Improvement in long-term survival with mesenchymal stem cell transplantation in systemic sclerosis patients: a propensity score-matched cohort study

Abstract

Background

The intricate and varied clinical presentations of systemic sclerosis (SSc) pose significant challenges for treatment. While several studies have investigated the therapeutic potential of mesenchymal stem cell transplantation (MSCT), the clarity of its long-term outcomes for SSc patients is still lacking.

Methods

Data on MSCT were extracted from the medical records of inpatients at Nanjing Drum Tower Hospital between January 2013 and December 2022. Additionally, Baseline characteristics and survival outcomes were ascertained from medical records and telephone follow-up. Propensity score matching (PSM) was employed to equalize the baseline characteristics of the patient groups, while survival analysis and multivariate Cox regression assessed the relationship between received MSCT and all-cause mortality and disease-specific survival rates in SSc patients.

Results

Of the 333 hospitalized SSc patients, 113 patients underwent MSCT. The log-rank test revealed significantly higher survival rates in the MSCT group compared to the control group (10-year survival rate 89.4% vs. 73.4%, P = 0.002). In the PSM cohort, receiving MSCT significantly reduced mortality (10-year survival 88.0% vs. 79.9%, P = 0.032). Multivariate Cox regression analysis indicated that MSCT was linked to a reduced mortality risk during the follow-up period (HR 0.38, 95% CI 0.19–0.75, P = 0.005). This finding was further confirmed in the matched cohort (HR 0.38, 95% CI 0.18–0.82, P = 0.014). Subgroup analyses revealed that treated with MSCT was correlated with reduced mortality in patients of various demographics, including younger age at diagnosis (≤ 47 years), female, diffuse cutaneous systemic sclerosis (dcSSc) subtype, concurrent arthritis, pulmonary arterial hypertension (PAH), and interstitial lung disease (ILD).

Conclusion

MSCT significantly enhances the survival rate of patients with SSc, with outcomes related to the age at diagnosis. MSCT is particularly indicated for patients with comorbid conditions, including PAH, ILD, digital ulcers, and arthritis, as well as those with severe disease presentations associated with the dcSSc subtype.

Introduction

Systemic sclerosis (SSc) is a rare and complex autoimmune disorder predominantly affecting young women, resulting from a convergence of vascular injuries, immune system hyperactivity, and fibrotic changes across multiple organs [1]. The elevated mortality and disability rates associated with the disease cause anxiety among patients and physicians [2]. To date, effective treatment options for SSc remain limited. Mycophenolate mofetil (MMF) or cyclophosphamide (CTX) are recommended for immunosuppression, particularly in the early stages of SSc [3]. Tocilizumab has recently shown promise for lung involvement in SSc, yet its impact on skin sclerosis warrants further investigation [4]. Furthermore, some studies indicate that Tripterygium wilfordii Hook F (TwHF) may exert beneficial effects on systemic sclerosis-associated interstitial lung disease (SSc-ILD) and skin sclerosis [5, 6]. Nonetheless, clinical research on TwHF's therapeutic efficacy in SSc is limited.

The pathogenesis of SSc is thought to involve vascular injury and dysfunction, attributed to Th2 cell imbalance, increased transforming growth factor-β, and oxidative stress [7, 8]. Mesenchymal stem cells (MSCs) can facilitate tissue repair, possess multilineage differentiation potential, and display immunomodulatory effects. They mitigate skin and organ fibrosis in SSc mouse models through the inhibition of inflammatory factor expression, reduction of reactive oxygen species, and enhancement of T regulatory (Treg) cell proliferation [7, 9]. Furthermore, our previous study showed that allogeneic mesenchymal stem cell transplantation (MSCT) alleviates osteopenia, skin tightness, and immune symptoms in SSc mice by transferring miR-151-5p to recipient bone marrow MSCs, thereby inhibiting IL-4 receptor α expression and down-regulating the mTOR pathway activation [9, 10].

