Multiroute administration of Wharton’s jelly mesenchymal stem cells in chronic complete spinal cord injury: A phase I safety and feasibility study

Abstract

BACKGROUND

Traumatic spinal cord injury (SCI) is a life-altering condition that results in long-term complications, including progressive neurodegeneration and cord atrophy. It presents a significant unmet medical need with extensive social and economic burdens.

AIM

To evaluate the safety and preliminary efficacy of allogeneic mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs) in patients with chronic complete SCI. The primary objective was to assess whether WJ-MSCs could facilitate neurological recovery and improve the quality of life in this patient population.

METHODS

This open-label, multicenter phase I study investigated the effects of administering WJ-MSCs via three delivery routes: Intrathecal (for localized spinal targeting); intramuscular (for targeting end organ); and intravenous (for systemic immunomodulation). While all three routes were used concurrently to enhance therapeutic synergy, neurological, sensory, and functional scales were used to assess overall efficacy. Participants with chronic SCI (duration of at least 6 months) who had significant impairment and disability were eligible for inclusion. WJ-MSCs were administered twice monthly for 2 months, with each route receiving a dose of 1 × 10⁶ cells/kg. Patients were closely monitored for 1 year following treatment.

RESULTS

At baseline, participants displayed considerable functional deficits, as indicated by the following scores: Functional independence measure of 77.5 ± 2.26; Modified Ashworth Scale of 15.83 ± 4.83; American Spinal Injury Association (ASIA) Motor score of 1.67 ± 2.66; ASIA Light Touch and Pin-Prick scores of 62 ± 18.42 each; Wexner Incontinence Score of 20; and Qualiveen Short Form, a validated questionnaire specifically designed to assess the impact of urinary dysfunction on quality of life in individuals with SCI, score of 32. Following WJ-MSC therapy, significant improvements were observed in all neurological functions over the 1-year follow-up. Notably, the ASIA Motor score improved significantly (χ² = 23.938, P < 0.001), and Qualiveen Short Form scores demonstrated a substantial enhancement in quality of life (z = -2.214, P < 0.05).

CONCLUSION

This phase I study, conducted without a control group, suggests that the administration of WJ-MSCs through multiple routes is both safe and potentially effective in patients with chronic complete SCI. However, the observed neurological improvements cannot be solely attributed to WJ-MSC therapy, as concurrent pharmacological and rehabilitative interventions were not controlled. These findings indicated that WJ-MSC therapy may offer a promising approach for enhancing neurological function and quality of life in this challenging patient population. Further research with larger cohorts and extended follow-up is necessary to validate these preliminary results.

Key Words: Spinal cord injury; Mesenchymal stem cell; Transplantation; Wharton’s jelly; Neurological recovery

Core Tip: Spinal cord injury causes severe disability with significant impairments in sensory and motor functions and dysfunction in multiple organs. We investigated the therapeutic potential of Wharton’s jelly-derived mesenchymal stem cells administered through a triple-route delivery system (intrathecal, intramuscular, and intravenous). The treatment resulted in significant improvements in motor and sensory functions. Patients experienced an improved quality of life. The procedure was well-tolerated with no significant side effects or safety concerns. No adverse events were reported highlighting the potential of Wharton's jelly-derived mesenchymal stem cell therapy as a safe and effective treatment option for individuals with chronic spinal cord injury.

INTRODUCTION

Traumatic spinal cord injury (SCI) remains one of the most devastating conditions, primarily affecting young adults and resulting in life-altering neurological deficits. The incidence of SCI is estimated at approximately 12500 new cases annually in North America, with a substantial burden on both individuals and healthcare systems[1]. SCI disrupts sensory and motor pathways, leading to permanent disability and secondary complications that compromise the function of multiple organ systems[2]. The global incidence varies significantly, ranging from 20.7 to 83.0 cases per million in North America and from 8.0 to 130.6 cases per million in Europe[3]. Despite significant advances in acute management, including early surgical decompression and intensive rehabilitation, these interventions have largely focused on enhancing survival rather than restoring meaningful neurological function[4,5].

The pathophysiology of SCI is complex and occurs in two distinct but interrelated phases: The primary phase, characterized by mechanical trauma leading to immediate axonal and neuronal injury; and the secondary phase, marked by a cascade of events including excitotoxicity, oxidative stress, inflammation, and apoptosis that exacerbate the initial damage[6-9]. Current therapeutic strategies, such as the administration of high-dose methylprednisolone, aim to mitigate secondary injury processes but have not consistently demonstrated long-term efficacy in functional recovery[10]. Given these limitations, there is an urgent need for novel therapeutic approaches that address both the acute and chronic phases of SCI.

Recent advances in regenerative medicine, particularly in the application of mesenchymal stem cells (MSCs), offer a promising avenue for the treatment of SCI. MSCs have shown the potential to modulate the immune response, promote neuroprotection, and enhance tissue regeneration in preclinical models of SCI. For example, studies by Aras et al[11] and Kabatas et al[12] have reported that MSCs derived from adipose tissue and dental pulp, respectively, can improve motor function and reduce lesion size in rodent models of SCI, suggesting a potential role for MSCs in promoting spinal cord repair. Additionally, our previous studies have demonstrated the safety and feasibility of Wharton’s jelly-derived MSCs (WJ-MSCs) for treating various neurological disorders, including SCI[13,14].

