Regenerative Medicine for Spinal Cord Injury Using Induced Pluripotent Stem Cells

Abstract

Spinal cord injury (SCI) is a devastating injury that causes permanent neurological dysfunction. To develop a new treatment strategy for SCI, a clinical trial of transplantation of human-induced pluripotent stem cell-derived neural precursor cells (NPCs) in patients in the subacute phase of SCI was recently initiated. The formation of synaptic connections with host neural tissues is one of the therapeutic mechanisms of cell transplantation, and this beneficial efficacy has been directly demonstrated using a chemogenetic tool. This research focuses on the establishment of cell therapy for chronic SCI, which is more challenging owing to cavity and scar formation. Thus, neurogenic NPC transplantation is more effective in forming functional synapses with the host neurons. Furthermore, combinatory rehabilitation therapy is useful to enhance the efficacy of this strategy, and a valid rehabilitative training program has been established for SCI animal models that received NPC transplantation in the chronic phase. Therefore, the use of regenerative medicine for chronic SCI is expected to increase.

Spinal cord injury (SCI) is a devastating injury that causes a sudden onset of neurological dysfunction. A survey conducted in 2018 reported that the incidence of traumatic SCI was 49 per million in Japan¹⁾. Moreover, SCI was more common in young patients due to their involvement in high-energy accidents and contact sports. However, elderly patients can easily injure their spinal cord; moreover, cervical canal stenosis in elderly patients is increasing in the aging Japanese society. In fact, individuals in their 70s currently comprise the largest age group among SCI patients, and the most frequent cause of the injury is fall on level surface¹⁾.Currently, surgical intervention and subsequent rehabilitation are the only treatment options for SCI. Methylprednisolone is used in the acute phase of injury; however, a therapeutic consensus has not been reached regarding its safety and effectiveness^2,3).

Recent developments in stem cell biology have the potential for addressing the lack of treatment strategies for SCI. Many preclinical studies have demonstrated the effectiveness of neural precursor cell (NPC) transplantation in SCI^4-10). A culture method was recently established to generate human-induced pluripotent stem cells (iPSCs) into NPCs^11-13). Furthermore, transplantation of these cells into the injured spinal cord of animal models resulted in functional recovery^13,14). These achievements from basic research led to an ongoing clinical trial for the transplantation of NPCs to SCI patients in the acute phase¹²⁾.

This review introduces recent studies that demonstrated the mechanisms of functional recovery of the injured spinal cord after NPC transplantation and provides an overview of current research into the use of regenerative medicine as a viable treatment option for more challenging chronic phase of SCI.

Mechanisms Underlying the Efficacy of iPSC-derived NPC Transplantation

Several studies demonstrated the efficacy of NPC transplantation in SCI treatment and reported several mechanisms underlying the observed functional recovery^5,15-20). The first is the secretion of neurotrophic factors by the grafted NPCs to attenuate secondary damage, consistent with the evidence of early functional improvement before the transplanted cells become fully mature^19,20). The second is the differentiation of transplanted NPCs into neurons that establish synaptic connections with host axons^16,17). Finally, transplants have been reported to generate oligodendrocytes and myelinate host axons by the transplants to recover saltatory conduction along neuronal axons^5,18).

The iPSC-derived NPCs, generated by Nori et al. and Kobayashi et al., presented characteristics of approximately 50%-70% neurogenic differentiation. Furthermore, histological analysis revealed that graft-derived neurons made synaptic connection to the host neurons, but there was no direct evidence regarding the formation of a functional synapse linked to the locomotor behavior^13,14). Mechanisms underlying this functional recovery after cell transplantation were elucidated using the designer receptor exclusively activated by designer drugs (DREADD) system²¹⁾. DREADD is a chemogenetic engineered protein that permits control of neuronal activity via administration of the ligand clozapine N-oxide (CNO). This system has been used to evaluate the therapeutic efficacy on neuronal activity in graft-derived cells (Fig. 1)^22,23). Initially, an inhibitory DREADD receptor (hM4Di) was introduced to the NPCs using a lentiviral vector, and these NPCs were then transplanted into the injured spinal cords of mice²²⁾. During administration of CNO, the locomotor function of host animals temporarily declined without any adverse effect. The results of loss-of-function experiments directly demonstrated the contribution of graft neuronal activity to locomotor function recovery.

