Background

Contusive spinal cord injury (SCI) can induce permanent impairment of the sensory and motor nervous systems, leading to restricted muscle movement. Moreover, patients with SCI have a lower quality of life in comparison to their uninjured peers due to low life expectancy and lower employment rates [1, 2]. The pathophysiologic mechanisms of SCI may be primary or secondary phase injury. Primary SCI is caused by direct traumatic injury to the spinal cord (SC). On the other hand, secondary SCI can be categorized into acute, subacute, and chronic phases according to the time and pathological mechanisms after the primary injury [3]. Secondary injury mechanisms include neuronal and glial cell death, primarily due to apoptosis and autophagy in the injured regions. In addition, secondary injuries activate astrocytes over time, resulting in reactive gliosis and subsequent glial scar formation, which acts as a physical and chemical barrier inhibiting axonal regeneration [4, 5]. Therefore, the goal of SCI treatment in clinical practice is to minimize secondary injuries after the primary injury. Therapeutic attempts to overcome SCI are generally based on two pathophysiological mechanisms: neuroprotective and neuroregenerative approaches [3, 5, 6].

Stem cell therapy has demonstrated both neuroprotective and neuroregenerative effects in SCI by minimizing the pathophysiological mechanisms that occur during primary and secondary injuries [7,8,9]. The neuroprotective mechanisms of the transplanted cells include secondary injury mitigation by protecting the injured region of the SC. The basic mechanism has been reported to be preservation of the adjacent tissues at the injured site by stem cells in comparison to the control group [5, 10]. Remyelination after SCI is a therapeutic target in regenerative trials. Myelination plays a critical role in effective action potential propagation along with survival of axons and corresponding neurons in the SC tissue. Enhanced approaches for cell transplantation and endogenous repair processes are being actively investigated to improve remyelination in SCI [3]. Numerous cell types have been transplanted and studied for their neuroprotective and neuro-regenerative effects in SCI. Several cell types, including Schwann cells, neural stem and progenitor cells (NSPCs), mesenchymal stem cells (MSCs), olfactory ensheathing cells (OECs), and oligodendrocyte precursor cells, are being studied in the context of promoting neuroprotection in SCI [10,11,12]. The therapeutic effects of different types of differentiated cells derived from multipotent or pluripotent cell types are also being investigated in SCI [5, 13]. Till date, various cell types have been successfully differentiated from induced pluripotent stem cells (iPSCs) and transplanted into animal models of SCI, suggesting their potential for neuroprotection and neuroregeneration [5, 14]. Moreover, several studies have reported the efficacy of differentiated cell types derived from iPSCs in preclinical and clinical SCI trials [15,43]. Based on previous studies, we confirmed the possibility of stem cell therapy using a rat model of contusive SCI.

Secondary SCI can be categorized into acute, subacute (or intermediate), and chronic phases depending on the time elapsed after injury and the pathological mechanism. Acute SCI phase is expressed in the last 48 h after the initial physical insult to the SC. The main phenomena observed in the acute SCI phase are vascular disruption, hemorrhage, and subsequent ischemia of the injured SC. After disruption of the microenvironment in the injured SC, pathological changes such as ion dysregulation, excitotoxicity, excessive production of free radicals, and inflammatory responses cause further damage to the neurons and glial cells at the lesional site [3, 44, 45]. The sub-acute (intermediate) phase is considered to last up two weeks in the injured SC. Characteristic features of the subacute phase are phagocytic response and reactive proliferation of astrocytes at the lesional site. Increase in the number of reactive astrocytes at the lesional site results in the formation of a glial scar. Glial scarring is a major cause of limited neuroregeneration and axonal regeneration in SCI [3, 46, 47]. A characteristic feature of chronic SCI phase is the maturation of the lesion, including scar formation and development of a syrinx at the lesional site. Endogenous regenerative capacity is affected by the release of growth-inhibitory molecules and glial scarring around the epicenter of the lesion [3, 44].

