Coordinated Actions of Neurogenesis and Gliogenesis in Nerve Injury Repair and Neuroregeneration

Abstract

The failure of endogenous repair mechanisms is a key characteristic of neurological diseases, leading to the inability to restore damaged nerves and resulting in functional impairments. Since the endogenously regenerative capacity of damaged nerves is limited, the enhancement of regenerative potential of quiescent neural stem cells (NSCs) presents as a therapeutic option for neural diseases. Our previous studies have shown exciting progress in treating sciatic nerve injury in mice and rats using NSCs in conjunction with neurotrophic factors such as fibroblast growth factor 1 (FGF1). Additionally, a recently discovered neurotrophic factor, IL12p80, has shown significant therapeutic effects in sciatic nerve injury repair via myelinating oligodendrocytes. IL12p80 induces oligodendrocyte differentiation from NSCs through phosphorylation of Stat3. Therefore, it might be possible to alleviate the myelination defects of oligodendrocytes in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and even schizophrenia through the administration of IL12p80. These applications could shed light on IL12p80 and FGF1, not only in damaged nerve repair, but also in rectifying the oligodendrocytes’ defects in neurodegenerative diseases, such as ALS and MS. Finally, the synergistic effects of neurogenesis-induced FGF1 and myelination-induced IL12 might be able to supplant the need of NSCs for nerve repair and neuroregeneration.

1. Introduction

The nervous system comprises the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS consists of the brain and spinal cord, while the PNS comprises cranial nerves, spinal nerves, and associated ganglia. Neurological injury and diseases, including sciatic nerve injury, spinal cord injury (SCI), stroke, Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Huntington’s disease (HD), cause inconvenience or even severe disability and fatal diseases for millions of people worldwide [1]. The pathogenesis of these diseases is related to various pathological mechanisms, including neuroinflammation [2,3], oxidative stress [4,5], immune dysregulation [6], neurotransmitter disruption [7], synaptic plasticity damage [2,8], and neuroendocrine disorders [5], leading to the gradual loss of neurons and glial cells. Neurological injury and diseases affect memory, thinking, orientation, comprehension, calculation, learning capacity, language, and judgement. The impairment is commonly accompanied or preceded by changes in mood, emotional control, behavior, or motivation. Neurological injury and diseases have physical, psychological, social, and economic impacts, not only for patients, but also for their caretakers, families, and society at large [9]. Under these conditions, the loss of neurons and glial cells highlights the urgent need for effective neuroregeneration strategies. Neurogenesis (the production of new neurons) and gliogenesis (the formation of glial cells) are critical processes for repairing and restoring nervous system function. Therefore, understanding and enhancing these processes are crucial steps for developing treatments for neurological injuries and diseases, with the expectation of at least slowing disease progression and improving the quality of life for patients.

AD is a progressive neurodegenerative disorder with a poor prognosis. AD is associated with the dysregulation of several biological pathways due to genetic, epigenetic, and environmental causes [10]. The mechanisms underlying AD are highly complex and may involve factors such as Aβ, tau, TDP43, and mitochondrial dysfunction [11]. In multiple AD research models, aggregated Aβ damages neurons and disrupts neurotransmission, induces neuroinflammation via IL12 [12], and promotes neurodegeneration [13,14]. Other aggregates like Tau and TDP43 could result in similar damaged neurons in AD/dementia [15,16]. Therefore, the therapy used to prevent or effectively treat AD might be targeting these aggregates using monoclonal antibodies for Aβ, Tau, or TDP43. However, clinical trials using the monoclonal antibodies approach have been discouraging. The most recent trial using lecanemab, although moderately slowing the deterioration, was also associated with adverse events [17]. It is most likely that the neurons in AD brains are irreversibly damaged by the aggregates, thus dissolving or neutralizing the aggregates might not be the most favorable approach.

Neurogenesis persists in the adult mammalian brain throughout life, occurring mainly in two regions, as follows: the subventricular zone (SVZ) of lateral ventricles (LV) and the dentate gyrus [18] of the hippocampus. Although neurogenesis declines in the adult brain, it can be marginally stimulated in response to ischemia and trauma. Since the endogenously regenerative capacity of a damaged brain is limited, the enhancement of its quiescent regenerative potential presents as a therapeutic option for neural diseases. The therapeutic effects of neural stem cells (NSCs) for damaged neurons, such as those caused by cerebral infarction, spinal cord injury, and sciatic nerve injury, have been studied [19]. We have shown that the brain-specific FGF1 gene promoter is active in the SVZ of cerebral ventricles [20,21,22]. Interestingly, the mood stabilizer valproate could activate FGF1 gene promoter through inhibiting histone deacetylase (HDAC) and glycogen synthase kinase 3 (GSK-3) activities [23]. Furthermore, we showed that intranasal delivery of nanoparticle-encapsulated FGF1 can restore cognition, memory, and learning function of the APP-PSEN1 transgenic mice of the AD animal model. This study further elucidates the mechanism, demonstrating that FGF1 activates neuroprotective pathways, including PI3K-CREB-IRE1α/XBP1, to upregulate ADAM10, reduce Aβ burden, and enhance synaptic plasticity. These findings highlight a novel, non-invasive therapeutic approach with potential for treating AD [24]. In this review, we use a monoplegic animal model to evaluate NSCs and neurotrophic factors to promote sciatic nerve injury repair. This model system also allows us to gauge the effectiveness of other treatments, including neurotrophic factors, in neuroregeneration.

