effect. In addition, further work is needed to establish whether key autophagy genes are involved in the functional implementation of various cells in SCI.
Based on the above, controlling the damaged cell microenvironment, activating anti-inflammation processes, reducing the excessive oxidative stress response, and maintaining intracellular autophagy homeostasis may also benefit stem cell repair. Oxidative stress and inflammatory factors are currently being widely studied in SCI, and the regulation, defense, and protection of autophagic flux levels in SCI may become potential neuroprotective targets for SCI regeneration and repair (Figure 2).
Figure 2: The crosstalk of oxidative stress, inflammation, and autophagy.Oxidative stress directly affects autophagy through AMPK, mTOR, etc. Activation of autophagy reduces the production of ROS and is beneficial in maintaining cellular homeostasis. Concurrently, activation of the NF-κB signaling pathway produces substantial inflammatory factor infiltration and activates autophagy through AMPK to reduce inflammation. AKT: Protein kinase B; AMPK: adenosine 5′-monophosphate-activated protein kinase; ARE: antioxidant response elements; ER: endoplasmic reticulum; HIF-1α: hypoxia-inducible factor-1α; HO-1: heme-oxygenase-1; IL-1β: interleukin-1β; Keap1: Kelch-like ECH-associated protein 1; LC3: microtubule-associated protein light chain 3; mTOR: mammalian target of rapamycin; NF-κB: nuclear factor-κB; NQO-1: NAD(P)H quinone oxidoreductase-1; Nrf2: nuclear factor E2-related factor 2; PI3K: phosphatidylinositide-3-kinases; ROS: reactive oxygen species; ULK: mammalian autophagy-initiating kinase.
Research models of SCI
At present, SCI is divided into three main types: transection, contusion, and compression injury. Complete transection injury is relatively rare in the clinic, but contusion and compression injuries are more common (Briona and Dorsky, 2014; Sabin et al., 2015; Roberts et al., 2017; Koffler et al., 2019; Zou et al., 2020; Klatt Shaw et al., 2021; Xu et al., 2021; Yuan et al., 2021). Contusion is caused by applying a short focused force to the dorsal side of the spinal cord, and the model for this injury usually uses a special mechanical device to apply a certain amount of force to the dorsal side of the spinal cord (Cusimano et al., 2012; Dell’Anno et al., 2018; Allahdadi et al., 2019; Hakim et al., 2019; Wang et al., 2020; Wu et al., 2020; Zhou et al., 2020c; Kong et al., 2021). This method imitates human cases of SCI caused by heavy impact and has high repeatability that can ensure the same pressure applied to model animals, which is convenient for simulating mild or severe injury. Compression injury is a crushing injury caused by applying compressive force to the spinal cord (Li et al., 2020). Compared with the contusion model requiring special devices, compression injury can be produced by applying force from the dorsal or lateral side of the spinal cord in a variety of ways (Yang et al., 2018; Lee et al., 2021). Methods of compression injury include calibration of forceps, aneurysm clips, etc. (Plemel et al., 2008). The method of calibrating forceps to compress the spinal cord was recently used to produce a repeatable SCI model without special instruments, which simplifies the SCI modeling surgery (McDonough et al., 2015). Mice are a valuable and highly repeatable animal model for studying SCI and regeneration and repair and have provided an experimental basis for research. In addition, other animals such as rats, zebrafish, salamanders, and rabbits can be used as SCI research models. In mammals, the robustness of the rat model of SCI allows extensive analysis of SCI pathology (Kjell and Olson, 2016), while some measures such as intramedullary pressure measurements would be more accurate in larger animals such as rabbits (Zhang et al., 2020). Although zebrafish and salamander are not mammals, their spinal cord transection operation is simple and easy to repeat. Moreover, the regeneration process of these model animals is rapid and their unique physiological structure permits visualization of the regeneration process in real time (Tazaki et al., 2017), thus they are good research models of spinal cord regeneration (Table 1).
Table 1: Model and repair mechanism of spinal cord injury
Repair of SCI in animal models
Currently, most SCI patients are lifelong disabled and have to bear high costs of treatment and nursing. In the treatment of SCI, re-establishing neural relays in the injured nervous system and promoting the recovery of physiological function are major research targets. The exploration of nerve repair and reconstruction has led to stem cell populations with differentiation potential entering the research field, with the goal of using the physiological function of stem cells for functional recovery after SCI.