Given these promising preclinical findings, it is crucial to investigate the clinical implications of MSCT in SSc patients. Evidence suggests that allogeneic MSCT may alleviate clinical symptoms and arrest disease progression in SSc patients [11,12,13]. However, most of these studies concentrated on MSCT's effects on lung involvement and skin sclerosis, with a limited number of subjects and a brief duration. Studies comparing the long-term survival rate of MSCT patients with traditional treatment were scarce. Consequently, this study aimed to enroll SSc patients over a nearly decade-long period, to assess if MSCT enhances long-term survival outcomes relative to conventional treatments.

Methods

Study population

We screened SSc patients (aged > 18 years) who were hospitalized in the Department of Rheumatology and Immunology, the Affiliated Drum Tower Hospital of Nanjing University Medical School, between January 2013 and December 2022. They met the 1980 ACR diagnostic criteria for SSc or the 2013 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria [14, 15]. Patients in the trial group underwent MSCT because they were unresponsive or dissatisfied with conventional immunosuppressant therapy, and those who did not undergo MSCT were automatically included in the control group. Following the exclusion of patients with incomplete medical records or lacking follow-up information, the study ultimately enrolled 333 patients, including 113 in the MSCT group and 220 in the control group (Fig. 1). The study protocol was approved by the Ethics Committee of Nanjing Drum Tower Hospital (reference number 2009004).

Fig. 1

The screening flowchart for SSc patients in this investigation. SSc: systemic sclerosis

Criteria for non-response or significant dissatisfaction with conventional immunosuppressive therapy include: continuous or concurrent use of CTX at 500–750 mg/m² per month, MMF at over 1000 mg/day, and other immunosuppressants for over six months without response. Furthermore, the mRSS had increased by at least 20%, and there was significant organ involvement related to SSc.

MSCT protocols

MSCs utilized in this study were extracted from the bone marrow (BM) and umbilical cord (UC) of healthy donors. BM donors, who were healthy family members aged from 18 to 40 years old, were HLA-matched. Isolated bone marrow cells were cultured at a density of 1 × 10⁶ cells/cm² in low-glucose DMEM-LG medium supplemented with 10% FBS. Non-adherent cells were discarded after 72 h, and the medium was refreshed twice weekly. Umbilical cords were harvested from healthy mothers’ post-normal delivery and washed in PBS with antibiotics. Following umbilical cord blood removal, the cords were sectioned into 1 mm² pieces and resuspended in low-glucose DMEM with FBS. Culture conditions were maintained at 37 °C in a 5% CO₂ atmosphere, reaching 80% confluence before passaging at 1 × 10⁴ cells/cm².

All MSCs utilized for transplantation adhered to clinical standards, encompassing morphology, cell viability (> 92%), sterility, and absence of viral contamination. The infused cell concentration was defined as 1 × 10⁶ MSCs per kilogram of body weight, which was determined based on our previous clinical studies in SSc as well as in SLE [13, 16]. The MSC administration carrier consisted of albumin and compound electrolyte solution, with the calculated MSC count suspended within it. The cell suspension was administered slowly via the cubital vein within 30 min.

Clinical variables

Demographic and clinical data, including gender, age at diagnosis, skin subtype, mRSS, comorbidities, and antibody profiles, were collected from patient visit records. Post-diagnosis treatments with immunosuppressants and glucocorticoids were also documented. For patients lacking documented visits in the six months leading up to December 2022, follow-up was conducted via telephone to ascertain their survival status. Demographic and laboratory indicators were compared using data obtained during the initial patient visits. Data from all follow-up visits were utilized to assess and compare complications and the efficacy of immunosuppressant therapies.

Age at diagnosis was defined as when initial non-RP-SSc symptoms were observed, with survival time being the interval from the age at diagnosis to the final visit or death. skin subtype was determined using the classification criteria set forth by LeRoy et al. [17]. The same qualified researcher utilized the mRSS to measure the severity of skin sclerosis. Data on patients receiving prednisone and its equivalents at ≥ 15 mg/day at the final follow-up, as well as those on immunosuppressant therapies for ≥ 6 months, including CTX, MMF, TwHF, and hydroxychloroquine (HCQ), were compiled [13, 18].