Nerve injury repair of the central and peripheral nervous systems poses a major challenge in modern clinics. Grasping a good handle on the neurotrophic proteins and molecular mechanisms that stimulate the regeneration of neurons will not only benefit patients with neural damage, but also could provide an opportunity to potentially treat neural degenerative disorders, including AD, PD, and ALS. The three most common causes of peripheral nerve injury are stretch-related [25], lacerations, and compressions [26]. Sciatic nerve injury serves as a typical example. Sciatic nerve injury results in nerve fiber degeneration and degradation of the surrounding tissues. Nerve degeneration leads to reduced motor and sensory activities, and ultimately sarcopenia. The regeneration of injured peripheral nerves is a multiplex process. When nerve injury occurs, resident Schwann cells are reprogrammed to differentiate into proliferating cells, which could secrete neurotrophic factors and attract macrophages to clear cellular debris. Although the conventional method of peripheral nerve repair involves the use of suturing, it is difficult when axons must traverse more than one coaptation site bridged with nerve grafts in the cases of nerve gap reconstruction. In these situations, alternative devices, such as nerve conduits, have been considered. Nerve conduits provide direct axonal sprouting and mechanical support between the injured nerve stumps. Conduits have been shown to retain neurotrophic factors recruited by or secreted from the damaged cells. They could also prevent the ingrowth of fibrous tissue at the injury site. However, these implanted nerve conduits do not contain Schwann cells and must be repopulated with endogenous Schwann cells migrating from adjacent nerves. Additional neurotrophic factors might facilitate such a recruiting process.

Most neurodegenerative diseases have complex pathogenic mechanisms. Despite extensive ongoing research, effective treatments remain elusive. However, recent studies have suggested that myelin repair could be a novel therapeutic approach. Although we know that myelin is formed by Schwann cells in PNS [27] and oligodendrocytes in the CNS [28], there are still various treatment modalities being proposed. Each Schwann cell forms a myelin sheath around an axon. In contrast, each oligodendrocyte forms multiple sheaths around different axons. Their primary function is to insulate axons from the surrounding environment, support saltatory conduction of nerve impulses, and significantly increase nerve conduction velocity [29,30]. Therefore, the fragmentation of myelin can be observed in SCI [31,32] and neurodegenerative diseases such as AD [33,34], FTD [35,36], PD [37,38], ALS [35,39], MS [40,41], and HD [42,43]. This raises the question of whether myelin repair could be another viable treatment option. However, despite both PNS and CNS myelin being primarily composed of cholesterol, phospholipids, glycolipids, and sphingolipids [44,45,46], specific proteins related to myelinated nerve fibers still exist in either the CNS or PNS [47,48,49,50]. Examples of such specificity include PNS myelin protein zero [51] or periaxin [52], as well as CNS-myelin-associated glycoprotein [53] or claudin-11 [54]. Therefore, understanding how to promote myelin regeneration in different neural systems or diseases remains a worthwhile research topic. At this juncture, NSCs offer a potential solution. By providing specific neurotrophic factors to NSCs, they can differentiate into both Schwann cells and oligodendrocytes, potentially treating neurodegenerative diseases [55,56,57,58]. However, the corresponding mechanisms for this treatment have only just begun to be elucidated. Nevertheless, this approach at least provides an opportunity to slow disease progression and improve patients’ quality of life.

2. FGF1 Protein Is Crucial to NSCs for Self-Renewal and Multipotency In Vitro

Fibroblast growth factor (FGF) has been shown to be essential in culturing embryonic stem cells (ESCs) and NSCs. Among them, acidic FGF, also known as FGF type 1 (FGF1), is the first of the 22 members of the FGF family [59,60,61,62]. All proteins in this family have 30–50% amino acid sequence similarity. Their encoding genes also have a similar exon/intron structure in the protein-coding region of the gene [63,64]. FGF1 is a unique member of the FGF family, which could bind to both IIIb and IIIc variants of FGF receptor 2 to stimulate the growth of epidermal cells and mesenchymal cells, respectively [65]. Consequently, FGF1 is involved in a series of physiological processes, including wound healing, metabolic balance, and neural development [65]. FGF1 is a neurotrophic growth factor that maintains NSCs proliferation and survival, particularly during the process of myelination and axon formation [66,67,68,69,70,71,72,73]. FGF1 is a neural growth factor that maintains neural stem cell proliferation and survival [74,75,76,77,78,79]. FGF1 is mainly expressed in the brain and retina and exerts its effect through high-affinity cell surface receptors, which are distributed densely throughout the nervous system. In peripheral nervous system cell cultures, FGF1 has been shown to promote neuronal regeneration [80] and survival [81]. Within the central nervous system, FGF1 has been shown to stimulate glial mitogenesis [82,83] and to have trophic effects [84,85,86]. The human FGF1 gene spans over 120 kb and has three coding exons and a long 3′ untranslated region [87]. As shown in Figure 1, it also has four alternative 5′ untranslated exons (1A, 1B, 1C, and 1D) that are spliced to the first coding exon [87,88,89]. These exons are regulated by different promoters and confer tissue-specific expression of FGF1 [87,90,91,92,93]. FGF1A is abundant in the kidney, brown adipose tissue, and the heart [90,94]; FGF1B in the brain and retina [20,63,90]; and FGF1C and FGF1D in vascular smooth muscle cells and fibroblasts [95]. FGF1B is the only transcript detected in the central nervous system, and it is enriched in the SVZ, where NSCs reside [94,96].

