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Introduction
Critical limb ischemia (CLI), the most severe manifestation of peripheral arterial disease characterized by ischemic rest pain, non-healing ulcers, or gangrene (1), presents a significant challenge in management, with approximately 5%∼20% of patients not being candidates for conventional revascularization techniques and facing a 6-month amputation rate of 10%∼40% (2, 3). These sobering statistics underscore the urgent need for novel therapeutic approaches.
To address these challenges, stem cell therapy has emerged as a promising approach for therapeutic angiogenesis in patients with CLI who are not suitable for conventional revascularization (4). Various types of stem cells, including bone marrow-derived mononuclear cells (BM-MNCs) and mesenchymal stem cells (MSCs), have demonstrated potential in promoting neovascularization and improving limb perfusion. However, existing stem cell therapies face several limitations. Autologous BM-MNCs require invasive harvesting procedures and may have reduced therapeutic potential in patients who have comorbidities (5). Moreover, the use of single-cell suspensions of MSCs often results in poor cell retention and survival at the target site, limiting their therapeutic efficacy (6).
Adipose tissue-derived mesenchymal stem cell clusters (ADMSCCs) offer several advantages over other stem cell types, including abundance, ease of harvest, and potent angiogenic properties (6). Allogeneic ADMSCCs provide immediate availability, standardized quality, and potentially higher potency in patients with comorbidities (7). To address the limitations of existing therapies, our study employs a novel approach using allogeneic ADMSCCs. These cell clusters have demonstrated improved cell retention and survival compared to single-cell suspensions, potentially leading to more effective therapeutic efficacy (6, 8).
Building on these promising characteristics, we conducted a Phase 1/2a clinical trial to evaluate the safety, tolerability, and efficacy of ADMSCCs in patients with CLI due to peripheral artery stenosis and occlusive disease. Our hypothesis is that the administration of ADMSCCs will be safe and well-tolerated, and may lead to improvements in limb perfusion, pain relief, and wound healing in patients with CLI. This study represents a crucial step in the clinical translation of advanced cell therapy for peripheral vascular diseases, potentially overcoming the limitations of current stem cell therapies by improving cell retention, survival, and therapeutic efficacy.
Materials and Methods
Patient selection
Patients aged 19 years or older diagnosed with CLI due to peripheral artery stenosis and occlusive disease (Rutherford category 4, 5, or 6) that had not improved after at least 3 months of drug treatment were eligible for enrollment. The exclusion criteria included: expected life expectancy less than 6 months; surgery or interventional procedure for CLI within 3 months before screening; candidacy for interventional procedures or surgery; history of other cell therapies; systemic immunosuppressive treatment within 3 months; history of malignancy within 5 years (with some exceptions); major bleeding or predisposing hematological conditions within 3 months; pregnancy or inadequate contraception; participation in another clinical trial within 3 months; prohibited concomitant medications; alanine aminotransferase or aspartate aminotransferase>2x upper limit of normal; estimated glomerular filtration rat<30 mL/min/1.73 m2; history of allergy to the investigational product; and investigator judgment of unsuitability.
Study design
This study was approved by the Institutional Review Board of Samsung Medical Center (IRB No. 2020-04-033, No. 2021-12-115). Written consent for the procedures was obtained from all patients. This was a Phase 1/2a, open-label, single-center clinical trial of allogeneic ADMSCCs in patients with CLI. A control group was not implemented in this Phase 1/2a study as the enrolled patients had exhausted all conventional therapeutic options and were ineligible for standard revascularization procedures. The absence of viable alternative treatments for this specific patient cohort required the adoption of a single-arm design.
The study consisted of two sequential phases: Phase 1 utilized a 3＋3 dose-escalation design to evaluate tolerability. After screening and a 2-week washout period, eligible subjects received a single administration of the investigational product and were monitored for adverse events. Follow-up evaluations occurred at 4, 12, and 24 weeks. Dose-limiting toxicity (DLT) was assessed based on grade≥3 adverse reactions (Common Terminology Criteria for Adverse Events [CTCAE] version 5.0). The maximum tolerated dose (MTD) was defined as the highest dose where ≤1 of 6 subjects experienced a grade ≥3 adverse reaction (CTCAE version 5.0).