PAH was diagnosed when the systolic pressure in the pulmonary artery exceeded 35 mmHg, as determined by echocardiography, or when the mean pulmonary artery pressure (mPAP) was greater than 25 mmHg and the pulmonary capillary wedge pressure (PCWP) was less than 15 mmHg, as assessed by right-heart catheterization (RHC). Experienced radiologists analyzed the presence of ILD in high-resolution computed tomography (HRCT) images according to the internationally recognized criteria established by the American Thoracic Society-European Respiratory Society in 2013 [19]. Laboratory assessments encompassed decreased complement 3(C3) (< 0.8 g/L), and decreased complement 4(C4) (< 0.2 g/L). Additionally, proteinuria was identified by a positive urinary protein test and a quantitative measure exceeding 0.5 g over 24 h.

Statistical analysis

This study used rigorous statistical methods to compare the MSCT and control groups. We utilized independent t-tests for normally distributed continuous variables and Mann–Whitney U tests for those that were not. The chi-square test assessed differences in categorical variables between the groups. Univariate and multivariate Cox regression analyses identified risk factors for all-cause SSc mortality, with results presented as hazard ratios (HR) and 95% confidence intervals (CI). To mitigate potential confounding factors, we conducted propensity score matching (PSM) to ensure comparable risk profiles between the MSCT and control groups [20]. Equilibrium variables in the PSM included age, gender, mRSS, skin subtype, and the use of glucocorticoids and immunosuppressants. The PSM process employed a nearest-neighbor algorithm with a 1:1 matching ratio and a caliper value of 0.2. Covariate balance was assessed using the standardized mean difference (SMD), with an absolute value below 0.1 indicating successful matching.

To verify the stability of our results, we conducted a sensitivity analysis to assess the impact of received MSCT on patient mortality. Initially, we re-analyzed the data, incorporating various covariates to refine the multivariate model. Subsequently, we performed subgroup analyses and interaction tests to explore relationships within patient subsets. Lastly, we calculated the E value to determine the minimum potential impact of unmeasured confounding factors on the observed association, quantifying the risk of result attenuation [21]. The P-value was two-tailed, with statistical significance defined as P < 0.05. Statistical analyses were conducted using R software, version 4.2.2.

Results

Patient characteristics

In this study, the median age of the entire patient cohort was 47.0 years (IQR, 32.0–56.0 years), the median mRSS was 13.0 points (IQR, 7.0–20.0 points), and 273 (82.0%) individuals were female. Table 1 shows the baseline patient characteristics for the MSCT group before and after PSM. Prior to matching, patients in the MSCT group exhibited higher mRSS (IQR 10.0–24.0 vs. 6.0–18.0, P < 0.001) and a higher prevalence of diffuse cutaneous systemic sclerosis (dcSSc) subtype (52.2% vs. 28.6%, P < 0.001) and digital ulcers (28.3% vs. 18.2%, P = 0.033). In contrast, control group patients were older (IQR 37.0–58.0 years vs. 27.0–49.0 years, P < 0.001), had a higher incidence of PAH (39.6% vs. 23.0%, P = 0.003), and were more inclined to receive glucocorticoids (70.0% vs. 55.8%, P = 0.010) in the later stages of the disease.

Table 1 Characteristics of study patients

Following PSM, the study included 204 patients, with 102 in the MSCT group and 102 in the control group. Baseline covariates were well-matched between the two groups (Table 1). The PSM process effectively reduced the SMD in included variables to below 10%, demonstrating a marked enhancement in covariate balance between the treatment groups (Supplementary Fig. 1).

MSCT enhanced long-term survival in SSc patients

We analyzed the impact of MSCT on the long-term survival of SSc patients. Before PSM, the median follow-up time was 4.0 years (IQR, 3.0–7.0 years) for the MSCT group and 3.0 years (IQR, 1.0–6.0 years) for the control group (P = 0.007). We recorded 77 deaths, with causes detailed in Table 2. Among these deaths, six patients were attributed to malignancies, specifically four from lung cancer, one from colon cancer, and one from ovarian cancer; the cause of death remained undetermined in five cases. Following PSM, the total follow-up time between the MSCT and control groups was well-balanced (IQR, 3.0–7.0 years vs. 2.0–6.0 years, P = 0.238) (Table 2). There was no difference in the proportion of causes of death between the two groups. The results of the survival analysis revealed a noteworthy contrast in survival rates between the MSCT group and the control group (10-year overall survival rate 89.4% vs. 73.4%, P = 0.002, Fig. 2A). Furthermore, the matched cohort, treated with MSCT continued to demonstrate a significant decrease in mortality (10-year overall survival 88.0% vs. 79.9%, P = 0.032, Fig. 2B).