Repair effect of endogenous stem cells
NSCs are pluripotent stem cells that can self-renew, meaning that they can produce different types of mature cells. NSCs exist in all major branches of the CNS of adult mammals, including the spinal cord (Stenudd et al., 2015). The adult spinal cord has always been believed to have very little regenerative capacity, which limits the self-healing of patients after injury. However, a study has shown that a group of cells resident in the spinal cord ependyma of adult rats migrate to the spinal cord parenchyma after SCI (Decimo et al., 2011). This group of cells express the neural stem/precursor markers that contribute to the formation of glial scars after injury (Decimo et al., 2011). Multidirectional differentiation of ependymal cells can treat SCI to a certain extent (Meletis et al., 2008). In addition, endogenous adult neural progenitor cells exhibited enhanced proliferation and migration abilities in early acute SCI in mice, indicating that neural progenitor cells try to restore neuronal dysfunction caused by SCI (Ke et al., 2006). Endogenous NSCs that were originally static in the spinal cord can be activated by SCI, and their proliferation increases after SCI stimulation (Johansson et al., 1999). However, NSCs that migrate to the injured site after SCI tend to differentiate into astrocytes to participate in the formation of glial scars (Meletis et al., 2008) rather than into functional neurons to restore the gap in the spinal cord; that is, in the SCI microenvironment, the neuronal differentiation of NSCs is limited, but neuronal differentiation is essential in functional recovery. It is necessary for newly differentiated neurons to replace damaged neurons and form new neuronal relays at both ends of the injured site so they can promote the reconstruction and functional recovery of new neural circuits (Bonner and Steward, 2015). Therefore, the latest research proposes a strategy to enhance the intrinsic neuronal differentiation ability of NSCs by using biomaterials (Xue et al., 2021) to help repair SCI. However, it is still worth exploring how the pluripotency of endogenous resting NSCs can be harnessed to ensure their differentiation ability in the repair of SCI.
Regeneration and repair application of stem cell transplantation
Regeneration of the CNS of adult mammals was previously believed to be very difficult owing to the silencing of adult stem cells. The latest research progress in SCI shows that although NSCs have more inherent regeneration ability (Barnabé-Heider and Frisén, 2008; Xie et al., 2022), their differentiation ability is limited and they do not have regeneration ability in the peripheral nervous system (Ahuja et al., 2017b). Therefore, complementary repair by exogenous transplantation was once favored as a potential treatment option for SCI. Traumatic SCI causes a series of inflammatory and pathological processes and finally forms glial scars that include various cellular and extracellular components (Bradbury and Burnside, 2019). Glial scar formation can prevent the spread of cell damage, but it also limits the growth and repair of new neurons and tissue. Therefore, using stem cell transplantation or biomaterial transplantation to facilitate the regeneration and repair of damaged parts of the spinal cord has attracted considerable attention.
Stem cells have the ability to differentiate into various cells, including neurons. In SCI, stem cells exert therapeutic effects by differentiating into neurons to replace damaged cells, secreting neurotrophic factors, and inhibiting secondary inflammatory responses (Shao et al., 2019). Stem cell transplantation in SCI has predominantly focused on NSCs, MSCs, embryonic stem cells, induced pluripotent stem cells, and other cell types (Gao et al., 2020). Here, we will primarily focus on the effect of NSCs and MSCs on SCI repair.
Long-term NSCs are present in the lateral ventricle, hippocampal dentate gyrus, and central canal of the spinal cord in mammals, but these cells are inactive most of the time in adults. NSC transplantation has been suggested to drive neurological recovery after SCI (Suzuki et al., 2017; Zhang et al., 2018b). Neural differentiation of transplanted NSCs after SCI can be tracked by using Dil dye-labeling, and transplanted NSCs were observed to regulate the inflammatory response after SCI by reducing macrophage activation and neutrophil infiltration to prevent secondary injury (Cheng et al., 2016, 2017). After severe SCI (complete transection), NSC transplantation can disrupt the long-distance growth and axonal regeneration connection (Lu et al., 2012), form a new axonal relay, and improve the electrophysiological and functional state. However, completely transected SCI accounts for only a small number of SCI cases. At present, the application of NSC transplantation and repair in SCI animal models is concentrated in spinal cord crush injury models (Kadoya et al., 2016). NSCs are transplanted into the injured spinal cord, and the regenerated corticospinal axons form a functional contact with the transplanted neurons, which effectively promotes functional recovery of the body (de Freria et al., 2021). Newly transplanted NSCs also contribute to the differentiation of oligodendrocytes to enhance myelination (Hofstetter et al., 2005) and improve motor and sensory functions. Moreover, the function of glial cells cannot be ignored in SCI regeneration and repair (Tai et al., 2021) as it results in a healing effect in neonatal mice after SCI (Li et al., 2019), which may provide ideas for the directional induction and differentiation of transplanted stem cells. In addition, research on the differentiation of neurons and the establishment of functional synapses after NSC transplantation indicates that many growth-promoting factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell-derived neurotrophic factor, etc.) secreted by NSCs to promote the survival of damaged neurons (Kerr et al., 2010) warrant further investigation. Changes in the SCI microenvironment caused by these cytokines may directly affect the neuronal differentiation of NSCs newly transplanted to the damaged site (Xue et al., 2021). In summary, progress on the transplantation and regeneration of NSCs has been made but identifying ways to enhance the survival and differentiation of transplanted NSCs remains a huge challenge.