NSCs are self-renewing, multipotent cells that generate differentiated cell types of the nervous system, including neurons, astrocytes, and oligodendrocytes. Understanding how the identity of NSCs is established and maintained, as well as what regulates their proliferation and differentiation, is vital to realizing their potential in regenerative medicine. Thus, NSCs with self-renewal and multipotent capacities were isolated from human glioblastoma tissues or adult mouse brains via fluorescence-activated cell sorting [96,97,98,99,100]. Flow cytometry and fluorescence-activated cell sorting (FACS) have been widely used to study the lineage complexity of hematopoietic systems and NSC biology, comprising different precursor and progenitor populations from the central and peripheral nervous systems. Following the mouse model, we first characterized the neurosphere-forming capacity of adherent cells isolated from glioblastoma multiforme (GBM) in neurosphere medium. NSCs were isolated from brain tissues using cell-surface markers (such as CD133) or GFP expression driven by NSC-specific promoters [96,101]. These cells were cultured with growth factors and formed neurospheres, indicating self-renewal capabilities. Upon growth factor withdrawal or induction, they differentiated into multiple neural lineages, including neurons, astroglial cells, and oligodendrocytes, demonstrating multipotency. Notably, the 1B promoter of the human FGF1 gene from −540 to +31 (F1B) was conserved in the mouse FGF1 gene, and it could drive the expression of F1B-Tag/F1B-GFP in a subset of NSCs, suggesting the important role of F1B activation in neurosphere formation (Figure 2) [96]. Consequently, this feature allows for the isolation and purification of NSCs with high self-renewal capacity and neural differentiation potential [96,102]. These GFP-positive neural stem/progenitor cells were then used to test for their functionality in repairing the behavioral deficit of rats whose sciatic nerve had been surgically severed [97,103].

3. Functionality of F1B-GFP+ Mouse NSCs and FGF1 in Repairing Sciatic Nerve Injury Results

Schwann cells are crucial for peripheral nerve regeneration after injury [104]. They secrete neurotrophic factors and help to recruit macrophages to clear cellular debris [105,106,107]. In therapeutic applications, nerve conduits could provide direct axonal sprouting and mechanical support to join the stumps of the injured nerve [107]. They also retain neurotrophic factors and prevent fibrous tissue ingrowth at the injured site [107]. Previous studies have shown that implanting NSCs enhances peripheral nerve regeneration. The implanted NSCs could differentiate into Schwann cells, which in turn produce neurotrophic proteins, enrich the microenvironment, and assist in myelination [103,104,108,109]. GFP-positive NSCs were used to repair sciatic nerve transection in rats [103]. In addition, microlithography and solvent casting were utilized to fabricate the micropatterned poly (D, L-lactide) (PLA) nerve conduits. Consequently, the PLA conduits were seeded with the novel adult mouse NSCs that specifically express green fluorescent protein (GFP) [96]. Approximately 85% of the cultured NSCs could be shown to align on the conduits with microgrooves successfully within 72 h. The cultured NSCs were shown to express genes that are relevant to the synthesis of neurotrophic proteins. To study the role of adult NSCs in peripheral nerve regeneration, we performed the following steps under sterile conditions: First, we used microscissors to excise a 10 mm segment of the sciatic nerve in rats’ left hind legs. Next, the PLA nerve conduits with microgrooves that were seeded with the mouse NSCs (Figure 3, Top Left) or without NSCs (Figure 3, Bottom Left) were inserted into the 10 mm nerve gap. The proximal nerve was secured within the conduit using 7-0 nylon sutures. The distal end was then sutured to the other end of the conduit. Both nerve stumps were sutured into the conduit, with a length of approximately 1 mm. Finally, we layered the wound using 3-0 Dexon sutures. The observations in the first week showed footprints of monoplegic rats (Figure 3). In rats transplanted with NSCs (Figure 3, Top Left), the footprints of the left foot are noticeably more prominent compared to those of the rats without NSC transplantation (Figure 3, Bottom Left). After four weeks, the footprints of the left foot with NSCs (Figure 3, Top Right) compared to the group without NSC transplantation (Figure 3, Bottom Right) became clearly visible, with noticeable toe extension and no signs of atrophy. Thus, nerve repair was facilitated, and functional recovery was improved in the group with NSCs. As shown in Figure 3, the results indicated that the F1B-GFP transfected cells not only have the self-renewal capacity of NSCs in vitro, but are also capable of repairing a transected sciatic nerve in rats [97].