Phase 2a commenced if tolerability was confirmed in Phase 1. Using sequential assignment, subjects received a single administration of the investigational product at the determined dose. Follow-up evaluations occurred at 4, 12, and 24 weeks. Phase 1 subjects continued efficacy evaluations at 12 and 24 weeks and were included in the Phase 2a efficacy analysis.
Materials and cellular characteristics
Human adipose-derived mesenchymal stem cells (hAd-MSCs) were isolated from donor tissue following the Ministry of Food and Drug Safety guidelines and expanded under the Good Manufacturing Practices conditions. For 3D spheroid formation, hAd-MSCs (10,000/well) were seeded onto MBP-FGF2-coated plates and cultured for 24 hours. The ADMSCCs were manufactured in two formulations: low dose (1,000 spheroids) and high dose (10,000 spheroids).
The ADMSCCs demonstrate multipotent differentiation capacity into mesenchymal lineages, including adipogenic, osteogenic, and chondrogenic cells. Flow cytometric analysis confirmed positive expression of characteristic MSC surface markers (CD29, CD73, CD90, CD105) and absence of hematopoietic markers (CD14, CD19, CD34, CD45). The cells consistently secreted angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, hepatocyte growth factor, and interleukin-8. Genetic stability was confirmed through both karyotype analysis and short tandem repeat testing, with no chromosomal abnormalities or genetic alterations detected across multiple passages, supporting their suitability for clinical application.
Procedure
Under general or spinal anesthesia, 1 mL of the cell product was withdrawn from a vial and diluted with saline to a total volume of 20 mL. This solution was injected into 15∼20 intramuscular sites along the course of the tibial and peroneal arteries below the knee in the ischemic region. Subjects were monitored for adverse events until the following day and discharged if no reactions were observed.
Endpoints
The primary endpoints were safety-related, focusing on the determination of the MTD based on DLT occurrence in Phase 1, and the incidence and severity of adverse events in both phases. Secondary endpoints included efficacy measures: changes in ischemic pain intensity (100 mm visual analogue scale [VAS]), pain-free walking distance (PFWD) (treadmill test), toe-brachial index (TBI), ankle-brachial index (ABI), and ulcer size. These were assessed at 4, 12, and 24 weeks post-treatment and compared to baseline. Additional safety endpoints included changes in laboratory tests (hematology, chemistry, urinalysis, immunological tests), vital signs, and physical examination findings.
Statistical analysis
All adverse events were coded using the Medical Dictionary for Regulatory Activities and graded according to the CTCAE version 5.0. Treatment-emergent adverse events were categorized as local or systemic and analyzed by treatment group.
Continuous variables including laboratory parameters, vital signs, and physical examination findings were summarized using descriptive statistics. Changes from baseline were analyzed using paired t-tests or Wilcoxon’s signed-rank test based on normality assumptions. Between-group comparisons employed two-sample t-tests or Wilcoxon’s rank-sum test as appropriate.
For efficacy endpoints (ischemic pain intensity VAS, TBI, ABI, PFWD, and ulcer area), longitudinal analyses were conducted at baseline, 4, 12, and 24 weeks using mixed models with autoregressive covariance structure. Categorical variables were analyzed using McNemar’s test for within-group changes and chi-square or Fisher’s exact test for between-group comparisons. Statistical significance was set at p<0.05 for all analyses. All statistical analysis was performed with SAS version 9.14 (SAS Institute Inc.).
Results
Patient characteristics
Between November 4, 2020, and April 22, 2024, 20 patients diagnosed with CLI were treated with ADMSCCs. Patient characteristics are presented in Table 1. Of the total participants, 19 (95.0%) were male. The mean age was 44.6±11.56 years in group 1 and 52.90±12.57 years in group 2, with no significant difference between the groups. All patients were classified as Rutherford category 4, except for one patient in group 2 who was classified as category 5. There were no significant differences between the two groups in baseline characteristics.