These findings have set the stage for the present phase I clinical trial, which aimed to evaluate the safety and preliminary efficacy of WJ-MSCs administered via multiple routes in patients with chronic, complete SCI[14]. This study represents a pivotal step toward translating the regenerative potential of WJ-MSCs into a clinically viable therapy for SCI, with the ultimate goal of improving neurological outcomes and quality of life for affected patients.

MATERIALS AND METHODS

Study design

This open-label, multicenter phase I clinical trial was designed to evaluate the safety and preliminary efficacy of WJ-MSC transplantations using a triple-route administration strategy. The study cohort, as detailed in Table 1, included adults with radiologically confirmed chronic SCI accompanied by substantial functional deficits. Participants were monitored for a duration of 1 year following WJ-MSC infusion. During the follow-up period, no restrictions were placed on concurrent pharmacological or rehabilitative interventions. Informed consent was obtained in accordance with the Declaration of Helsinki, and the study protocol was approved by the Ministry of Health. Patient demographics and clinical characteristics are provided in Table 2.

Table 1 Enrollment criteria and assessment timeline.

Criteria	Details
Inclusion criteria	Age ≥ 18 years
	Radiologically confirmed chronic SCI
	No chronic illnesses (e.g., cancer, organ failure)
	Life expectancy > 12 months
	Severe disability (wheelchair-bound, assistance required)
	No significant functional improvement for at least 3 months
	Commitment to standard care and follow-up
	Provide written informed consent
Exclusion criteria	Significant medical/psychiatric conditions affecting safety
	Recent severe infections (meningitis, sepsis, etc.)
	Uncontrolled seizure disorder
	CNS neoplasm or cancer history (last 5 years)
	Abnormal blood cell counts (WBC ≥ 15000/μL, platelets ≤ 100000/μL)
	Abnormal liver/kidney function (AST/ALT > 3 × ULN, creatinine > 1.5 × ULN)
	Prior participation in other investigational stem cell trials
	Patient decision to withdraw or death during study
Treatment timeline	Baseline assessment (week 0): Neurological and functional evaluations
	MSC therapy initiation (week 1-8): 4 rounds of triple-route administration
	Short-term follow-up (weeks 1-12): Safety and early functional outcomes
	Mid-term assessment (month 6): Progress evaluation
	Long-term follow-up (month 12): Final outcome assessment

SCI: Spinal cord injury; CNS: Central nervous system; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ULN: Upper limit of normal; MSC: Mesenchymal stem cell.

Table 2 Study population.

Characteristic		n	%
Age, years	19	1	16.7
	20	1	16.7
	29	3	50.0
	39	1	16.7
Sex	Female	1	16.7
	Male	5	83.3
Cause of SCI	Fell from tree	1	16.7
	Firearm injury	2	33.3
	Ski accident	1	16.7
	Traffic accident	2	33.3
Level of injury	T11 burst fracture	1	16.7
	T12 burst fracture with T11-T12 and T12-L1 traumatic disc herniations	1	16.7
	T12 burst fracture with dislocation	1	16.7
	T5 burst fracture	1	16.7
	T7-8 burst fracture	1	16.7
	T8-9 burst fracture	1	16.7
Pre-transplantation treatments	Only physiotherapy	3	50.0
	T11 laminectomy + physiotherapy	1	16.7
	T12 corpectomy + posterior instrumentation + laminectomy + physiotherapy	1	16.7
	T12 corpectomy + posterior instrumentation + laminectomy + physiotherapy	1	16.7
Comorbidity	None	6	100.0
Duration of SCI and first transplantation	6 months	1	16.7
	9 months	2	33.3
	12 months	1	16.7
	4 years	1	16.7
	12 years	1	16.7

SCI: Spinal cord injury.

Procedure

Ethics and consent: Umbilical cords were procured from a facility adhering to Good Manufacturing Practice standards. Prior to collection, ethical protocols were observed, including obtaining informed consent from the donors and approval from an institutional review board (protocol number: 56733164-203-E.2659). The umbilical cords were donated by individuals who had undergone full-term pregnancies. Written informed consent was secured from all study participants[13,14].

Processing and quality control of umbilical cords: The cord was washed with PBS to ensure cleanliness. Following the removal of blood vessels, the cord tissues were cut into small pieces (5-10 mm³) known as explants. These tissue explants were then placed in culture dishes and incubated under humanized culture conditions (5% CO₂, 37 °C, and high humidity) until the cells migrated from the tissue pieces. When the cells reached approximately 70% to 80% confluency in passage 3, they were harvested, and characterization assays were conducted[13,14].

Characterization of WJ-MSCs by flow cytometry: Flow cytometry analysis revealed that WJ-MSCs were positive for surface markers CD44, CD73, CD105, and CD90 and negative for hematopoietic lineage markers CD34, CD45, and human leukocyte antigen DR. Telomerase activity was maintained throughout cell culture, and cells exhibited a characteristic flattened morphology[13,14]. The flow cytometry assay was performed using the human MSC Analysis Kit (BD, NJ, United States, Cat no: 562245) on the BD FACS CANTO II device. Telomerase activity assay was performed using TRAPEZE^® RT Telomerase Detection Kit (Sigma-Aldrich, MO, United States, Cat No: S7710). The protocol stipulated by the kit was followed meticulously without any deviations.