Neuroprotective and neuroregenerative approaches are being used to treat SCI [3]. Transplantation of various types of stem cells reportedly has great potential to preserve damaged tissues and promote the recovery of nerve function [46, 48]. Previous studies have reported that bone marrow-derived MSC (BM-MSC) transplantation in acute and chronic SCI rat models increased clinical improvement, neuroprotection, and neuroregeneration via differentiated astrocytes and oligodendrocytes at the lesional sites [7,8,9, 27]. The findings of the aforementioned studies on transplantation of BM-MSCs in SCI suggests the potential for cell therapy in acute and chronic SCI models. It has been shown that the densities of astrocytes and oligodendrocytes return to near-normal levels in the residual white matter within several weeks after SCI. However, chronic SCI causes loss of gray matter neurons due to damage at the lesional site and permanent damage to motor function. In chronic SCI, a disorder characterized by progressive loss of motor neurons, a feasible treatment approach is to replace lost or degenerated motor neurons [20].

Recently, high-purity iMNPs and iMNs were successfully established, and their transplantation in SCI was studied [19, 30]. Another study reported that transplantation of human embryonic stem cell (hESC)-derived motor neurons progenitor (MP) enhanced astrogliosis at the lesional site four-months after SCI. In addition, it was confirmed that increased astrogliosis at the lesional site favored the survival and differentiation of hESC-derived neurons and correlated with improved motor function recovery [21]. In another study, when a high-purity population of human motor neuron progenitors (hMNP) derived from hESC was transplanted into an SCI rat model, it was reported that hMNP at the lesional site suppressed the intracellular signaling pathway associated with SCI pathogenesis, which correlated with greater endogenous neural survival and neurite branching [22]. However, studies on their therapeutic effects in SCI are lacking. We aimed to confirm the increased differentiation of gray matter neurons and recovery of motor function at the lesional site by transplantation of iPSC-derived motor neuron progenitor cells (iMNP) in a chronic SCI model. In the context of subtopic 1 of the study, we confirmed the efficacy of iMNP in neuroregeneration in a chronic SCI model. We generated iPSC-derived iMNPs and iMNs in vitro and selected iMNPs with high proliferation for successful transplantation into the SC of chronic SCI model (Fig. 2a and b). The iMNPs transplanted at the lesional site showed possible in vivo MN differentiation and maturation. Moreover, the cells also showed behavioral recovery through BBB locomotor scale scores (Fig. 2c-g). Our findings suggest successful engraftment and MN differentiation of implanted iMNP in chronic SCI. However, the subsequent formation of glial scars and their microenvironment could not be effectively decreased at the injured site. In addition, the transplanted iMNP did not decrease the expression of reactive gliosis in the round cystic area in chronic SCI (Additional file 1: Fig.S1a-g). However, subtopic 1 of the study had a limitation in that few animals were used due to the pilot study concept.

Transplantation in chronic SCI results in low rate of engraftment and functional restoration due to the subsequent formation of glial scars [8, 29]. Therefore, the timing of transplantation must be taken into consideration and alterations in the microenvironment of chronic SCI tissues should be induced to enhance the effectiveness of transplantation [29, 49]. MSC transplantation can prevent the secretion of various inflammatory cytokines, apoptosis, and inflammation to exert neuroprotection during acute SCI [7, 50,51,52]. Based on the results of previous studies and our initial findings, we attempted to increase the transplantation effect at the lesional site through multiple hMSC injections for acute SCI (Subtopic 2), and found that it increased the cell transplantation efficacy in comparison to single hMSC injection by increasing neuronal cells via NGF and axonal regeneration at the lesional site (Fig. 3f and g). Interestingly, multiple hMSC injections promoted clinical recovery by increasing neuronal cell differentiation, whereas a single hMSC injection promoted clinical recovery by increasing astrocyte and oligodendrocyte differentiation in the injured SC (Additional file 2: Fig.S2a-b). C3 is a specific marker of A1 neuroinflammation-reactive astrocytes in SCI, and evaluation of the phenotype of reactive astrocytes in SCI have been reported by several studies [23, 32, 33, 53]. One research reported that an IV injection of MSC-derived exosomes reduced the number of C3- or GFAP-positive astrocytes at the lesional site in acute SCI [53]. Our findings suggest that multiple and single injections of hMSCs can effectively promote functional behavioral recovery by decreasing C3 expression at the lesional site in acute SCI (Additional file 3: Fig.S3a-c).