Table 2 Comparison of the causes of death between the MSCT and the control groups before and after PSM

Fig. 2

Kaplan–Meier survival curves of SSc patients with or without MSCT before (A) or after (B) PSM were compared. MSCT: mesenchymal stem cell transplantation, HR: Hazard Ratio, CI: Confidence Interval, PSM: propensity score matching

Factors affecting the survival of SSc patients

Employing a multivariate Cox regression model, we evaluated the risk factors for all-cause mortality. This model categorized age and mRSS by median values. Factors with significant impacts in univariate regression or deemed clinically relevant to SSc patient mortality were incorporated into the multivariate model (Supplementary Table 1). The multifactorial analysis indicated that MSCT was linked to a 62% reduced risk of death during the follow-up period (HR 0.38, 95% CI 0.19–0.75, P = 0.005) (Fig. 3A). Furthermore, the study revealed lower mortality among women and patients receiving immunosuppressant medications. Conversely, factors such as age above 47 years, dcSSc, comorbid PAH and dysphagia, and glucocorticoid use were correlated with higher mortality rates in SSc patients. Consistently, in the matched cohort, receiving MSCT significantly reduced mortality among SSc patients during the follow-up period (HR 0.38, 95% CI 0.18–0.82, P = 0.014) (Fig. 3B). Notably, E-values of 3.29 (95% upper CI: 1.74) and 3.29 (95% upper CI: 1.56) for MSCT in the unmatched and matched cohorts, respectively. This suggests that unmeasured confounding factors must possess a substantial effect size to obscure the derived effect estimates (Supplementary Fig. 2).

Fig. 3

Multivariate Cox regression analysis examined forest plots of predictors of mortality in SSc patients before PSM (A) or after PSM (B). mRSS: modified Rodnan skin score, dcSSc: diffuse cutaneous systemic sclerosis, PAH: pulmonary arterial hypertension, ILD: interstitial lung disease, MSCT: mesenchymal stem cell transplantation, SSc: systemic sclerosis, PSM: propensity score matching, HR: Hazard Ratio, CI: Confidence Interval

Independent influence of MSCT on mortality across subgroups

The relationship between underwent MSCT and mortality was noted in a broad subgroup of SSc patients (Table 3). Received MSCT was linked to lower mortality rates among patients with a female, age ≤ 47, the dcSSc subtype, and concurrent digital ulcers, arthritis, dysphagia, PAH, and ILD. Furthermore, the protective effect of treatment with MSCT was evident in patients without proteinuria, and those not receiving glucocorticoid therapy. In laboratory assessments, patients testing abnormal C3 and C4, negative for anti-Scl-70, ANA, and positive for anti-Ro52KD appeared to exhibit a better response to MSCT. Concurrently, significant interactions were observed between MSCT and C4 reduction (P for interaction = 0.031). This result indicates that the efficacy of MSCT could be influenced by C4 levels, offering a novel angle for the development of personalized treatment approaches and highlighting the necessity for further investigation into the underlying biological mechanisms.

Table 3 The mortality risk ratio of patients with SSc using MSCT before PSM

Sensitivity analyses

In select sensitivity analyses, there was a modest attenuation in the risk of death. Nonetheless, most of the results demonstrated remarkable consistency across all sensitivity analyses. For instance, initial analyses were confined to minimal models including only age and gender, with subsequent incremental inclusion of covariates such as skin subtype, medication, comorbidities, and laboratory tests (Supplementary Table 3). Additionally, we conducted analyses treating age and mRSS as continuous variables (Supplementary Table 4). The associations observed in these sensitivity analyses were consistent within the patient cohort following PSM, showing no indication of effect moderation by these factors.