Safety
Intramuscular injection of ADMSCCs was well tolerated by all patients. No deaths or serious adverse events were observed during the Phase 1 clinical trial. Adverse events are summarized in Table 2. The table provides a comprehensive overview of the events, management, and result of adverse events observed during the study period. There were two treatment-related adverse events, both involving injection site pain, which resolved with medication. In terms of tolerability, DLT was observed, leading to the determination of the MTD as 1×108 cells/1 mL/vial. Post-treatment safety assessments, including laboratory tests, vital signs, physical examinations, and electrocardiograms, revealed no notable concerns regarding tolerability and safety.
Efficacy
PFWD and VAS: Baseline ischemic pain intensity (VAS) was measured at 49.10±20.17 mm in group 1, 52.90±19.87 mm in group 2, and 51.00±19.59 mm for all subjects. At 24 weeks, ischemic pain intensity was measured at 7.70±9.57 mm in group 1, 1.50±3.10 mm in group 2, and 4.60±7.62 mm for all subjects. VAS scores decreased significantly at 4, 12, and 24 weeks post-ADMSCCs administration compared to baseline (p<0.05), demonstrating a reduction in ischemic pain intensity (Fig. 1). This effect was maintained up to 24 weeks, with no significant differences between the experimental groups (p>0.05), suggesting no dose-response correlation.
Baseline PFWD was measured at 195.70±68.38 m in group 1, 199.60±69.48 m in group 2, and 197.65±67.13 m for all subjects. At 12 weeks, PFWD was measured at 221.90±51.07 m for all subjects. PFWD showed a significant increase at the 12-week time point for all study subjects (p=0.029). An increasing trend was maintained from baseline through 4, 12, and 24 weeks, suggesting an improvement in limb ischemia following ADMSCCs administration (Fig. 2). There were no significant differences between the experimental groups, indicating no dose-response correlation.
ABI and TBI: No significant changes in ABI or TBI were observed compared to baseline (Fig. 3, 4) and there were no significant differences between the experimental groups (p>0.05).
Ulcer area: Of the 20 test subjects who participated in this clinical trial, only one developed an ulcer. Information regarding the ulcer area of this subject is presented in Table 3. It was observed that the ulcer area gradually decreased at 4, 12, and 24 weeks after administration of the investigational drug compared to the baseline. For the remaining 19 subjects who had no ulcers at baseline, no new ulcers developed in the area of CLI during the clinical trial.
Discussion
In the last decade, therapeutic angiogenesis has been explored as a treatment for patients with CLI who are ineligible for surgical or interventional options (9, 10). Previous studies, including randomized controlled trials and meta-analyses, have demonstrated that stem cell-based therapies are safe and effective for treating CLI (11-13). The therapeutic effects of these treatments appear to be primarily mediated by the angiogenic of stem cells (14, 15).
In this study, ADMSCCs were administered at two different doses to two groups of 10 patients each. Statistical analysis confirmed no significant demographic differences between the groups at baseline (p>0.05), including age, comorbidities, and smoking history. The demographic homogeneity between groups strengthens our ability to compare treatment outcomes. Furthermore, our comprehensive literature review and analysis suggest that these demographic factors would not be expected to significantly influence treatment outcomes.
We analyzed the safety profiles and therapeutic efficacy measures (TBI, ABI, VAS, and PFWD) between low-dose and high-dose groups. Our objective was to determine the optimal therapeutic dose for future clinical applications through comparative analysis. Statistical comparison between the groups showed no significant differences in therapeutic outcomes (p>0.05), while both doses demonstrated acceptable safety profiles. Notably, the low-dose treatment achieved satisfactory therapeutic efficacy comparable to the high dose across all measured parameters. This finding is particularly significant as it suggests that effective treatment can be achieved with a lower cell dose. Based on these results, we recommend the lower dose for future clinical applications, as it offers an optimal balance of efficacy, safety, and practical considerations.