It was shown that the combinatorial treatment of FGF1 and peripheral nerve grafts could restore hind limb function in the monoplegic adult rats [111]. It was also reported that similar treatments benefited the patients with common peroneal nerve lesions as well [112]. We have developed a series of patented technologies in which NSCs could be isolated as GFP-positive cells when adult mouse brain cells were transfected with the F1B-GFP plasmid. This F1B-GFP plasmid is composed of the GFP-coding sequences whose expression is driven by the human FGF1 promoter [113]. We showed that FGF1 and NSCs achieved better sciatic nerve injury repair than either one alone. In

Table 1. Regenerative effects of FGF1 in sciatic nerve injury repair in rats.

Regeneration Parameters	Conduit	Conduit+FGF1
SFI	−56.0 ± 4.9	−37.1 ± 1.5
No. of myelinated axons	1704 ± 14	3909 ± 136
Regeneration area (mm2)	10.3 ± 0.1	28.2 ± 1.9
No. of blood vessels	10.3 ± 0.1	28.2 ± 1.9
Regeneration area (mm2)	11.8 ± 0.1	36.2 ± 0.2
Muscle action potential (mV)	0.40 ± 0.05	1.01 ± 0.03
Nerve conduction velocity (m/s)	32.7 ± 1.1	36.5 ± 2.3

, we show that FGF1 alone, even in the absence of NSCs, exhibited significant improvement of sciatic nerve injury repair.

An air plasma treatment was employed to graft FGF1 onto the PLA nerve conduits (Cn) with designed micropores and surface microgrooves [97]. As noted in Figure 4, the best result was achieved from the group of Cn+FGF1+NSCs. Interestingly, only the conduit group of mice did not recover well. Furthermore, the Cn+NSCs group had a dramatic recovery during the period from 7 to 12 weeks, approaching those of Cn+FGF1+NSCs. The lag of the sciatic functional index (SFI) in the Cn+NSCs group versus the Cn+FGF1 and Cn+FGF1+NSCs groups during the 4th–8th weeks and the rapid rise subsequently suggested that additional paracrine factor(s), other than FGF1, could be secreted by the NSCs in the conduits. Moreover, it has been revealed that the transplanted NSCs could differentiate into myelinating Schwann cells and secrete neurotrophic proteins. These actions thereby enrich the microenvironment and assist in myelination, which might result in the increase in muscle action potential and nerve conduction velocity (Table 1). These observations imply that aligned NSCs in the conduits might either recruit neurotrophic factors or synthesize neurotrophic factors per se, and thereby contribute to the nerve repair observed during the 4th–8th weeks post-implant and speed up nerve regeneration during the 8th–12th weeks.

4. Mouse and Human IL12p80 Proteins Could Promote Nerve Regeneration Through Differentiation of NSCs Toward Oligodendrocytes and Myelinating Schwann Cells

To explore the nature of cytokines or growth factors that are involved in this process, the proteomic array was performed on mice [114]. Similar results were obtained using a 0.3 cm gap of sciatic nerve injury in mice and sutured with a 0.5 cm (length) × 0.1 cm (diameter) PLA conduit. The Conduit+NSCs group increased the expression levels of IL12p40 but not IL12p35. The levels of IL12p40 in these conduits were 1.6-fold higher than those in conduits without NSCs. This observation indicated that IL12p80, the IL12p40 bioactive homodimeric form, may be a crucial factor that existed in the Conduit+NSCs group involved in nerve regeneration.

IL12 is a cytokine with multifunctional activities that is produced by dendritic cells and macrophages naturally [115]. IL12p70 was originally reported as a protein of heterodimer, which comprises p35 and p40 subunits that are joined together using disulfide bonds. Homodimeric IL12p80, comprising two subunits of p40 monomers, can also be detected in rare situations. IL12p70, IL12p80, and the monomeric IL12p40 are all glycosylated proteins that could be secreted extracellularly. IL12p70 is associated with inflammatory responses, while IL12p80 is crucial in anti-inflammatory responses [116,117,118,119,120]. IL12p40 is the ligand for IL12 receptor β1 (IL12Rβ1) [121]. The affinity of IL12p40 to IL12Rβ1 could result in antagonizing effects of IL12p70-binding in both humans and mice [119,122,123,124]. IL12 was secreted by macrophages, during the Wallerian degeneration, for a process of axonal regeneration. It was also reported that IL12p40 mRNA expression could be detected between days 7 and 14 of Wallerian degeneration [125,126,127].

