Human multipotent mesenchymal stromal/stem cells (MSCs) have been utilized in cell therapy for various diseases and their clinical applications are expected to increase in the future. However, the variation in MSC-based product quality due to the MSC heterogeneity has resulted in significant constraints in the clinical utility of MSCs. Therefore, we hypothesized that it might be important to identify and ensure/enrich suitable cell subpopulations for therapies using MSC-based products. In this study, we aimed to identify functional cell subpopulations to predict the efficacy of angiogenic therapy using bone marrow-derived MSCs (BM-MSCs). To assess its angiogenic potency, we observed various levels of vascular endothelial growth factor (VEGF) secretion among 11 donor-derived BM-MSC lines under in vitro ischemic culture conditions. Next, by clarifying the heterogeneity of BM-MSCs using single-cell RNA-sequencing analysis, we identified a functional cell subpopulation that contributed to the overall VEGF production in BM-MSC lines under ischemic conditions. We also found that leucine-rich repeat-containing 75A (LRRC75A) was more highly expressed in this cell subpopulation than in the others. Importantly, knockdown of LRRC75A using small interfering RNA resulted in significant inhibition of VEGF secretion in ischemic BM-MSCs, indicating that LRRC75A regulates VEGF secretion under ischemic conditions. Therefore, LRRC75A may be a useful biomarker to identify cell subpopulations that contribute to the angiogenic effects of BM-MSCs. Our work provides evidence that a strategy based on single-cell transcriptome profiles is effective for identifying functional cell subpopulations in heterogeneous MSC-based products.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum-free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)-QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum-free expansion media with VEGF, Flt-3 ligand, TPO, IL-6, and SCF. The vascular regeneration capability of MNC-QQ cells pre- or post-QQc was evaluated with an EPC colony-forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre- and post-MNC-QQc cells from diabetic donors were injected into a murine wound-healing model using Balb/c nude mice. The percentage of wound closure and angio-vasculogenesis was then assessed. This study revealed vasculogenic, anti-inflammatory, and wound-healing effects of MNC-QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound-healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient-based vascular and regenerative therapy for patients with diabetes mellitus.