Figure 1.

Neuronal activity control using the DREADD system.

Using genetically engineered receptors, it is possible to transiently regulate neuronal activity in transplanted NPCs in a ligand (CNO)-dependent manner. Furthermore, hM4Di and hM3Dq inhibits neuronal and excitatory activities, respectively. These receptors were introduced into NPCs before transplantation, and motor function changes were evaluated by administering CNO to model animals. DREADD, Designer Receptors Exclusively Activated by Designer Drugs; CNO, clozapine N-oxide; NPCs, neural precursor cells

Conversely, how transplanted cells with enhanced neuronal activity affected the injured spinal cord was also examined²³⁾ by introducing excitatory DREADD receptor (hM3Dq) to the NPCs, using similar methodology as described above, and continuous CNO administration for 6 weeks after transplantation (Fig. 1). This consecutive and selective chemogenetic stimulation of transplanted NPCs enhanced the expression of synapse-related genes and proteins in the surrounding host tissues. Furthermore, CNO treatment prevented the atrophy of injured spinal cord and improved locomotor function. This result emphasizes the significance of enhanced neuronal activity in transplanted cells, which directly promoted synapse formation with host neurons, and provides a strategy for enhancing graft activity to improve the efficacy of cell transplantation therapy for SCI.

Most previous studies using the DREADD system have focused on the neural activity of transplanted cells. However, Ago et al. explored the activity of the host spinal tract²⁴⁾ by introducing the excitatory DREADD system into the corticospinal tract of host animals; however, stimulation of this tract enhanced the neuronal activity of the transplanted cells via the synapses that formed with the host tissues. This study used the bioluminescence imaging system composed of AkaLumine-HCl and Akaluc (AkaBLI), a novel technology that produces bright emission spectra and enables deep tissue imaging in living animals²⁵⁾, for visualization of neuronal activity and noninvasive monitoring of neuronal activity-dependent gene expression from graft cells in injured spinal cords.

Along with neuronal differentiation and synaptic formation after transplantation, NPC-derived oligodendrocytes play an important role in restoring saltatory conduction and in locomotor recovery. Kamata et al. established gliogenic NPCs from clinical-grade human iPSCs and transplanted in the subacute phase of SCI²⁶⁾. Approximately 40% of the grafted cells differentiated into oligodendrocytes, which myelinated the host demyelinated axons. These favorable histological results contributed to the electrophysiological restoration of neural conduction, and consequently, the locomotor ability.

A unique study used directly reprogrammed human NPCs (drNPCs), produced from bone marrow somatic cells via transient transfection of the factors Musashi-1, neurogenin-2, and methyl-CpG binding domain protein²⁷⁾. In this induction method, the cells did not pass through the pluripotent status, as iPSCs do, and could reduce the duration of NPC production and enhance the efficiency of the reprogramming process. A protocol has also been established to generate cells with an oligodendrogenic fate from drNPCs transplanted into a rat SCI model in the subacute phase. Approximately 30% of oligogenic NPCs survive at the lesion site, with myelinated host axons and spared white matter areas. These beneficial effects contributed to functional locomotor recovery without tumorigenicity.