MSCs are pluripotent progenitor cells of the mesoderm lineage that have rapidly entered the research field of stem cell transplantation for the treatment of SCI owing to their common sources and biological effects (Zhou et al., 2022b). At present, three types of MSCs—human umbilical cord-MSCs, bone marrow MSCs, and adipose-derived MSCs—are frequently used for research. Many MSCs can be obtained from bone marrow, umbilical cord, amnion, placenta, and adipose tissue (Honmou et al., 2011). Although MSCs derived from different tissues have different properties, they perform extremely well in neurological diseases in mouse models (Qu and Zhang, 2017; Volkman and Offen, 2017). For example, MSCs can effectively improve symptoms in animal models of Parkinson’s disease (Glavaski-Joksimovic et al., 2010), amyotrophic lateral sclerosis (Krakora et al., 2013), multiple sclerosis (Dadon-Nachum et al., 2011), and stroke (Zhang et al., 2022). Currently, the therapeutic effect of MSCs is mainly attributed to their secretory function. Anti-inflammatory factors, growth factors, and cell adhesion factors secreted by MSCs can improve the lesion microenvironment after SCI (Pang et al., 2022) and promote self-healing, with the main functions being immune regulation (Li et al., 2019; Zhou et al., 2020b) and neurotrophic and anti-apoptotic effects. MSCs have a high degree of multidirectional differentiation; however, their substitution and differentiation are not significant in SCI. Although MSCs have been tested in the clinic (de Araújo et al., 2022), the data are not optimistic, and the problem of SCI repair still needs to be overcome. Therefore, while it may be beneficial to transplant neural progenitor cells with regenerative potential to the lesion after SCI, the specific type of neural cells that is most effective in this situation remains to be elucidated (Figure 3).
Figure 3: Type of stem cells commonly used in traumatic spinal cord injury transplants.The cells include MSCs, ESCs, iPSCs, NSCs, and HSCs. MSCs consist of BM-MSCs, AD-MSCs, and HUC-MSCs. Transplanted stem cells release neurotrophic factors and form new neurons at the injury site. AD-MSC: Adipose-derived mesenchymal stem cell; BM-MSC: bone marrow mesenchymal stem cell; ESC: embryonic stem cell; HSC: hematopoietic stem cell; HUC-MSC: human umbilical cord-mesenchymal stem cell; iPSC: induced pluripotent stem cell; MSC: mesenchymal stem cell; NSC: neural stem cell.
Autophagy and Stem Cell Function
Autophagy, as a key intracellular quality control and repair mechanism, removes dysfunctional organelles and protein aggregates, maintains stem cell homeostasis, and participates in the self-renewal, differentiation, and reprogramming of cells during development (Li et al., 2022). An increasing number of studies have proven that autophagy is involved in the genesis and development of stem cells, and even plays a role in cell fate decisions.
Autophagy is involved in the degradation of Notch signaling, a classical signaling pathway regulating neural development, to regulate the development and neurogenesis of NSCs (Wu et al., 2016). The establishment of autophagy plays a vital role in the stemness maintenance of stem cells. In stem cell maintenance, senescence is the primary factor that causes cells to lose functions, and oxidative stress is a major contributor to cellular senescence (Wang et al., 2015b). Stem cells can maintain homeostasis by actively reducing oxidative stress, but under normal circumstances, the main source of ROS is the damaged mitochondria in cells, which can be effectively removed by autophagy in stem cells. Thus, it can be said that the fate of stem cells is affected by autophagy regulators (He et al., 2021; Adelipour et al., 2022).