In the sciatic nerve injury mouse model, the implantation of NSCs combined with nerve conduit and IL12p80 improved motor recovery and increased the diameter of the regenerated nerve up to 4.5-fold at the medial site (Figure 5). In NSCs, Stat3 phosphorylation has been shown to participate in astroglial differentiation processes [128]. Stat3 protein can be activated by a variety of growth factors or cytokines. It plays a crucial role in various physiological activities, including inflammation, immune response, cell proliferation, differentiation, and apoptosis [129]. However, its role in oligodendrocyte differentiation has not previously been reported [130]. In T cells, the IL12p40 subunit can bind to IL12Rβ1 and then induce Stat3 phosphorylation and the downstream signaling pathway [114]. Furthermore, in vitro studies have also revealed that IL12p80 stimulates the oligodendrocyte/Schwann cells differentiation of mouse NSCs through the phosphorylation of Stat3 in vitro. These results suggest that IL12p80 can trigger oligodendroglia differentiation of mouse NSCs through phosphorylation of Stat3 and improve the functional performance via the increase in regenerated sciatic nerves in a nerve injury mouse model [114].

The differentiated cells could secrete neurotrophic factors per se or create a microenvironment to enrich neurotrophic factors from milieu, and, in turn, assist in myelination [108,109,131]. The unique characteristic of oligodendrocytes is that they produce myelin, a multilayered and lipid-rich sheath that covers and insulates neuronal axons, enhancing the speed of transmission of electrical signals and providing metabolic support to neurons. Impairments in the metabolic support of oligodendrocytes contribute to disease onset and progression [132]. To pave the way for clinical trials, we repeated the experiments with human IL12p80. As shown in Figure 6, we demonstrated that the treatment with human IL12p80 could generate more PZO (marker for myelinating Schwann cells) staining, consistent with the notion that IL12 could enhance oligodendrocyte differentiation and thereby improve neuroregeneration [133].

5. Coordination of Neurogenesis and Myelinogenesis Might Be Required for Efficient Neuroregeneration

The interaction of cytokines and growth factors that are involved in neuroregeneration remains incompletely understood. The molecular mechanism for Schwann cell differentiation of the implanted NSCs into newly regenerated axons is also not well established. When the transcription factor Sox9 is ablated from NSCs, knockout mice develop deficiencies in differentiation into astrocytes, accompanied by defects in glial lineage cells in the central nervous system. This is coupled with a dramatic decrease in early progenitor cells and a transient increase in myelin-forming oligodendrocytes and motor neurons, indicating the contribution of other cooperating proteins. However, the loss of both Sox9 and Sox10 transcription factors leads to a further reduction in oligodendrocyte progenitor cells. Astroglial cell numbers are also severely reduced in the absence of Sox9 and do not recover at the later stages of spinal cord development. Thus, Sox9 is shown to be a major molecular component of the neuron–glia switch during spinal cord development [134]. In this context, exploration of whether the enhanced nerve repair can differentiate into myelinating Schwann cells (facilitated by IL12) and neurons (facilitated by FGF1) through the ability of implanted NSCs is crucial not only in the repair of damaged nerve cells, but also in the field of neurodegenerative diseases.

In recent years, research on the treatment of AD has demonstrated the efficacy of stem cell transplantation in promoting myelin regeneration in both in vitro and in vivo models [135,136,137,138,139]. Neural stem cell transplantation has been shown to effectively replace defective myelin while also promoting the remyelination of axons [140]. Experimental evidence further suggests that, when transplanted into mice with myelin formation disorders, human neural stem cells can differentiate into oligodendrocytes and facilitate functional myelin formation [141,142]. However, it is important to note that in AD, ALS, MS, and other CNS diseases, ongoing inflammatory responses in the lesions or areas of neuronal damage may reduce the effectiveness of NSC transplantation, as the differentiated cells might be compromised by the inflammatory environment. This underscores the need for anti-inflammatory strategies, including treatment with IL12p80, to support the survival and function of transplanted cells. Therefore, utilizing NSCs in conjunction with FGF1 or IL12p80 for promoting myelin repair may also offer valuable intervention strategies for AD. ALS is known as a rare neurological disease that affects motor neurons—those nerve cells in the brain and spinal cord that control voluntary muscle movement [143]. This neurological disease is typically characterized by the loss of motor neurons, leading to progressive muscle wasting and eventual death. This complex multifactorial disorder involves anomalies of astrocytes, oligodendrocytes, and microglia [144]. Importantly, all ALS oligodendrocyte lines tested could induce motor neuron death through conditioned media and in co-culture [145]. The accumulating data established that the contribution of non-neuronal cells to the disease is a primary event, and ALS pathogenesis is driven by both cell-autonomous and non-cell-autonomous mechanisms [146]. Given the inflammatory responses in ALS lesions, additional interventions to modulate inflammation may be necessary to enhance the effectiveness of NSC transplantation. Through our own studies, we have learned that neuroregeneration will require the rejuvenation of oligodendrocytes [114,133]. Our demonstration that IL12p80-enhanced oligodendrocyte differentiation contributes to the enhanced axon growth in sciatic nerve injury up to 4.5-fold further buttresses this notion [114,147]. As motor neurons degenerate and die, they stop transmitting messages to the muscles, causing them to weaken, start to twitch (fasciculations), and waste away [148]. Eventually, the brain loses its ability to initiate and control voluntary movements, causing difficulties in speech, swallowing, and breathing, and, ultimately, death. The causes of most ALS cases are still largely unknown. However, it is argued that the defective oligodendrocytes in ALS patients contribute to the loss of motor neurons.