Clinical Trial for Using iPSC-derived NPCs in SCI Treatment

Based on the promising results observed in the studies on cell transplantation therapy, Sugai et al. conducted a clinical study using human iPSC-NPCs in SCI patients^12,28). For the clinical application, researchers collaborated with the Center for iPS Cell Research and Application (CiRA) at Kyoto University, which has established clinical-grade, integration-free human iPSC lines²⁹⁾. NPCs were successfully generated from the iPSCs obtained from CiRA, and their characteristics and quality were thoroughly evaluated using various endpoints, including marker expression and in vitro functional, genomic, and safety analyses^30,31). Moreover, an in vivo study involving the transplantation of cells into animal models of SCI has been conducted to assess functional recovery¹²⁾ To address the safety concerns, Sugai et al. grafted the cells into rodent CNS, and the animals were observed to evaluate tumor formation over a period of 3 months after transplantation¹²⁾ After clearing all subitems, the Certified Special Committee for Regenerative Medicine approved the trial at our institution in November 2018.

In December 2021, our group conducted the first in-human iPSC-derived NPC transplantation in patients with SCI in the subacute phase (Fig. 2). The protocol of this clinical study was designed to target SCI patients with American Spinal Injury Association impairment scale A and to transplant 2 × 10⁶ iPSC-NPCs at the lesion epicenter within 14 to 28 days after injury. The cells were manually delivered under a surgical microscope using a Neuros syringe (Hamilton Company, USA) and a needle by a spine surgeon. This study enrolled four patients and provide 1 year of follow-up with appropriate neurological and imaging evaluations and rehabilitation²⁸⁾.

Figure 2.

Overview of first-in-man clinical trial for the treatment of SCI using human iPSC-derived NPCs.

SCI, spinal cord injury; iPSC, induced pluripotent stem cell; NPC, neural precursor cells

In an actual clinical setting, autologous cell transplantation is ideal for preventing immune rejection. However, this approach is prohibitively expensive as it requires a considerable number of trials and quality assessment of iPSC-NPCs before transplantation. Therefore, allogeneic grafting is currently conducted using iPSC stocks^28,29,32). Although allogeneic transplantation causes immunological issues, Ozaki et al. performed a combination of lymphocyte reaction (MLR) assay to evaluate the importance of human leukocyte antigen (HLA) matching in-human iPSC-NPC transplantation³³⁾ In the study, when HLA-mismatched iPSC-NPCs were cultured with peripheral blood mononuclear cells (PBMCs), the immune response was surprisingly low, and the reaction levels were comparable between HLA-matched and HLA-mismatched NPCs. Moreover, iPSC-NPCs suppressed the proliferation of allogeneic HLA-mismatched PBMCs in a dose-dependent manner. As iPSC-NPCs exhibit low antigen-presenting function even under stimulation by inflammatory cytokines³³⁾, these results indicate that the cells themselves possess immunosuppressive effects even under allogenic leukocyte antigen-mismatch conditions.

Regenerative Therapy for the Chronic Phase of SCI

Most SCI studies have focused on the acute to subacute phases as the points of therapeutic interventions due to their plasticity and reactivity. However, >90% of patients with SCI experience functional impairment and disability in the chronic phase. At this stage, a substantial loss of neural tissue leads to cavity formation, which is surrounded by glial scar tissue, and the suppression of axonal elongation occurs due to neurite inhibitory molecules^30,34). In this unfavorable environment for regeneration, transplanted NPCs cannot exert their beneficial effects, exhibiting low survival rates and failure to achieve histological and neurological restoration^20,35).

Despite the complicated pathology, numerous studies have reported the efficacy of cell transplantation therapy for chronic SCI^36-40). Okubo et al. reported a modest but significant functional recovery using a gamma secretase inhibitor (GSI) for transplanting iPSC-NPCs³⁸⁾. The GSI plays a role in inhibiting Notch signaling, which controls the proliferation of undifferentiated NPCs. Inhibition of this signaling pathway promotes the maturation and neuronal differentiation of NPCs. Since transplantation of GSI-treated iPSC-NPCs in the subacute phase of SCI demonstrated functional recovery in the previous study⁴¹⁾, the cells were transplanted into the spinal cord of NOD/SCID mice at 6 weeks after injury³⁸⁾. Results revealed that grafted cells predominantly differentiated into mature neuronal cells, whereas the number of undifferentiated immature NPCs was reduced 12 weeks after transplantation. The differentiated neuronal axons formed inhibitory synaptic connections with the host tissues, indicating that the suppression of spasticity by the grafts contributed to improved motor coordination. Interestingly, inhibition of Notch signaling by GSI resulted in enhanced phosphorylation of p38 MAPK, which plays an important role in axonal regeneration. Thus, treatment of NPCs with GSI yielded favorable outcomes for functional recovery, even in the chronic phase.