Autophagy is involved in cell reprogramming during early development and the transition period from zygote to embryo (Hanna et al., 2010; Chen et al., 2011; Jopling et al., 2011), and is also involved in vertebrate embryonic development (Lu et al., 2014, 2016; Wang et al., 2015a, 2018). Many studies have investigated the role of autophagy in NSCs. Core autophagy-related genes are expressed in the developing CNS, and knockout of genes such as Atg7 and Atg5 affects embryonic neurogenesis. Blocking autophagy in adult NSCs can lead to defects in adult neurogenesis and astrogenesis (Fleming and Rubinsztein, 2020), and blocking autophagy in vitro and in animal models decreases synaptic plasticity, leading to learning and memory deficits (Jung et al., 2020).
Autophagy is also involved in the differentiation of stem cells. In the early stage of mouse embryonic olfactory bulb development, the autophagy genes Atg7, BECN1, and LC3 were reported to increase in parallel with neuronal markers, indicating that neural stem/progenitor cells meet the energy required for cell remodeling during differentiation by activating autophagy (Vázquez et al., 2012). In an NSC culture model, autophagy increased with the differentiation of NSCs (Ha et al., 2019). Autophagy is not limited to NSCs, but also has recognized roles in MSCs (Deng et al., 2021), embryonic stem cells (Xu et al., 2020; Zhou et al., 2022a), and adult stem cells (Adelipour et al., 2022). For example, chaperone-mediated autophagy can maintain hematopoietic stem cell function (Dong et al., 2021). Chaperone-mediated autophagy is necessary for the control of protein quality in stem cells and the upregulation of fatty acid metabolism when hematopoietic stem cells are activated. Autophagy regulates oligodendrocyte differentiation and myelination to a certain extent (Bankston et al., 2019). Inhibition of the mammalian target of rapamycin (mTOR) pathway by rapamycin in embryonic stem cells significantly reduces the levels of the pluripotent transcription factors Oct4, Sox2, and Nanog, and promotes the expression of markers for mesoderm (Brachyury and MESP1), endoderm (GATA4, GATA6 and Sox17), and so on (Zhou et al., 2009). In an Alzheimer’s disease model, MSCs enhanced autophagy by upregulating the expression of BECN1 (Shin et al., 2014), which promoted the formation of autophagic lysosomes and cleared misfolded proteins. Since autophagy and autophagy-related genes are crucial for the maintenance and differentiation of stem cells, it has been questioned whether they can be combined with stem cells for the treatment of SCI. Regardless of the potential of endogenous stem cells or pretreatment, exogenous transplanted stem cells are stimulated to commit to the repair of SCI, improve the autophagic flux and the microenvironment of SCI, and enhance the survival and functions of stem cells, which may provide an attractive target for stem cell transplantation and repair (Figure 4).
Figure 4: The role of autophagy in the maintenance of stem cell homeostasis.Autophagy is crucial for stem cell proliferation, differentiation, and self-renewal. As a protective mechanism, autophagy also empowers cells to resist external stress.
Function of Autophagy and Transplanted Stem Cells in Spinal Cord Injury
Stem cell renewal and differentiation require strict control of protein turnover in the stem cells to complete the process of cell remodeling. Autophagy, as a highly conserved “gatekeeper” of cell homeostasis, regulates cell morphological remodeling by precisely controlling protein turnover in cells (Qi et al., 2017; Gong et al., 2021; Lv et al., 2021; Ordureau et al., 2021). The mechanism by which autophagy in SCI affects stem cell function is complex and multifactorial. Traumatic SCI induces ROS production, which leads to the accumulation of misfolded proteins, inflammatory factor infiltration, nerve cell apoptosis, and impaired autophagy function leading to inability to clear cellular debris. The resulting complex microenvironment is responsible for an unsatisfactory survival rate and differentiation ability of transplanted stem cells after SCI. Mitophagy—the autophagic removal of damaged or redundant mitochondria—is a key biological process that regulates mitochondria-related antioxidation and inflammation. For NSCs, the maturation of mitophagy is particularly important because NSCs are highly dependent on glycolysis to maintain their own growth and homeostasis (Lange et al., 2016; Zhang et al., 2018a). In addition, the maturation of mitophagy increases the utilization of fatty acids and thus reduces the utilization of glucose (Gong et al., 2015), which is beneficial to the proliferation of NSCs. In a recent SCI study, rosiglitazone was found to regulate the survival and proliferation of transplanted NSCs via the PTEN-induced kinase 1 pathway, which regulates the mitophagy process (Meng et al., 2022). It was established that enhancing autophagy through the serine/threonine kinase AMP-activated protein kinase, mTOR, and transcription factor EB signaling pathway after SCI can inhibit the occurrence of cell death in SCI (Wu et al., 2021). For SCI, the activation of autophagy plays a unique role in reducing tissue damage. Damaged and nonfunctional cytoplasmic organelles must be removed to provide a corresponding environment for the function of stem cells. Targeting Beclin-1 through the delivery of small extracellular vesicles of NSCs enhances autophagy to reduce apoptosis and inflammation caused by SCI (Rong et al., 2019b, 2022). The activation of chaperone-mediated autophagy contributes to the elimination of abnormal intracellular proteins and exerts neuroprotective effects after SCI (Handa et al., 2020).
