Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes

• Immortalized BMSC clones were generated for in-depth functional and biophysical analysis
• Distinct differentiation-competent and incompetent BMSC subsets are defined
• Trilineage-competent BMSC clones exhibit enhanced vascular interaction gene sets
• A non-differentiating, “immune-primed,” CD317+/IL-7hi BMSC subset was identified in vivo

SummaryBone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis of functional markers for BMSC subsets. All clones expressed typical BMSC cell-surface antigens; however, clones with trilineage differentiation capacity exhibited enhanced vascular interaction gene sets, whereas non-differentiating clones were uniquely CD317 positive with significantly enriched immunomodulatory transcriptional networks and high IL-7 production. IL-7 lineage tracing and CD317 immunolocalization confirmed the existence of a rare non-differentiating BMSC subtype, distinct from Cxcl12-DsRed+ perivascular stromal cells in vivo. Colony-forming CD317+ IL-7hi cells, identified at ∼1%–3% frequency in heterogeneous human BMSC fractions, were found to have the same biomolecular profile as non-differentiating BMSC clones using Raman spectroscopy. Distinct functional identities can be assigned to BMSC subpopulations, which are likely to have specific roles in immune control, lymphopoiesis, and bone homeostasis.