However, to enhance functional improvement, a combination therapy with rehabilitation should be considered for spinal cord regeneration. Furthermore, Tashiro et al. have evaluated the effectiveness of mouse CNS-derived NPC transplantation for chronic SCI combined with rehabilitation therapy⁴²⁾. In that study, the cells were transplanted 49 days after SCI, and the recipient mice continued to perform treadmill training for 8 weeks. Remarkably, this combination therapy promoted neuronal differentiation of the grafted cells, increased serotonergic neuronal and axonal regeneration, and enhanced coordination gait. Moreover, these interventions ameliorated sensory abnormalities, such as thermal allodynia and tactile hyperalgesia⁴³⁾. Therefore, it was concluded that treadmill exercise with NPC transplantation promoted neuronal differentiation, regeneration, and maturation of neural circuits and enhanced the recovery of motor and sensory functions.

Shibata et al. have obtained similar results in a human cell transplantation study. Prior to starting the transplantation of human iPSC-derived NPCs, a treadmill training protocol was validated based on the overload principle for chronic SCI⁴⁴⁾. Using quadrupedal treadmill training for mice with incomplete thoracic SCI, the treadmill speeds at which the mice were able to run based on the severity of paresis were examined and the impact of the protocol on functional recovery was investigated. Assessment of changes in running speed during the treadmill training period revealed that treadmill speeds were faster in mice with mild paresis than in those with severe paresis. Notably, training parameters, such as speed and distance traveled, positively correlated with changes in motor function. These results suggest that the most suitable running speed during treadmill training differs according to the level of motor dysfunction and running longer distances has a positive impact on motor functional recovery.

Thus, based on the results of this treadmill protocol, Shibata et al. transplanted human iPSC-derived NPCs at 6 weeks after SCI and performed the training based on the overload principle³⁹⁾. The mice that underwent rehabilitative training showed better transplanted NPC survival rates and neuronal differentiation at the lesion site. In the lumbar area, which contains a central pattern generator, synaptic formation was more abundant in the transplantation with treadmill training group than in the graft-only group. Moreover, the rehabilitation group showed regeneration of serotonergic neurons in a larger area than the graft-only group. The favorable histological findings were corroborated by the secretion of trophic factors such as brain-derived neurotrophic factor and neurotrophin 3 in the lesion area. These results comprehensively demonstrated enhanced locomotor recovery in the transplantation with rehabilitation group than in the transplantation-only group. To the best of our knowledge, this was the first study to demonstrate the efficacy of the combined use of human iPSC-NPC transplantation and rehabilitation for the treatment of chronic SCI, making this finding a significant achievement in establishing a therapeutic strategy using regenerative medicine for chronic SCI in actual clinical settings.

Furthermore, chondroitin sulfate proteoglycans (CSPGs), which are produced from reactive astrocytes and are distributed in glial scar tissue during the chronic phase of SCI, are inhibitory molecules of neuronal regeneration⁴⁵⁾. C-ABC is a bacterial enzyme that detaches the GAG from CSPG proteins, and this degradation allows neural axons to grow^45,46). Shinozaki et al. evaluated the efficacy of C-ABC treatment at the chronic phase of SCI combined with treadmill exercise and demonstrated functional motor recovery with a substantial increase in regenerating neurite axons⁴⁷⁾. Other groups have performed NPC transplantation with C-ABC infusion and rehabilitation and reported enhanced cell survival rates and increased motor-related neurons with functional improvement^36,37,40). Together, regenerative capacity can be preserved even in the chronic phase of SCI, and appropriate therapeutic interventions could provide beneficial effects to enhance this potential.