In contrast, MS is a potentially disabling disease of the brain and spinal cord. It is thought to be an autoimmune disorder, a condition in which the body attacks itself for unknown reasons. The proposed causes for this include genetics and environmental factors, such as viral infections. In MS, the immune system attacks the protective myelin that covers nerve fibers and causes communication problems between the brain and the rest of the body. This loss of myelin forms scar tissue called sclerosis. These areas are also called plaques or lesions. When the nerves are damaged in this way, they cannot conduct electrical impulses to and from the brain. MS is an unpredictable disease that affects people differently. Some people with MS may have only mild symptoms. Some people with severe MS may lose the ability to walk independently. Other individuals may experience long periods of remission without any new symptoms, depending on the type of MS they have.

Induced pluripotent stem cell (iPSC) lines from ALS patients have been established, and the isogenic, reverted wildtype iPSC lines were generated using CRISPR/Cas9 [148,149]. With the availability of iPSC lines from ALS patients, it is possible to co-culture the iPSC-derived neurons with iPSC-derived oligodendrocytes. Interestingly, when co-culturing iPSC-derived neurons from ALS patients with iPSC-derived oligodendrocytes from healthy individuals, there is no manifestation of defects in the neurons. On the contrary, when co-culturing iPSC-derived neurons from healthy individuals with iPSC-derived oligodendrocytes from ALS patients, the defective neuronal phenotypes of typical ALS neurons were manifested. This observation allowed the speculation that the defects observed in the ALS patients’ neurons are likely the result of the incapacity of the oligodendrocytes to protect neurons.

ALS and MS exhibit varying compositions of myelin sheaths in both central and peripheral nerves [150]. Currently, extensive research is ongoing regarding stem-cell-based therapies. Among these, NSCs possess the remarkable ability to differentiate into neurons, astrocytes, and oligodendrocyte progenitor cells. Stem cells can protect damaged or remaining neurons from oxidative stress, apoptosis, and inflammatory injury by secreting brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), and insulin-like growth factor-1 (IGF-1) [151,152,153,154,155]. Additionally, stem cells can replace dysfunctional or deceased cells by differentiating into neurons or glial cells, thereby restoring the structural and functional integrity of neural networks. Furthermore, stem cells modulate immune responses by influencing the activation, differentiation, migration, and secretion of immune cells, suppressing neuroinflammation and mitigating immune-mediated damage in neurodegenerative diseases [152,153,156,157]. Therefore, the discovery of IL12p80 neurotrophic factor may offer an additional avenue that complements existing stem cell therapies, providing a novel possibility. To recapitulate the cytopathies of ALS patients’ motor neurons, iPSC lines from patients with the SOD1^G85R mutation were established. The isogenic SOD1^G85G revertant with a corrected protein sequence was also generated through CRSPR/CAS9 techniques. These iPSC lines could be differentiated toward motor neurons [148]. It was further demonstrated that SOD1^G85R motor neurons recapitulated ALS-specific nerve fiber aggregates. Neurotransmitter-induced calcium hyper-response and neurite degenerations were also manifested. Moreover, coactivation of the GSK3β and IGF-1 pathways was a mechanism involved in the therapeutic effects of ALS through the reduction in nerve fiber cytopathies in ALS motor neurons. It is demonstrated that oligodendrocytes derived from ALS samples could induce motor neuron death via distinct mechanisms of toxicity modulated by soluble factors and cell-to-cell contact, even though no sign of oligodendrocyte degeneration can be detected [145]. It was reported that Sox10 can induce the differentiation of iPSC lines to oligodendrocytes [158]. The IL12p80 homodimer may rectify the defective ALS oligodendrocytes’ protective effect on neurons using a scheme as devised [132], such that the co-culture of ALS oligodendrocytes and wildtype neurons will protect the neurons from exhibiting cytopathies. This is an innovative approach that could incorporate two cutting-edge accomplishments, as follows: (1) the discovery of the myelinogenic IL12p80 protein and (2) the available ALS-iPSC lines and reverted isogenic iPSC lines.

Although the therapeutic role of GSK3β activation on ALS motor neurons in this study was different from the role previously reported in research using cell-line- or transgenic animal-based models, it is possible that in vitro ALS-specific nerve fiber and neurofunctional markers would be useful for exploring effective therapeutic drugs. More importantly, this iPSC-based model could reveal thus-far unknown therapeutic mechanisms [149] that may serve as potential targets for ALS therapy. Separately, microglia play key roles in neurogenesis and synaptic remodeling. Therefore, their disease-relevant cell models are key to the success of neuroimmune and neurodegeneration research. Precision reprogrammed microglial models developed from human iPSC lines offer a robust solution to accelerate research, providing important new insight into human neurological disease [159,160,161]. As we have identified IL12p80 as a differentiation factor for oligodendrocyte differentiation, this discovery has paved the way for its role in therapeutic approaches for not only neuronal diseases, but also oligodendrocyte diseases.