We used allogeneic ADMSCCs for therapeutic angiogenesis in our study, but other published studies on therapeutic angiogenesis have used bone BM-MNCs or MSCs (16, 17). Autologous BM-MNCs require invasive harvesting procedures and may have reduced therapeutic potential in patients with comorbidities (5). Moreover, the use of MSCs single-cell suspensions often results in poor cell retention and survival at the target site, limiting their therapeutic efficacy (6). ADMSCCs offer several advantages over other stem cell types, including abundance, ease of harvest, and potent angiogenic properties (6). Allogeneic ADMSCCs provide immediate availability, standardized quality, and potentially higher potency in patients with comorbidities (7).
The trial demonstrated no significant safety concerns related to the ADMSCCs, as evidenced by the absence of serious adverse events and dose-limiting toxicities across both dose groups. Local adverse events included injection site pain in two participants (20%) in the high-dose group. No dose-limiting toxicities were observed, and the MTD was determined to be 1×108 cells/mL/vial. These adverse events are consistent with the safety profiles reported in similar trials using human umbilical cord blood-derived mesenchymal stem cells or autologous bone marrow-derived stem cells, indicating a favorable safety profile for future clinical use (8, 18).
The efficacy results, evaluated through several key clinical measures, suggest that allogeneic ADMSCCs therapy could be an effective alternative for patients with CLI who are not candidates for traditional revascularization techniques. A significant reduction in ischemic pain, as measured by the VAS, was observed at 4, 12, and 24 weeks following treatment, with P-values indicating strong statistical significance (p<0.0001 for most comparisons). The increase in PFWD, particularly the significant improvement at the 12-week timepoint, further highlights the potential benefits of this intervention for patients suffering from CLI. Importantly, these improvements persisted through the 24-week observation period. These findings suggest potential benefits in daily activities and reduced pain medication dependence for patients who have been unresponsive to conventional treatments, potentially leading to improved quality of life for those who previously showed little to no progress with other therapeutic approaches. Similar findings have been reported in previous studies, including those by Kim et al. (14), which demonstrated increased collateral vessel formation following autologous stem cell transplantation. Additionally, while the reduction in ulcer area observed in one participant and the lack of new ulcer formation in other participants suggest potential benefits for tissue regeneration, the limited number of participants with ulcers makes it challenging to draw firm conclusions. Future studies with larger sample sizes will be necessary to validate these findings.
In this study, no significant changes were observed in ABI and TBI. Previous studies have also reported inconsistent findings regarding these parameters, with some showing improvements while others did not. Tateishi-Yuyama et al. (15) initially observed significant changes in ABI, but Matoba et al. (19) found that these effects diminished in the long-term outcome. Matoba et al. (19) suggests that while ABI effectively reflects large arterial status, it may not adequately capture the microvascular changes induced by stem cell therapy. Consequently, despite meaningful clinical improvements, ABI may fail to reflect therapeutic benefits, highlighting its limitations as a reliable marker for assessing long-term treatment effects. Similar to previous studies, our research demonstrated significant clinical improvements, including reduced ischemic pain and positive trends in both PFWD and ulcer size. However, despite these improvements, ABI and TBI measurements did not show significant changes, suggesting that traditional vascular indices may not fully capture the microvascular benefits of ADMSCCs therapy. Future studies should consider incorporating more sensitive assessments, such as tissue oxygenation metrics or advanced imaging techniques, to better evaluate the therapeutic impact on microcirculation.
To address the limitations of this study, including the small sample size and lack of a control group, future research should focus on larger, randomized trials. These trials should specifically assess improvements in key outcomes such as pain reduction, walking distance, ulcer healing, and overall quality of life, in comparison to standard care options. Long-term follow-up is needed to evaluate the durability of the therapeutic benefits and to identify any late-onset adverse effects.
In conclusion, this clinical trial provides initial evidence supporting the safety and potential efficacy of allogeneic ADMSCCs therapy for CLI. These findings align with previous studies indicating the value of stem cell-based interventions for improving patient outcomes in severe peripheral artery diseases. The observed reduction in ischemic pain, increase in PFWD, and overall safety profile suggest that this therapy could become a viable new treatment option for patients who have limited alternatives. Future Phase 3 trials should employ a double-blind, placebo-controlled design with a larger and more diverse patient population. This more robust study design will be essential to validate the promising trends observed in our current study and enable broader generalization of results through increased statistical power.
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