Safety of MSCT

Following MSCT, 11 patients experienced short-term adverse reactions within 48 h post-infusion: One patient developed hematuria the following morning, likely due to hemorrhagic cystitis; one experienced a mild headache during the infusion process. One patient reported dizziness, blurred vision, and elevated blood pressure post-treatment. One patient experienced occasional tinnitus on the first day following MSC infusion; one was diagnosed with an acute upper respiratory tract infection, presenting with fever, vomiting, and a mildly sore throat on the first day post-infusion. One patient developed oral herpes; one complained of cough and expectoration; one reported abdominal distension; three experienced post-treatment fever and chills. In addition, the records showed that one patient had developed cervical cancer two years post-infusion, another patient was diagnosed with rectal adenocarcinoma two years later, and a third patient had gastric cancer three years following the MSC infusion. A comprehensive list of adverse reactions is provided in Supplementary Table 2.

Discussion

The significant efficacy of MSCT on the long-term survival of SSc patients was confirmed in our single-center, PSM cohort, controlled observational study involving hospitalized patients. A noteworthy finding was that, across predefined primary endpoints, MSCT use seemed to yield a better prognosis for patients with more severe disease and concurrent ILD and PAH. This association remained independent of all identified confounders and demonstrated reliability in extensive sensitivity analyses. The results suggest the potential effectiveness of MSCT in a broader patient population, beyond those typically included in highly selective and enriched randomized controlled trial (RCT) populations.

Mortality among SSc patients has decreased in recent years owing to improved diagnostics and therapeutics, yet significant heterogeneity persists in disease management [22]. In a prospective study examining mortality in high-risk or early dcSSc, 86% received immunosuppressant therapy, predominantly MMF, Still, skin deterioration occurred in approximately 20%, and a continuous decline in forced vital capacity (FVC) was observed in nearly 20% [22]. Homberg et al. [23] administered monthly intravenous CTX to 75 patients with SSc-ILD, observing no statistically significant changes in FVC at 12, 24, and 36 months. Consequently, most rheumatologists concur on the urgent need to explore innovative and effective treatment strategies for SSc patients experiencing rapid disease progression or a marked decline in quality of life [24]. This situation has propelled MSCs into the spotlight due to their distinctive immunomodulatory capabilities.

Infusion of MSCs has been reported to secrete various cytokines and growth factors that regulate the immune response. For example, MSCs inhibit T cell and B cell proliferation, reduce pro-inflammatory cytokine release, and thus mitigate the autoimmune response [9]. Additionally, MSCs play a crucial role in fibrosis regulation. Studies demonstrate that MSCs attenuate fibroblast activation and inhibit the increase in collagen content by suppressing pro-inflammatory cytokines, specifically interleukin (IL)−33, thereby reducing fibrosis [25]. In the HOCl-induced SSc mouse model, MSC treatment significantly reduced skin and lung fibrosis, highlighting its potential for anti-fibrosis therapy [7]. Most importantly, MSCs secrete pro-angiogenic factors, such as vascular endothelial growth factor, which promote the formation of new blood vessels and improve tissue oxygen supply [26]. This effect is particularly important for SSc patients, as vascular disease is a key factor in causing tissue hypoxia and subsequent fibrosis. Thus, we believe that MSCT could have a safe and effective impact on SSc patients.

Our study established that MSCT was a viable therapeutic alternative for SSc patients who do not respond well or are highly dissatisfied with traditional immunosuppressant treatments. Recent retrospective studies demonstrate that MSCT can continuously improve or stabilize the symptoms of skin, ILD, and PAH in SSc patients [13]. Nonetheless, numerous aspects of MSCT remain unclear, such as its efficacy in treating specific complications and ensuring long-term safety. For over a decade, MSCT has been implemented at Nanjing Drum Tower Hospital to address a variety of autoimmune diseases [27, 28]. Our study encompassed the largest cohort of SSc patients treated with MSCT in the past decade. The study showed that MSCT could lower the mortality rate among SSc patients, regardless of the uniformity of their baseline conditions. This finding was further corroborated by a subsequent multifactorial Cox regression analysis. Further subgroup analyses revealed that the mortality-reducing effect of MSCT was associated with age at diagnosis and patient gender, demonstrating higher survival rates among younger patients (≤ 47 years) and females, highlighting the applicability of MSCT in different populations. Concurrently, patients with dcSSc subtypes and complications appear to derive greater benefits from MSCT. This aligns with previous findings that individuals with severe disease exhibit a more robust response to MSCT [13, 22, 27].