Strategy to Overcome Complete Paralysis due to SCI at the Chronic Stage

Even in cases where functional improvement was achieved in chronic SCI, the studies largely focused on incomplete injury, wherein the continuity of the spinal cord tissue is maintained. Conversely, the treatment of complete transection injuries is far more challenging (Fig. 3). Hashimoto et al. recently reported functional recovery under this severe neurological status⁴⁸⁾. To compensate for the gap in the transected space in SCI, they placed a clinically relevant collagen scaffold that secreted hepatocyte growth factor (HGF) in the gap. HGF is a powerful mitogen for mature hepatocytes⁴⁹⁾; however, it also exerts neuroprotective effects. In preclinical studies on acute SCI, administration of recombinant human HGF promoted angiogenesis and anti-inflammatory effects, which resulted in axonal regeneration and motor functional recovery^50,51). Phase I/II clinical studies have been conducted on the use of HGF in patients with SCI in the acute phase and favorable outcomes were reported in the HGF group⁵²⁾. Thus, it can be hypothesized that even if there was a gap in the severed chronic SCI, an HGF-secreting scaffold could be used to fill and modify the spinal cord environment, thereby augmenting the efficacy of cell transplantation therapy.

Figure 3.

Pathological difference between incomplete and complete spinal cord injuries at the chronic phase.

Compared with the incomplete injury, complete injury presents as an avascular cavity surrounded by scar tissue and inflammatory cells. Host axons rupture and demyelination proceeds.

Thus, based on this hypothesis, Hashimoto et al. induced SCI transection in rodent models and embedded an HGF-releasing scaffold into the injury site at 6 weeks after injury⁴⁸⁾. Following this procedure, angiogenesis and anti-inflammatory action were observed at the site of chronic SCI. Notably, inflammatory macrophages and microglial cells themselves had the potential to secrete HGF, which amplified the efficacy of the beneficial environmental modulation. When iPSC-derived NPCs were transplanted into the scaffold and spinal cord cephalocaudal stumps, their survival rate drastically increased and the scar and cavity areas were significantly reduced. Host neuronal axons regenerated and extended beyond the lesion site, leading to motor and bladder function improvements, even in the chronic phase. As all materials used in the study were clinically relevant, this novel strategy holds promise for the establishment of a regenerative medicine strategy for severe chronic SCI.

Conclusions

Although iPSC-NPCs have been transplanted in patients in the acute phase of SCI, they have only been used in clinical studies to a limited extent and their efficacy awaits further evaluation. Moreover, for the treatment of chronic SCI, clinical validation will require additional time even as basic research progresses. Thus, further studies are necessary to establish robust regenerative medicine strategies for SCI from the standpoint of both basic and clinical research.

Conflicts of Interest: M.N. declared the role of consultant with K-Pharma. H.O. is a compensated scientific consultant for San Bio Co., Ltd. and K Pharma Inc. N.N. and K.S. declare no relevant conflicts of interests.

Sources of Funding: This work was supported by the Research Center Network for the Realization of Regenerative Medicine of AMED Japan (grant nos. JP23bm1223008, JP23ym0126118, JP22bk0104114, JP21bm0204001, JP20bm0204001, JP19bm0204001, JP20bk0104017, and JP19bk0104017 to H.O. and M.N.) and the Japan Society for the Promotion of Science (JSPS) (KAKENHI grant number 22H03205 to N.N.).

Author Contributions: N.N. designed the study and wrote the manuscript and K.S., H.O. and M.N. supervised the study.

Ethical Approval: The Keio University Certified Special Committee for Regenerative Medicine approved the procedures for the trial (November 2018) (R2016001).

Informed Consent: Informed consent for publication was obtained by all participants in this study.