Report of a phase 1 clinical trial for safety assessment of human placental mesenchymal stem cells therapy in patients with critical limb ischemia (CLI)

Abstract

Background: Critical limb ischemia (CLI) is associated with increased risk of tissue loss, leading to significant morbidity and mortality. Therapeutic angiogenesis using cell-based treatments, notably mesenchymal stem cells (MSCs), is essential for enhancing blood flow to ischemic areas in subjects suffering from CLI. The objective of this study was to evaluate the feasibility of using placenta-derived mesenchymal stem cells (P-MSCs) in patients with CLI.

Methods: This phase I dose-escalation study investigated P-MSCs in nine CLI patients who were enrolled into each of the two dosage groups (20 × 10⁶ and 60 × 10⁶ cells), delivered intramuscularly twice, two months apart. The incidence of treatment-related adverse events was the primary endpoint. The decrease in inflammatory cytokines, improvement in the ankle-brachial pressure index (ABI), maximum walking distance, vascular collateralization, alleviation of rest pain, healing of ulceration, and avoidance of major amputation in the target leg were the efficacy outcomes.

Results: All dosages of P-MSCs, including the highest tested dose of 60 × 10⁶ cells, were well tolerated. During the 6-month follow-up period, there was a statistically significant decrease in IL-1 and IFN-γ serum levels following P-MSC treatment. The blood lymphocyte profile of participants with CLI did not significantly differ, suggesting that the injection of allogeneic cells did not cause T-cell proliferation in vivo. We found clinically substantial improvement in rest pain, ulcer healing, and maximum walking distance after P-MSC implantation. In patients with CLI, we performed minor amputations rather than major amputations. Angiography was unable to demonstrate new small vessels formation significantly.

Conclusion: The observations from this phase I clinical study indicate that intramuscular administration of P-MSCs is considered safe and well tolerated and may dramatically improve physical performance and minimize inflammatory conditions in patients with CLI.

Trial registration: IRCT, IRCT20210221050446N1. Registered May 09, 2021.

Keywords: Buerger; Cell therapy; Clinical trial; Critical limb ischemia; Mesenchymal stem cell; Peripheral artery disease; Placenta.

Fig. 1

Clinical trial methodology for intramuscular administration of P-MSC. A P-MSC injected to the ischemic limb following isolation and in vitro expansion, B P-MSCs suppress inflammation and promote immunomodulation by secreting immunomodulatory cytokines, which stimulate the induction of M2 macrophages and increase the number of circulating regulatory T cells, resulting in an increase in interleukin IL-10 and resolution of inflammation. Additionally, MSCs release factors that promote angiogenesis directly. C Efficacy outcomes of P-MSC administration in patient with CLI

Fig. 2

Flowchart: 15 patients recruited in the study and allocated to two groups (low-dose group = 3; high-dose group = 8)

Fig. 3

Change in patients’ serum inflammatory cytokines levels on days 0 (baseline) and 6 months after the first infusion. Analysis of biomarkers on before (baseline) and 6 months after the first infusion demonstrated a significant reduction in IL-1, and interferon-γ (IFN-γ) in all patients with CLI. Serum levels of tumor necrosis factor alpha (TNF-α) decreased in four cases, but the differences were not statistically significant. *P < 0.05; ns, not significant

Fig. 4

Measured markers of CD4 and CD8 and CD25 on patients with CLI at screening and 6-month follow-up after P-MSC implantation

Fig. 5

Changes in collateral artery development on angiography 6 months following implantation of placenta-derived mesenchymal stem cells (P-MSC). A Collateral vessel formation was increased at 6 months after P-MSC administration. B The degree of collateral vessel formation was ranked as 0, + 1, + 2, and + 3 according to the number of collaterals (P value = 0.0625)

Fig. 6

Efficacy parameters: A Ischemic rest pain score measured by verbal analogue scale: 0 = no pain, 10 = worst pain in patient’s life, P < 0.0001. B Ankle brachial index of the treated limb, P = 0.1228. C Walking distance, P = 0.0017. D Vascular quality of life questionnaire-6, P < 0.0001

Fig. 7

Representation of the treated limbs showing the progression of ischemic ulcer/gangrene throughout 6-month follow-up. Patient 3: a 41-year-old man with thromboangiitis obliterans (TAO), Rutherford IV presented with non-healing ulcers on the ankle and lateral midfoot healed completely at 6 months after P-MSC implantation. Patient 4: a 55-year-old man with TAO presented with gangrene of the first and second toes, and a history of amputation of the 3th and 4th toes showed clear healing of ischemic necrosis on foot after amputation at 6 months after P-MSC implantation. Patient 7: A 66-year-old men, TAO with Rutherford-III, initially advanced gangrene and wound infection who diagnosed as needing major amputation, get minor amputation after cellular therapy and whole ulcer surface measurement was largely decreased at 6 months after P-MSC implantation. Patient 9: a 62-year-old man with Rutherford IV (TAO) presented with non-healing ulcers at dorsum of the foot and gangrene of the toe, showed clear healing of ulcer. Gangrene did not progress during the 6-month follow-up