Pulmonary involvement is consistently the most prevalent complication and the primary cause of mortality in SSc patients [29]. In the study by Hu et al. regarding mortality causes in SSc patients, despite 85.5% having ILD, the majority maintained lung function, suggesting that PAH is the predominant cause of death rather than ILD [30]. In keeping with prior studies, our research likewise observed no significant variation in mortality rates among patients with ILD. An RCT indicated that HSCT could enhance the survival rate of SSc patients in comparison to CTX [11]. Consequently, the 2017 EULAR guidelines recommended HSCT for patients with rapidly progressive SSc-ILD [31]. However, considering the treatment-related side effects and early TRM, the guidelines suggest cautious patient selection for this treatment approach. In SSc-PAH, primary treatments include endothelin receptor antagonists (ERAs) and phosphodiesterase-5 (PDE5) inhibitors [32]. However, the efficacy of these medications is variable, and safety profiles differ [33]. Consequently, our investigation centered on the impact of MSCT on SSc-ILD and SSc-PAH. Multivariate analysis indicated that MSCT significantly reduces mortality in patients with pulmonary complications. It also offers potential treatment options for these serious conditions in patients with SSc.

Musculoskeletal involvement significantly impairs mobility and diminishes the quality of life for SSc patients [34]. Wu et al. found that miR-140-3p carried by mesenchymal stem cell extracellular vesicles (MSC-EVs) inhibited chondrocyte apoptosis and cell-like synovial cell proliferation by down-regulating SGK1 expression and ultimately reduced musculoskeletal injury in mice [35].Our findings also indicate that MSCT can yield more favorable outcomes for SSc patients with arthritis. On the other hand, subgroup analysis indicated that MSCT could decrease the glucocorticoid dosage required in later stages of the disease, consequently lowering the mortality risk. A study on the association between copy number (CN) variations in the C4 and SSc risk suggested that increased C4 CN may confer protection against SSc [36]. This finding partially accounts for the observed significant interaction between C4 variations and MSCT effects on the survival of SSc patients in our study. Nonetheless, due to the sample size limitations, the precise effect of this interaction requires further confirmation. Despite these considerations, adjusting for C4 as a covariate in our sensitivity analyses did not obscure the protective effect of MSCT on SSc patients. In addition, our long-term follow-up demonstrated no severe adverse reactions among MSCT recipients, with all transient effects resolving following appropriate treatment or without intervention. Although three patients developed tumors within three years post-MSCT, no correlation was found with the treatment, considering the inherent high cancer risk in SSc patients [37, 38].

Acknowledging the limitations of this study, despite verifying the stability of outcomes through PSM and multiple sensitivity analyses, certain mortality associated complications in the control group, potentially elevate the risk of death. Nonetheless, by performing multivariate Cox analysis, results continue to demonstrate a favorable outcome for MSCT. Additionally, the hypothesis regarding the efficacy of MSCT in a broader patient population warrants validation through a large RCT. The principal strength of this study is overcoming the constraints of a small sample size and including a diverse participant pool, thereby enhancing the validation of the results. Furthermore, the study's long-term follow-up contributed to the comprehensiveness of the data, while the substantial control group enhanced the precision of the interpretation of the findings.

Conclusion

In summary, this single-center, propensity score-matched, retrospective study confirms that MSCT significantly enhances long-term survival in SSc patients. Furthermore, the study's findings were uniformly positive across the majority of subgroups, demonstrating the efficacy of MSCT in reducing mortality among patients diagnosed at a younger age (≤ 47 years), with the dcSSc subtype, and those having concurrent PAH, ILD, digital ulcer, and arthritis, when compared to the control group. Additionally, MSCT elicited a more favorable response among female patients. These findings offer robust evidence of the efficacy and safety of MSCT for a diverse patient population.