6. Discussion

Neural injuries and diseases, including sciatic nerve injury, SCI, stroke, AD, FTD, PD, ALS, MS, and HD, are generally complex in terms of their pathogenesis. The deficiency of endogenous repair mechanisms is a key characteristic of neurological diseases, leading to the inability to restore damaged nerves and resulting in functional impairments. However, treatment approaches continue to evolve and improve. The discovery of stem cells has provided new therapeutic options for these conditions. Currently, we know that both ESCs [162,163] and iPSCs [164] possess self-renewal capabilities. These non-specialized, immature cells can generate nearly identical copies of themselves over an extended period without differentiation. Moreover, they have the potential to differentiate into various cell lineages. While ESCs are well-suited for cell replacement therapy, due to their unlimited proliferation capacity, they come with risks such as teratoma formation, immune rejection, and ethical concerns. The use of derivatives from iPSCs (such as midbrain dopaminergic progenitors [165,166,167]) may continue to raise concerns about safety and feasibility. Therefore, the feasibility and safety of applying NSCs combined with neurotrophic factors for treating these neurodegenerative diseases have been improved. Currently, NSCs have demonstrated efficacy in treating various preclinical neurodegeneration models (such as ALS [168] and PD [169]) and in developing CNS-related cell types [170]).

NSCs could differentiate into various types of neural cells, including neurons, astrocytes, and oligodendrocytes. As a result, NSCs functionally contribute to neuronal connections in specific brain regions through neurogenesis, whereas gliogenesis ensures myelination by generating new oligodendrocytes and astrocytes [171]. Based on these characteristics, NSCs play a significant role in nerve repair and neuroregeneration. In many pathological conditions affecting the nervous system, alterations occur in neurogenesis and gliogenesis, such as in neuropsychiatric diseases [172], neurodegenerative diseases [173,174,175,176], and demyelinating disorders [177]. Understanding the extent to which adult neurogenesis and gliogenesis can be regulated to compensate for functional losses has garnered attention. This article focuses on exploring the potential of differentiating NSCs into neurons or oligodendrocytes to repair or enhance myelin formation, thereby delaying the progression of the aforementioned diseases. It was reported that FGF1 has neurite-promoting effects in vitro [178]. FGF1 not only promotes neurite outgrowth, but also encourages the survival of various types of central and peripheral neurons in vivo [80,179]. As a powerful modulator of biological functions, FGF1 mainly promotes neuroprotection and neurogenesis through the activation of the MAPK/ERK and PI3K/AKT signaling pathways [180,181], which are crucial for neuron survival and differentiation. Because FGF1 is such a powerful regulator of biological functions, its presence in tissues is highly regulated in both time and space. FGF1 prevents oligodendrocyte death [182], promotes remyelination [183], and directly accelerates myelin sheath formation [183]. FGF1 has also been studied in other neurodegenerative disease treatments. For example, in an AD model, FGF1 has been shown to enhance hippocampal neurogenesis, potentially alleviating cognitive decline [181]. Its anti-inflammatory properties also help to create a favorable environment for neuronal growth and function. In PD, FGF1 supports the survival of dopaminergic neurons and reduces the accumulation of α-synuclein aggregates [180]. In ALS research, FGF1 has been found to promote NSCs differentiation into oligodendrocytes, aiding motor neuron survival and enhancing myelination. This helps to maintain motor function and slow disease progression. However, FGF1 itself has cell-proliferation-promoting properties, raising concerns about tumorigenesis with prolonged use [184,185,186]. Therefore, we propose combining it with IL12p80 to mitigate these risks and concerns.

On the other hand, using ciliary neurotrophic factor (CNTF) and thyroid hormones (T3) to treat NSCs that differentiate into oligodendrocytes serves as a positive control group [96]. IL12p80 stimulates NSCs differentiation into oligodendrocytes, leading to increased levels of Sox10 and Olig1 proteins and decreased levels of Sox2 protein. This response is similar to the differentiation of oligodendrocytes from NSCs induced by CNTF+T3. Sox10 is primarily expressed by oligodendrocyte precursor cells and mature oligodendrocytes, regulating their maturation [187]. The synergistic action of Sox10 and Olig1 induces the expression of myelin basic protein. Therefore, IL12p80 can promote NSCs differentiation into oligodendrocytes, potentially contributing to neural repair or regeneration

Based on the results summarized in this review, FGF1 has been shown to be crucial for the proliferation of ESCs and NSCs. FGF1 is primarily expressed in the brain and retina, and the FGF1B mRNA has been identified as the sole transcript detected in the human CNS and enriched in the SVZ where NSCs reside. In the CNS of mice, the distribution of FGF1 is entirely explained by the FGF1B transcript [188,189]. FGF1B expression was detected in the trigeminal ganglia at E16, the superior ganglia at E18, and the trigeminal motor nucleus and facial nucleus in the postnatal mouse brain [188]. Using transgenic mice with the SV40 T antigen (Tag) reporter gene controlled by the F1B promoter, we found that the F1B promoter was active in the ventral tegmental area [76,190]. Further sequence analysis revealed 76.2% sequence homology between the human and mouse FGF1 1B promoter sequences. F1B-Tag brain cells from F1B-Tag transgenic mice expressed features of neural stem cells, including self-renewal. Additionally, using the F1B-GFP reporter gene, we isolated F1B-GFP(+) cells from the adult mouse brain, and these cells exhibited significantly higher neurosphere formation efficiency than the F1B-GFP(−) cells. In summary, FGF1 appears to be useful not only for culturing mature neuronal cells [191,192,193,194], but also for isolating another subset of NSCs from the embryonic mouse brain. Additionally, brain cells from F1B-Tag transgenic mice exhibit self-renewal and pluripotency. In a rat model of sciatic nerve injury, the animals showed significant improvement in recovery when NSCs were present in nerve conduits after four weeks. Furthermore, in a mouse model of sciatic nerve injury, combining nerve conduits with IL12p80 and NSCs improved motor recovery and increased the diameter of the regenerated nerves by 4.5 times. IL12p80 was found to trigger the differentiation of mouse NSCs into oligodendrocytes/Schwann cells via the phosphorylation of Stat3, enhancing functional performance in the nerve-injured mouse model.