Cell differentiation and karyotyping: Expression of key stem cell markers (TERT, POU5F1, SOX2, ZFP42) and differentiation markers (CD44, VCAM1, THY1, BMP2, RUNX-1) was assessed to confirm the pluripotency of WJ-MSCs. Differentiation assays demonstrated that WJ-MSCs could differentiate into osteoblasts, adipocytes, and chondrocytes. Karyotype analysis confirmed that the cells exhibited a normal chromosomal complement without any structural or numerical abnormalities[13,14]. In the gene expression test, RNA isolation was performed using the RNeasy^® Plus Mini Kit (QIAGEN, Netherlands, Cat No: 74104). Subsequently, the RT² First Strand Kit (QIAGEN, Netherlands, Cat No: 330401) was utilized for the translation of the RNA into cDNA. The protocol stipulated by the kit was followed meticulously without any deviations (Figure 1 and Table 3).

Figure 1 Wharton’s jelly derived mesenchymal stem cell flow cytometry, gene expression, telomerase activity, and karyotyping. A: Wharton’s jelly-derived mesenchymal stem cell (WJ-MSC) flow cytometry. Positive marker values (CD90, CD105, CD73, and CD44) are above 95%. Negative marker values (CD45, CD34, CD19, CD11B, human leukocyte antigen DR, and CD14) are below 2%; B: WJ-MSC gene expression. In the gene expression test for MSCs, SOX2, OCT4, TERT, REX1, NES, RUNX2, PPARG, JAG1, ICAM1, VCAM1, and VIM had Ct values below 29 and were strongly positive. BMP2 had a Ct between 30-37 and was positive; C: WJ-MSC telomerase activity. In the telomerase activity assay of WJ-MSCs, the telomerase activity of the cell was not as high as the positive control; D: WJ-MSC karyotyping. Chromosomal analysis of metaphases obtained from WJ-MSCs after short-term cell culture using GPL banding revealed no numerical or gross structural chromosomal abnormalities. HLA: Human leukocyte antigen.

Table 3 Wharton’s jelly-derived mesenchymal stem cell quality control parameters.

	QC parameters
Cell count and viability	Cell viability > 90. Cell count: Varies according to the patient
Immunophenotyping	CD90, CD44, CD105, CD73 ≥ 95%. CD45, CD34, CD19, CD11B, HLA-DR, CD14 ≤ 2% (Figure 1A)
Gene expression	SOX2, OCT4, TERT, REX1, NES, BMP2, RUNX2, PPARG, JAG1, ICAM1, VCAM1, VIM. It was concluded that there was strong positivity in genes with Ct values lower than 29, positive in genes with values between 30-37, and poor expression in samples with Ct values between 38-40 or there may be environmental contamination (Figure 1B)
Telomerase	The result should be lower than that of the positive control (Figure 1C)
Mycoplasma	Negative
Endotoxin	< 0.0625 EU/mL

HLA: Human leukocyte antigen; QC: Quality control.

Pre-transplantation process: Final preparations for WJ-MSC infusion were derived from passage 3 cultures. The cell preparations were resuspended in normal saline at concentrations of 1 × 10⁶ cells in 3 mL, 1 × 10⁶ cells in 20 mL, and 1 × 10⁶ cells in 30 mL[13,14].

Surgical procedure and WJ-MSC transplantation procedure: Prior to treatment, comprehensive evaluations were performed by anesthesiology, neurosurgery, physical therapy, and rehabilitation specialists. The WJ-MSC implantation was carried out under conditions of stable patient health, with no contraindications from internal medicine or cardiology that would preclude the use of sedation or general anesthesia. The absence of severe infections, such as sepsis, was also confirmed.

The standard protocol for MSC administration involved allogeneic WJ-MSCs being administered via intrathecal, intramuscular, and intravenous routes. The triple application route was applied together in each treatment round for each patient. Intrathecal injections were performed using lumbar puncture, guided by established protocols. Intramuscular injections were conducted with ultrasonographic guidance, and intravenous infusion was administered over 30 min. Post-procedure, patients were monitored in the intensive care unit for 1 day and then transferred to the Department of Neurosurgery for ongoing physical therapy and rehabilitation. Exercise programs were suspended on administration days, and the protocol was repeated before and after each infusion, as outlined in Table 4.

Table 4 Administration schedule.

Rounds	Route	WJ-MSC
Round 1	IT	1 × 106 cells/kg in 3 mL
	IV	1 × 106 cells/kg in 30 mL
	IM	1 × 106 cells/kg in 20 mL
Round 2 (week 1)	IT	1 × 106 cells/kg in 3 mL
	IV	1 × 106 cells/kg in 30 mL
	IM	1 × 106 cells/kg in 20 mL
Round 3 (week 4)	IT	1 × 106 cells/kg in 3 mL
	IV	1 × 106 cells/kg in 30 mL
	IM	1 × 106 cells/kg in 20 mL
Round 4 (week 6)	IT	1 × 106 cells/kg in 3 mL
	IV	1 × 106 cells/kg in 30 mL
	IM	1 × 106 cells/kg in 20 mL

WJ-MSC: Wharton’s jelly derived mesenchymal stem cells; IT: Intrathecal; IM: Intramuscular; IV: Intravenous.

Pre-treatment neurological examination: A multidisciplinary team performed a thorough pre-treatment assessment using the American Spinal Injury Association (ASIA) impairment scale to evaluate neurological and functional status. Spasticity was quantified using the Modified Ashworth (MA) scale. The impact of incontinence on quality of life was assessed with the Wexner Incontinence Score (WIS) and the Qualiveen Short Form (SFQ) score. Overall functional independence was evaluated using the Functional Independence Measure (FIM)[15,16].