Stem cell-derived MN and MNP offer promising strategies for cellular replacement in SCI. However, in our pilot experiment, the data suggested that iMNP cell transplantation confirmed the limitations of cellular replacement strategies in chronic stage astrocyte and Neurocan formation scar cavity. Thus, we used a stepwise cell therapy strategy for SCI to increase the efficacy of the transplanted cells at the lesional site. This main topic of the study included confirmation of the neuroprotective and neuroregenerative effects of multiple preemptive hMSC injections and increased MN differentiation at the lesional site through transplanted iMNP. Interestingly, we found that stepwise cell therapy promoted MN maturation and axonal regeneration at the lesional site. We also confirmed that cell transplantation with iMNP alone increased MN differentiation at the lesional site (Additional file 4: Fig.S4a-d). Another study reported that MNP cell transplantation resulted in MN lineage differentiation in the ventral horns at the lesional site. However, the failure of MNPs to mature in all other regions of the SC likely reflected the gliogenic nature of the SCI environment [22]. Our findings suggest that the increased neuroprotective effects of multiple preemptive hMSC injections in the acute SCI phase can enhance MN differentiation and maturation at the lesional site. More importantly, it was demonstrated in vitro that hMSC and iMN co-culture significantly increased neurite outgrowth during the MN maturation stage (Fig. 6b-g). Another study reported that differentiated Schwann cells (SC), human bone marrow-MSCs, and umbilical-cord-blood MSCs significantly promoted neurite outgrowth and elongation in comparison to untreated MSCs [54]. We found that 2D and 3D co-cultured hMSC and iMN induced neurite outgrowth and elongation compared to hMSC and iMN separately (Fig. 6f-g). However, hMSC alone did not significantly promote neurite outgrowth and elongation as compared to iMN. Another study suggested that hMSC promotes neurite outgrowth via a paracrine effect through growth factors including BDNF and NGF [55,56,57].

In summary, this study confirmed that clinical behavioral outcomes were restored through induction of mature motor neuron differentiation and axonal regeneration at the lesional site using stepwise combined cell transplantation of hMSCs and iMNPs in a contusive SCI model, suggesting the therapeutic efficacy of stepwise combined cell transplantation strategy in a severed contusion SCI rat model. The stepwise combined cell transplantation strategy has the advantage of not only suggesting ideal stem cell selection for each stage of SCI, but also confirming the function of the transplanted cells. However, a limitation of this study is the lack of an explanation for the mechanisms underlying the synergistic effect of stepwise combined cell transplantation in a contusion SCI model. In future studies, it will be necessary to confirm the synergistic effects of stepwise combined cell transplantation mechanisms using time-dependent RNA sequencing (RNA-seq) or single-cell analysis at the lesional site. In addition, selecting a sample size for animal experiments requires calculating a sample size sufficient for statistical analysis using a few free software packages (G power, power sample size). There is a need to overcome the limitations of stem cell therapy for SCI using a stepwise combined cell transplantation strategy with a 3D iPSC-derived motor neuron source.

Conclusion

Our study demonstrated that stepwise cell therapy increased MN differentiation and axonal regeneration compared to single-cell therapy in severed SCI model. Stepwise cell therapy increased behavioral recovery and the rate of BBB locomotor scale grade 3 (BBB score, 10–15). Moreover, it also induced alterations in the microenvironment for effective cell therapy in severed SCI model. These in vitro results suggest that co-cultured hMSC and iMN synergistically promoted induction of MN neurite outgrowth. Taken together, we report a proof-of-concept study showing that stepwise combined transplantation can increase cell engraftment and SC recovery based on cell type and transplantation timing in SCI.