Now that we have established that either FGF1 or IL12 alone could enhance the nerve regeneration in the sciatic nerve injury models in mice and rats, we further suggest that these neurotrophic factors might be applicable in neurodegenerative disorders. These neurodegenerative disorders are incurred not only as a consequence of the damaged neurons per se, but also could be due to a defect of the myelinating oligodendrocytes or an inflammatory attack from microglial cells. Here, we argue the possible applicability of FGF1 and IL12, using ALS as a specific example. ALS is known as a rare neurological disease that affects motor neurons [143]. This progressively degenerative disease is typically characterized by the loss of motor neurons leading to gradual muscle weakening and eventual death. This complex multifactorial disorder involves anomalies of astrocytes, oligodendrocytes, and microglia [144]. Importantly, all ALS oligodendrocyte lines tested could induce motor neuron death through conditioned media and in co-culture [145]. The established data suggested that non-neuronal cells, oligodendrocytes, or microglial cells contribute to neurodegeneration as a crucial event. Furthermore, the pathogenesis of ALS is a consequence driven by both cell-autonomous and non-cell-autonomous events [146]. Through the findings from our own lab, we learned that neuroregeneration requires the rejuvenation of oligodendrocytes [114]. Our demonstration that IL12p80-enhanced oligodendrocyte differentiation contributes to the significant enhancement of axonal growth in sciatic nerve injury further buttresses this concept [114,147]. When motor neurons degenerate and die due to demyelination, the muscles cease to receive messages transmitted from neurons, causing the muscles to weaken and eventually waste away [148]. Ultimately, the brain loses its ability to initiate and control voluntary movements, causing difficulties in speech, swallowing, and breathing, leading to eventual death. It is argued that the defective oligodendrocytes and malfunctioning myelination in the ALS patients contribute to the loss of motor neurons. In this aspect, our discovery that IL12p80 could facilitate nerve repair through efficient myelination could certainly be extended to neurodegenerative diseases.

With the availability of iPSC lines from ALS patients in our own laboratory [195], it is now possible to co-culture the iPSC-derived neurons with iPSC-derived oligodendrocytes. Importantly, when co-culturing iPSC-derived neurons from ALS patients with iPSC-derived oligodendrocytes from healthy individuals, there is no manifestation of defects in the neurons. On the contrary, when co-culturing iPSC-derived neurons from healthy individuals with iPSC-derived oligodendrocytes from ALS patients, the defective neuronal phenotypes of typical ALS neurons are manifested. This observation further supports the hypothesis that the defects observed in the ALS patients’ neurons are likely the result of the ineffectiveness of the oligodendrocytes to protect neurons, due to the fragmentation of myeline.

7. Conclusions and Perspective

Based on the results summarized here, the efficiency of myelination could stabilize the function of regenerating nerves in the central and peripheral nervous systems. The synergistic effects of IL12p80 and FGF1 in promoting neural repair, regeneration, and facilitating motor function recovery might serve as a foundation for future clinical applications. Notably, IL12p80, beyond its anti-inflammatory properties, can also enhance oligodendrocyte differentiation. Therefore, for neurodegenerative diseases caused by oligodendrocyte defects, such as ALS, MS, and even schizophrenia, administering NSCs+IL12p80 may potentially alleviate myelination defects in oligodendrocytes. Further investigations could explore whether it has an inhibitory effect on chronic inflammation during the progression of ALS and MS. According to recent research, IL12p80 has been confirmed as a novel neurotrophic factor [114,133]. The combinatorial usage of FGF1 and IL12, which might generate synergistic effects, might even supplant the need of NSCs for nerve repair and neuroregeneration.

In conclusion, the combination of NSCs with neurotrophic factors like FGF1 and IL12p80 represents a promising strategy for promoting nerve repair and regeneration in the CNS. This approach has the potential to address the critical need for effective treatments for neurodegenerative disorders. However, significant challenges remain, and further research is needed to fully realize the therapeutic potential of these strategies. By advancing our understanding of the mechanisms of neurogenesis and gliogenesis and developing optimized delivery methods, we can pave the way for innovative therapeutic strategies and improved clinical outcomes for patients suffering from neurodegenerative conditions.