Single-cell Transcriptomic Analysis Reveals the Cellular Heterogeneity of Mesenchymal Stem Cells

Ex vivo-expanded mesenchymal stem cells (MSCs) have been demonstrated to be a heterogeneous mixture of cells exhibiting varying proliferative, multipotential, and immunomodulatory capacities. However, the exact characteristics of MSCs remain largely unknown. By single-cell RNA sequencing of 61,296 MSCs derived from bone marrow and Wharton’s jelly, we revealed five distinct subpopulations. The developmental trajectory of these five MSC subpopulations was mapped, revealing a differentiation path from stem-like active proliferative cells (APCs) to multipotent progenitor cells, followed by branching into two paths: 1) unipotent preadipocytes or 2) bipotent prechondro-osteoblasts that were subsequently differentiated into unipotent prechondrocytes. The stem-like APCs, expressing the perivascular mesodermal progenitor markers CSPG4/MCAM/NES, uniquely exhibited strong proliferation and stemness signatures. Remarkably, the prechondrocyte subpopulation specifically expressed immunomodulatory genes and was able to suppress activated CD3⁺ T cell proliferation in vitro, supporting the role of this population in immunoregulation. In summary, our analysis mapped the heterogeneous subpopulations of MSCs and identified two subpopulations with potential functions in self-renewal and immunoregulation. Our findings advance the definition of MSCs by identifying the specific functions of their heterogeneous cellular composition, allowing for more specific and effective MSC application through the purification of their functional subpopulations.