Safety evaluation criteria: During the transplantation procedure, safety was monitored rigorously. Criteria included surveillance for infection (fever, elevated C-reactive protein levels, leukocytosis), complications related to anesthesia and analgesia, wound infections, and potential allergic reactions or shock. Immediate safety was assessed for 1-2 weeks post-procedure, with long-term follow-up extending 1 year to monitor for infection, cancer development, neuropathic pain, and neurological deterioration[13,14].

Follow-up assessment of treatment success: Follow-up evaluations included comprehensive assessments of neurological and functional outcomes. Spasticity was measured using the MA scale, and the impact of incontinence was evaluated with the WIS and SFQ score. Functional recovery was assessed using the FIM score. Additionally, the incidence of neuropathic pain, urinary tract infections, secondary infections, and pressure ulcers was monitored. These assessments provided a detailed understanding of patient progress and treatment efficacy.

Statistical analysis

Non-parametric statistical methods, including the Friedman test and the Wilcoxon signed-rank test, were employed to analyze changes in FIM scores, MA scale, ASIA Motor and Sensory scores, WIS, and SFQ values before and after treatment. Non-parametric tests were utilized due to the small sample size and non-normal distribution of the data, making parametric tests unsuitable for analysis. While non-parametric tests were chosen based on the characteristics of the data, we acknowledge that mixed-effects models could provide additional insights by accounting for repeated measures and potential variability between subjects. However, given the sample size and the exploratory nature of this phase I study, the use of non-parametric methods was deemed most appropriate.

RESULTS

FIM score

The FIM Motor scores demonstrated a statistically significant progressive improvement following the intervention, indicating enhanced motor function and independence over time. In contrast, the FIM Cognitive scores exhibited stability, showing no substantial changes post-intervention, as expected. A detailed statistical analysis revealed a significant difference between pre-intervention and post-intervention FIM Motor Scores (χ² = 29.505, P < 0.001), confirming the effectiveness of the intervention in improving motor function. To further elucidate the temporal effects of the intervention, Wilcoxon signed-rank tests were conducted at various time points (Table 5 and Figure 2).

Figure 2 Changes in the Functional Independence Measure Motor scores following intervention. This figure shows significant improvements in the Functional Independence Measure Motor scores at 1 month, 2 months, 4 months, and 1 year following intervention (χ² = 29.505, P < 0.001). Notable improvements were observed at 1 month (P = 0.039), 2 months (P = 0.026), and 4 months (P = 0.027), with sustained gains through 1 year (P = 0.027). FIM: Functional Independence Measure.

Table 5 Results of the Friedman test for changes in Functional Independence Measure Motor score values before and after intervention.

	n	Mean	SD	Mean rank	χ2	df	P value
Preoperative	6	42.50	2.26	1.50
Postoperative week 1	6	43.00	1.90	1.67
Postoperative month 1	6	45.67	1.97	2.83	29.505	5	0.000
Postoperative month 2	6	48.00	1.90	4.00
Postoperative month 6	6	50.50	2.17	5.00
Postoperative month 12	6	56.17	2.99	6.00

SD: Standard deviation.

First week: No significant change was observed in FIM Motor scores during the first week post-intervention (z = -1.00, P = 0.32), suggesting that early postoperative recovery might not reflect the full impact of the intervention.

One month: Significant improvement was observed at 1 month (z = -2.060, P = 0.039), indicating an initial positive response to the intervention.

Two months: The improvement continued with significant gains observed at 2 months (z = -2.226, P = 0.026), further supporting the efficacy of the intervention in enhancing motor function.

Four months: At 4 months, the significant improvement in FIM Motor scores was sustained (z = -2.207, P = 0.027), highlighting the ongoing positive effects of the intervention.

One year: The significant improvement persisted through the 1-year follow-up (z = -2.207, P = 0.027), demonstrating the long-term benefits of the intervention in motor function recovery. While the mean FIM Motor score before the application was 42.50 ± 2.26, this value increased to 56.17 ± 2.99 in the first year after the application. Overall, these findings underscore the potential of the intervention in significantly improving motor function over time.

MA scale

The MA scale assessments indicated a statistically significant reduction in spasticity following the intervention, as evidenced by decreased scores for both the right and left lower extremities (Figure 3).

Figure 3 Changes in spasticity as measured by the Modified Ashworth scale. This figure illustrates the significant reduction in spasticity in both the right and left lower extremities following intervention. Statistically significant decreases in the Modified Ashworth scale scores were observed at 1 month, 2 months, 4 months, and 1 year for both extremities (right: χ² = 28.452, P < 0.001; left: χ² = 29.030, P < 0.001).

Right lower extremity spasticity: Statistical analysis demonstrated a significant difference in the MA scale right values between pre-intervention and post-intervention assessments (χ² = 28.452, P < 0.001). Wilcoxon signed-rank tests revealed no significant differences in the right MA Scale scores during the first week post-intervention (z = -1.890, P = 0.059). However, significant reductions in spasticity were observed at 1 month (z = -2.271, P = 0.023), 2 months (z = -2.207, P = 0.027), 4 months (z = -2.214, P = 0.027), and 1 year (z = -2.207, P = 0.027). These findings suggest that while initial changes were not apparent within the first week, substantial and sustained improvements in spasticity were achieved over the subsequent months (Table 6). While the mean MA scale for the right lower extremity before the application was 8.00 ± 2.45, this value decreased to 2.17 ± 1.94 in the first year after the application.

Table 6 Results of the Friedman test for changes in the Modified Ashworth scale (right values) before and after intervention.

	n	Mean	SD	Mean rank	χ2	df	P value
Preoperative	6	8.00	2.45	5.83
Postoperative week 1	6	7.17	3.06	5.17
Postoperative month 1	6	5.33	2.66	3.67	28.452	5	0.000
Postoperative month 2	6	3.83	2.48	3.00
Postoperative month 6	6	3.00	2.19	2.08
Postoperative month 12	6	2.17	1.94	1.25

SD: Standard deviation.