Overall, the extensive impairment after SCI leads to the loss of cell functions, including some basic cell functions such as autophagy. The loss of basic functional homeostasis in the complex environment generated after SCI results in cells at the injured site being unable to resist adverse pathological factors and experiencing difficulty in maintaining normal functions; this includes stem cells transplanted to the damaged site (Chen et al., 2013; Sotthibundhu et al., 2016; Ho et al., 2017; Wang et al., 2017; Casares-Crespo et al., 2018). Whether through macroautophagy, mitophagy, or chaperone-mediated autophagy, activation of autophagy after SCI is committed to removing numerous damaged cytoplasmic apparatus components, inflammatory factors, and other substances and reducing the occurrence of oxidative stress, which inhibits neuronal apoptosis and promotes the survival of transplanted stem cells. Moreover, autophagy is vital for the maintenance and function of stem cells. In the treatment and repair of SCI, autophagy may play an indispensable role. We can enhance autophagy at the injury site following SCI, encourage the survival of stem cells transplanted into the injury site, and offer a new target for the therapy and repair of SCI by using autophagy as the “gatekeeper” (Table 2 and Figure 5).
Table 2: Stem cells and autophagy in spinal cord injury repair
Figure 5: Protective effect of autophagy activation in SCI on stem cell transplantation.Whether in the form of mitophagy, CMA, or macroautophagy, activation of autophagy promotes the proliferation and differentiation of transplanted stem cells in the SCI microenvironment and reduces neuronal death arising from the poor intracellular microenvironment. AMPK: Adenosine 5′-monophosphate-activated protein kinase; CMA: chaperone-mediated autophagy; HSC70: heat-shock cognate protein 70; LAMP-2A: lysosome-associated membrane protein 2A; mTOR: mammalian target of rapamycin; PINK1: PTEN-induced kinase 1; SCI: spinal cord injury; TFEB: transcription factor EB; ULK: mammalian autophagy-initiating kinase.
Conclusion
Using the biological characteristics of stem cells to treat SCI is currently considered to be a promising treatment. However, it has yet to be determined how to ensure the survival of transplanted stem cells at the damaged site, stimulate the potential of stem cells, and guarantee the differentiation ability of stem cells. Since its discovery, autophagy has been considered a very coordinated cell protection mechanism. It can remove unnecessary or dysfunctional components of cells through orderly degradation and recycling and maintain the steady state of cells under basic conditions. Stem cells have long-term renewal and differentiation abilities. Autophagy also plays an important role in the maintenance and differentiation of stem cells. In the complex microenvironment of SCI, autophagy can remove damaged mitochondria to a certain extent, which reduces the production of ROS and the damage caused by oxidative stress, helping cells resist excessive inflammatory factors. Regulating autophagy may provide a maintenance scheme for stem cell repair, whether in the context of endogenous or exogenous transplanted stem cells after injury. Thus, regulating autophagy and activating the differentiation ability of endogenous stem cells or exogenous transplanted stem cells may provide new therapeutic targets for SCI repair. However, accurate utilization of autophagy characteristics to assist stem cells to function in different stages of SCI has not been fully discussed in this review and needs to be explored further (Figure 6).
Figure 6: Synergistic effect of autophagy regulation and stem cells in the treatment of traumatic SCI.Through activation of autophagy, the production of ROS can be reduced and misfolded proteins can be removed to decrease inflammation. Simultaneously, the steady state of the microenvironment is conducive to the survival, proliferation, and differentiation of stem cells, providing directions for the repair and treatment of traumatic SCI. ER: Endoplasmic reticulum; IL-1β: interleukin-1β; NF-κB: nuclear factor-κB; Nrf2: nuclear factor E2-related factor 2; ROS: reactive oxygen species; SCI: spinal cord injury.
Author contributions:Review design: YS, YPW, XC, XY, GW; data collection and figure preparation: YS; manuscript draft: YPW, XY, GW; manuscript revision: XC, XY, GW. All authors approved the final version of the manuscript.
Conflicts of interest:The authors declare no conflicts of interest.
C-Editor: Zhao M; S-Editors: Yu J, Li CH; L-Editors: Yu J, Song LP; T-Editor: Jia Y
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