Identifying NOTCH signaling-specialized hematopoietic supportive subpopulation from mesenchymal stem cells

Human umbilical cord mesenchymal stem cells (UC-MSCs) are widely studied as a promising cellular therapy for a variety of diseases due to their accessible sources, less of ethical concerns, and capacities for immunomodulation and tissue repair. However, cellular heterogeneity arising during in vitro expansion imposes challenges in quality control for clinical applications and transform, requiring further investigation. Two fetal umbilical cords were obtained from Sinogene C&G Stem Cells Co., Ltd. Primary UC-MSCs were isolated and subjected to continuous in vitro passaging culture. The harvested cells were sorted for single-cell RNA sequencing. A total of 78,178 cells and 14 subpopulations were screened for downstream bioinformatic analysis. Experimental validation was performed through both in vitro cell assays and in vivo animal studies. Mid-passage UC-MSCs showed more favorable functional performance, based on differential gene expression and functional enrichment analysis. Furthermore, we obtained an optimal subpopulation (C8) during clinical-grade cell culture by holistic evaluation of stemness, hematopoietic support capacity, and immunomodulatory potential. The stemness maintenance of C8 subpopulation was closely associated with NOTCH signaling pathway, as identified by cell–cell communication network analysis. Further analyses established NOTCH2 as the dominant receptor mediating NOTCH signaling in UC-MSCs. Notably, we identified MLPH and LPXN as signature markers specifically enriched in the C8 subpopulation. MLPH high LPXN high UC-MSCs displayed higher hematopoietic support and immunosuppression capabilities compared to MLPH low LPXN low UC-MSCs. Mid-passage UC-MSCs demonstrate favorable characteristics for clinical applications. The UC-MSC subpopulation with high NOTCH signaling activity exhibits enhanced hematopoietic support and immunosuppressive capabilities. MLPH and LPXN serve as ideal markers for isolating this functionally subpopulation.