Left lower extremity spasticity: Similarly, the MA scale left values demonstrated a significant difference between pre-intervention and post-intervention scores (χ² = 29.030, P < 0.001). The Wilcoxon signed-rank test showed no significant differences in left MA scale scores during the first week post-intervention (z = -1.633, P = 0.102). Significant decreases in spasticity were recorded at 1 month (z = -2.226, P = 0.026), 2 months (z = -2.207, P = 0.027), 4 months (z = -2.207, P = 0.027), and 1 year (z = -2.214, P = 0.027). This indicates that while the reduction in spasticity was not immediately detectable, there was a notable and progressive decrease in spasticity over time (Table 7). While the mean MA scale score for the left lower extremity before the application was 7.83 ± 2.40, this value decreased to 2.17 ± 1.84 in the first year after the application.

Table 7 Results of the Friedman test for changes in the Modified Ashworth scale (left values) before and after intervention.

	n	Mean	SD	Mean rank	χ2	df	P value
Preoperative	6	7.83	2.40	5.75
Postoperative week 1	6	7.17	2.64	5.25
Postoperative month 1	6	5.50	2.43	3.92	29.030	5	0.000
Postoperative month 2	6	4.17	2.64	2.92
Postoperative month 6	6	3.00	2.28	1.92
Postoperative month 12	6	2.17	1.84	1.25

SD: Standard deviation.

ASIA Motor score

The ASIA Motor scores demonstrated a statistically significant and progressive improvement following the intervention (Figure 4). While the mean ASIA Motor score for lower extremities before the application was 1.67 ± 2.66, this value increased to 15.00 ± 11.23 in the first year after the application. Initial analyses revealed a notable difference in ASIA Motor scores between pre-intervention and post-intervention evaluations, with a χ² statistic of χ² = 23.938 (P < 0.001), indicating a robust effect of the intervention on motor function (Table 8).

Figure 4 Changes in the American Spinal Injury Association Motor scores over time. This figure shows significant and progressive improvements in the American Spinal Injury Association Motor scores following intervention (χ² = 23.938, P < 0.001). ASIA: American Spinal Injury Association.

Table 8 Results of the Friedman test for changes in the American Spinal Injury Association Motor score values before and after intervention.

	n	Mean	SD	Mean rank	χ2	df	P value
Preoperative	6	1.67	2.66	2.00
Postoperative week 1	6	1.67	2.66	2.00
Postoperative month 1	6	3.33	3.93	2.75	23.938	5	0.000
Postoperative month 2	6	7.67	5.28	4.08
Postoperative month 6	6	12.00	8.10	4.75
Postoperative month 12	6	15.00	11.23	5.42

SD: Standard deviation.

Initial observations: During the first week post-intervention, the Wilcoxon signed-rank tests showed no statistically significant changes in ASIA Motor scores (z = 0.00, P = 1.00), suggesting that immediate post-intervention assessments did not reflect the potential benefits of the treatment. Similarly, no significant changes were observed in scores during the first month (z = -1.633, P = 0.102), which may be attributed to the time required for the intervention to manifest its effects.

Progressive improvements: Significant enhancements in motor function were first detected at the 2-month follow-up, with a Wilcoxon signed-rank test statistic of z = -2.060 (P = 0.039). This was followed by further significant improvements at the 4-month mark (z = -2.023, P = 0.043) and sustained progress up to the 1-year follow-up (z = -2.023, P = 0.043). These results highlight a delayed but substantial improvement in motor function, suggesting that the benefits of the intervention accrue progressively over time.

Long-term effects: The observed increase in ASIA Motor scores underscores the potential of the intervention to induce meaningful and lasting improvements in motor function. The delayed onset of significant changes may reflect the time required for cellular or physiological adaptations to the intervention, leading to improved motor outcomes in the longer term. Overall, the data indicate that while immediate effects were not apparent, the intervention resulted in a marked and sustained enhancement in motor function, with notable improvements emerging within the first 2 months and continuing through the 1-year follow-up period.

ASIA Sensory score

Post-intervention assessments revealed significant improvements in ASIA Sensory scores for both Light Touch (LT) and Pin-Prick (PP) modalities (Figure 5).

Figure 5 Changes in the American Spinal Injury Association Sensory scores over time. This figure shows significant improvements in American Spinal Injury Association Sensory Scores for both Light Touch and Pin-Prick modalities following intervention (Light Touch: χ² = 28.299, P < 0.001; Pin-Prick: χ² = 28.838, P < 0.001). For both modalities, indicating a progressive improvement in sensory function over time. ASIA: American Spinal Injury Association; LT: Light Touch; PP: Pin-Prick.

LT: Statistical analysis showed significant differences between pre-intervention and post-intervention ASIA Sensory LT scores (χ² = 28.299, P < 0.001). While the mean ASIA Sensory LT score before the application was 62.00 ± 18.42, this value increased to 81.67 ± 22.50 in the first year after the application. Wilcoxon signed-rank tests indicated no significant differences in LT scores during the first week (z = -1.342, P = 0.180) and the first month (z = -1.826, P = 0.068). Significant improvements were noted at 2 months (z = -2.207, P = 0.027), 4 months (z = -2.201, P = 0.028), and 1 year (z = -2.201, P = 0.028), reflecting an enhancement in sensory function over time (Table 9).

