Background <p>Acute graft-versus-host disease (aGVHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), particularly if treatment is refractory. Mesenchymal stromal cells (MSCs) have demonstrated promising therapeutic effects in aGVHD, due to their well-described immunomodulatory properties. Based on the importance of maternal-fetal interface immune tolerance, the umbilical cord may provide a superior tissue source of MSC. This study aimed to investigate the safety and efficacy of human umbilical cord derived MSCs (hUC-MSCs) delivered as salvage therapy for steroid-refractory (SR)-aGVHD.</p> Methods <p>This phase Ib/IIa, multicenter, open-label clinical trial of a third-party, off-the-shelf preparation of hUC-MSCs enrolled grades II to IV SR-aGVHD patients. This trial consist of 2 parts: a phase Ib dose escalation part using a standard 3 + 3 design, which planed twice weekly i.v. infusions of hUC-MSCs, at 0.5 × 10⁶ per kg, 1.0 × 10⁶ per kg and 2.0 × 10⁶ per kg for 3 weeks, to determine the maximum tolerated dose and recommended phase 2 dose (RP2D). Phase IIa of this work is an expansion cohort study of hUC-MSCs at RP2D. The primary endpoint of the study was safety and RP2D. The key secondary endpoints were efficacy of the overall response rate (ORR) at Day 28.</p> Results <p>The study enrolled 25 patients with SR-aGVHD who received different doses of hUC-MSCs treatment. Safety analysis showed that no dose-limiting toxicity was observed in all dose groups. An ORR of 80.0% was achieved by day 28, with a complete response rate of 40.0% and a partial response rate of 40.0%. The incidence, severity, and type of adverse events (AEs) were similar across different dose groups, showing no obvious correlation with dose. The RP2D was determined to be 1.0 × 10⁶ per kg. The 1-year overall survival rate was 74.3%. The 1-year incidence of non-relapse mortality and relapse of the underlying malignancy after SCT was 17.0% and 8.4%, respectively. Biomarker analysis showed that in responding patients, the proportion of CD8 + central memory T cells and CXCR-10 levels significantly increased at 24&#xa0;h after hUC-MSCs infusion.</p> Conclusions <p>The remarkable efficacy and favorable safety profile of hUC-MSCs offer a promising salvage therapy for patients with severe SR-aGVHD.</p> Trial registration <p>CTR20221330. Registered 06 June 2022, <a href="http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml">http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml</a>.</p>