Table 9 Results of the Friedman test for changes in the American Spinal Injury Association Sensory score (Light Touch values) before and after intervention.

	n	Mean	SD	Mean rank	χ2	df	P value
Preoperative	6	62.00	18.42	1.50
Postoperative week 1	6	65.33	18.23	1.83
Postoperative month 1	6	70.33	18.26	2.75	28.299	5	0.000
Postoperative month 2	6	74.67	19.04	4.08
Postoperative month 6	6	78.67	20.73	5.08
Postoperative month 12	6	81.67	22.50	5.75

SD: Standard deviation.

PP: Statistical analysis revealed significant differences in ASIA Sensory PP scores between pre-intervention and post-intervention assessments (χ² = 28.838, P < 0.001). While the mean ASIA Sensory PP score before the application was 62.00 ± 18.42, this value increased to 81.33 ± 22.26 in the first year after the application. Wilcoxon signed-rank tests demonstrated no significant differences in PP scores during the first week (z = -1.342, P = 0.180) and the first month (z = -1.841,P = 0.066). However, significant improvements were observed at 2 months (z = -2.207, P = 0.027), 4 months (z = -2.201,P = 0.028), and 1 year (z = -2.201, P = 0.028), indicating a notable enhancement in sensory perception over the study duration (Table 10). Overall, the findings highlight the effectiveness of the intervention in reducing spasticity and enhancing motor and sensory functions over time, with significant improvements becoming evident at various stages post-intervention.

Table 10 Results of the Friedman test for changes in the American Spinal Injury Association Sensory Score (Pin-Prick values) before and after intervention.

	n	Mean	SD	Mean rank	χ2	df	P value
Preoperative	6	62.00	18.42	1.50
Postoperative week 1	6	63.67	18.13	1.83	28.838	5	0.000
Postoperative month 1	6	68.67	17.47	2.75
Postoperative month 2	6	74.33	19.82	3.92
Postoperative month 6	6	78.33	20.92	5.08
Postoperative month 12	6	81.33	22.26	5.92

SD: Standard deviation.

Incontinence scores (WIS and SFQ values)

The evaluation of incontinence using the WIS and the SFQ values revealed notable improvements following the intervention (Figure 6). These metrics were used to assess both gastrointestinal (GI) and urinary incontinence as well as overall quality of life related to these conditions.

Figure 6 Changes in the incontinence scores (Wexner Incontinence Score and Qualiveen Short Form) following intervention. This figure shows significant improvements in incontinence scores following intervention, as measured by the Wexner Incontinence Score (z = -2.226, P < 0.05) for bowel incontinence and the Qualiveen Short Form (z = -2.214, P < 0.05) for urinary incontinence. WIS: Wexner Incontinence Score; SFQ: Qualiveen Short Form.

WIS: The WIS is a validated instrument that quantifies the severity of GI incontinence, encompassing symptoms such as frequency, urgency, and leakage. Our data indicated a significant reduction in WIS scores post-intervention, reflecting an improvement in GI control and a subsequent enhancement in the patients’ quality of life. The statistical analysis demonstrated a significant decrease in WIS scores from baseline to follow-up, with a calculated z value of -2.226 and a P value < 0.05 (Table 11). This suggests that the intervention had a meaningful impact on alleviating symptoms of GI incontinence.

Table 11 Results of the Wilcoxon signed-rank test for changes in the Wexner Incontinence Score values before and after intervention.

Wexner Incontinence Score	Ranks	n	Mean rank	Sum of ranks	Z score	P value
Preoperative	Negative ranks	0	3.50	21.00	-2.226	0.026
	Positive ranks	6	0.00	0.00
Postoperative	Ties	0
	Total	6

SFQ: The SFQ is a widely used tool for assessing the impact of urinary incontinence on quality of life. It provides a comprehensive measure of how urinary incontinence affects daily functioning and psychological well-being. The results indicated a significant improvement in SFQ scores following the intervention. The statistical analysis yielded a z value of -2.214 and a P value < 0.05 (Table 12), signifying a substantial enhancement in the patients’ quality of life related to urinary control. This improvement reflects a reduction in the frequency and severity of urinary incontinence symptoms, leading to a better overall quality of life.

Table 12 Results of the Wilcoxon signed-rank test for changes in the Qualiveen Short Form values before and after intervention.

SFQ	Ranks	n	Mean rank	Sum of ranks	Z score	P value
Preoperative	Negative ranks	0	3.50	21.00	-2.214	0.027
	Positive ranks	6	0.00	0.00
Postoperative	Ties	0
	Total	6

SFQ: Qualiveen Short Form.

Overall impact on quality of life: Both WIS and SFQ score improvements underscore the potential benefits of the WJ-MSC intervention in managing incontinence. These results indicate that the intervention not only addressed neurological deficits but also contributed to a reduction in the adverse effects of incontinence on daily life. The significant decreases in both GI and urinary incontinence scores highlight the comprehensive nature of the therapeutic benefits observed, supporting the notion that MSC-based treatments may offer substantial improvements in various aspects of patient well-being beyond motor and sensory function.

Safety and adverse events

The WJ-MSC transplantation procedures were generally well-tolerated across all participants, with no serious adverse effects linked to the WJ-MSC injections. The majority of patients exhibited a favorable response to the intervention, without experiencing any major complications. However, 4 patients reported transient, minor complications post-intervention (Table 13): (1) Subfebrile episodes: A total of 4 patients experienced mild, transient fever, categorized as subfebrile. This response was effectively managed with standard antipyretics and resolved without further medical intervention; (2) Moderate headaches: Two patients reported moderate headaches following intramuscular injections. These headaches were managed with standard analgesics and were resolved within a short period; and (3) Localized muscle soreness: Mild muscle soreness at the injection sites was noted in 2 patients. This discomfort was self-limiting and was alleviated with minimal symptomatic treatment.

Table 13 Early and late complications.

Complications		Patient No. 11				Patient No. 21				Patient No. 31				Patient No. 41				Patient No. 51				Patient No. 61
		1st	2nd	3rd	4th	1st	2nd	3rd	4th	1st	2nd	3rd	4th	1st	2nd	3rd	4th	1st	2nd	3rd	4th	1st	2nd	3rd	4th
Early	Infection	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Fever	-	+	+	-	-	-	-	-	+	+	-	-	-	-	-	-	-	+	-	-	+	-	+	-
	Pain	-	-	-	-	-	-	-	-	+	+	+	-	-	-	-	-	+	-	-	-	-	-	-	-
	Headache	-	-	-	-	-	-	-	-	-	-	+	-	-	-	-	-	-	-	-	-	+	+	+	-
	Increased C-reactive protein levels	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Leukocytosis	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Allergic reaction or shock	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Perioperative complications	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Late	Secondary infections	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Urinary tract infections	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Deterioration of neurological status	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Neuropathic pain	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Carcinogenesis	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

¹Administration.

Throughout the 1-year follow-up period, no significant safety concerns or adverse events were reported. There were no instances of severe infections, complications related to anesthesia, allergic reactions, or episodes of shock. Additionally, no evidence of neurological deterioration or progression of SCI was observed. The absence of serious adverse effects and the manageable nature of the minor complications underscore the safety and feasibility of the WJ-MSC transplantation procedure for SCI treatment.

DISCUSSION

SCI is a life-altering event that leads to profound and multifaceted challenges. The neurological deficits resulting from SCI, ranging from complete paralysis to partial loss of function, significantly impact motor and sensory abilities. These deficits often give rise to secondary complications such as chronic pain, pressure ulcers, and autonomic dysfunction, which further exacerbate the patient’s condition. The psychological burden of SCI cannot be understated, as patients frequently grapple with heightened levels of depression, anxiety, and post-traumatic stress disorder. The ripple effects extend to family members and caregivers, who face emotional distress and substantial financial costs associated with long-term care and rehabilitation. Societally, SCI contributes to increased healthcare expenditures and a greater need for specialized care services. Although it was once thought that the central nervous system had limited regenerative capacity, contemporary research indicates that targeted modulation of the injury microenvironment can foster neuroprotection and stimulate repair mechanisms[17].

Historically, SCI treatments included the transplantation of fetal spinal cord tissue, peripheral nerves, Schwann cells, and fibroblasts, aiming to overcome inhibitory factors and create a supportive environment for nerve regeneration[18,19]. More recent strategies have shifted towards the use of MSCs, which exhibit remarkable self-renewal and differentiation abilities. MSCs can give rise to various mesodermal lineages such as bone, cartilage, and muscle[20,21]. The substantial increase in MSC-based clinical trials (over 1138 registered by June 2020) highlights their emerging role as a promising therapeutic approach for SCI and other complex disorders[22]. This rising interest underscores the potential of MSCs to address the challenges associated with traditional treatment modalities.

The dramatic increase in MSC-related clinical trials reflects an expanding exploration of their therapeutic potential. The number of registered trials surged from 220 in 2012 to over 1138 by mid-2020[22]. This escalation indicates a growing recognition of MSCs as a viable treatment for SCI, suggesting that ongoing research may reveal further benefits and optimize therapeutic strategies. Bone marrow (BM) has traditionally been the primary source of MSCs, but its use is limited by the invasive nature of BM aspiration and a decline in cell proliferation and differentiation potential with donor age. Recent research has identified alternative sources, such as fetal tissues including Wharton’s jelly from the umbilical cord, which offer distinct advantages. WJ-MSCs are characterized by their primitive state and reduced immunogenicity, which may enhance their therapeutic efficacy compared to BM-derived MSCs[23]. Wharton’s jelly, a unique connective tissue within the umbilical cord, provides a non-invasive, abundant source of MSCs, facilitating their application in both research and clinical settings[24-26].

Stem cell therapy (SCT) for SCI is designed to promote neuronal regeneration and neurotrophic factor production. This approach involves either endogenous recruitment of stem cells to the injury site or ex vivo manipulation and subsequent transplantation[27]. Research involving MSC-derived neurospheres in animal models has demonstrated improvements in motor function, suggesting that SCT can aid recovery through mechanisms such as neuroprotection, modulation of inflammation, and promotion of axonal growth[28]. These mechanisms highlight the potential for SCT to address the complex pathophysiology of SCI.

The therapeutic efficacy of MSCs is primarily attributed to their paracrine effects, which involve the secretion of a diverse array of growth factors, cytokines, and exosomes. These factors modulate the local microenvironment, promoting tissue repair and reducing inflammation. MSCs secrete anti-inflammatory cytokines and neurotrophic factors that mitigate inflammation and support neuronal survival[29]. Research has demonstrated that MSCs can reduce immune cell activation and lower proinflammatory cytokines such as interleukin-6, which are critical markers of spinal cord inflammation. Additionally, MSC treatment has been associated with increased levels of anti-inflammatory mediators like interleukin-1 receptor antagonist, which further supports the therapeutic potential of MSCs[30].

Study findings and functional improvements

In this study, patients with SCI received MSC injections via intrathecal, intravenous, and intramuscular routes approximately 38.00 ± 54.25 months post-injury. The combination of SCT and intensive neurorehabilitation led to moderate improvements in FIM scores across both motor and cognitive domains (77.50 ± 2.26). Notably, FIM Motor scores increased significantly, while FIM Cognitive scores showed minimal change. It is crucial to differentiate between treatment-induced improvements and spontaneous recovery in such studies[31]. The data indicate that SCT, when administered between 6 months and 12 years post-injury, can enhance neurological and functional outcomes, reinforcing the importance of integrating SCT with comprehensive physiotherapy for optimal results.

This study represents the largest trial to date evaluating the use of allogeneic WJ-MSCs through multiple administration routes and doses in SCI patients. It builds upon earlier pilot studies[13,14]. The treatment was generally well-tolerated, with adverse events being infrequent, mild, and transient. The target dose of 1 × 10⁶ cells/kg for each administration route was deemed safe, with only minor complications such as subfebrile fever, headache, and localized muscle pain, all resolving within 24 h.

The study demonstrated a 13.67-point improvement in the FIM Motor score and a 13.33-point improvement in the ASIA Motor score (Tables 5 and 8) among individuals with chronic SCI. These results are promising, but further validation through larger, controlled studies is necessary. Detailed physical examinations revealed a gradual enhancement in motor function, with ASIA Motor scores increasing from 1.67 ± 2.66 to 12.00 ± 8.10 at the 1-year endpoint. The percentage of patients achieving significant functional outcomes (total FIM Motor score ≥ 91) increased from 46.7% at baseline to 61.7% at 1 year (Table 5). Additionally, significant improvements were observed in sensory scores, with ASIA LT and PP scores showing notable increases.

Significant reductions in spasticity were observed, as indicated by the MA scale. The combined right and left scores improved from 15.83 ± 4.85 to 4.34 ± 3.78, with reductions of 5.83 points in the right values and 5.66 points in the left values. These improvements were sustained at the 1-year follow-up, suggesting that MSCs have a substantial impact on managing spasticity and enhancing spinal cord function. These findings warrant further validation through larger, controlled trials to confirm their robustness.

Chronic SCI often leads to GI dysfunction, which significantly impacts patients’ quality of life. Bowel disorders, which affect approximately 30% of SCI patients, often present greater challenges than bladder or sexual dysfunction[32]. The study utilized the WIS to assess GI function, revealing a 15% improvement (scores decreased from 20 to 17). Urinary dysfunction, prevalent in 70%-84% of SCI patients, was evaluated using the SFQ score, which indicated a 10.9% improvement in bladder function. These findings underscore the broader therapeutic impact of MSC therapy on various aspects of quality of life affected by SCI.

The therapeutic effects of WJ-MSCs in chronic SCI may be attributed to their unique biological properties and multifaceted mechanisms of action. WJ-MSCs are known to secrete a variety of trophic factors, including brain-derived neurotrophic factor, vascular endothelial growth factor, and transforming growth factor beta, which collectively promote neuroprotection, angiogenesis, and modulation of the inflammatory microenvironment[33,34]. These cells can also enhance the recruitment and differentiation of endogenous progenitor cells, potentially facilitating neuronal regeneration[35]. Furthermore, WJ-MSCs possess immunomodulatory capabilities, mitigating the chronic inflammation often observed in SCI and thus reducing secondary tissue damage[35]. Additionally, extracellular vesicles derived from WJ-MSCs play a significant role in intercellular communication, delivering bioactive molecules such as microRNAs and proteins that promote neural plasticity and repair processes[36]. These mechanisms, individually or synergistically, may contribute to the observed improvements in neurological function and quality of life, as evidenced in this study.

Study strengths and limitations

The study’s strengths include its focus on a large cohort of patients with chronic SCI, the use of allogeneic MSCs, and the application of multiple administration routes with restricted cell culture passages. Comprehensive safety assessments conducted over a 1-year period further enhance the credibility of the study. However, the absence of a control group limits the ability to accurately assess the observed benefits. Additionally, the study did not investigate the specific mechanisms underlying MSC therapy, such as growth factor release, anti-inflammatory effects, and exosome-mediated signaling. Future research should address these limitations by incorporating control groups and exploring the underlying mechanisms of MSC therapy. Effective restorative therapies are often enhanced by appropriate training, although this aspect was not fully implemented in the current safety-focused study.

Future research should focus on elucidating the mechanisms underlying MSC therapy, including the identification of key paracrine mediators and optimization of treatment protocols. Multicenter clinical trials are essential to validate the safety and efficacy of MSC therapies and refine treatment strategies for SCI. Further exploration of the mechanisms of action, such as the roles of growth factors, anti-inflammatory effects, and exosomes, will deepen understanding and potentially improve clinical outcomes for SCI patients. Enhanced understanding of these mechanisms will be critical for developing targeted therapies and improving patient care in the context of SCI.

CONCLUSION

This study underscored the potential of WJ-MSCs in treating chronic SCI. Notable improvements in motor and sensory functions, alongside reductions in spasticity and enhancements in quality of life, were observed following WJ-MSC treatment. These findings support WJ-MSC therapy as a promising option for SCI. However, given the absence of a control group and the influence of concurrent pharmacological and rehabilitative interventions, the observed improvements cannot be definitively attributed to WJ-MSCs alone. Further research with controlled trials is necessary to confirm efficacy and elucidate mechanisms of action.