miR-29b-3p–Tet2–E-cadherin axis regulates BMSC stemness

Loss of stemness during in vitro expansion limits the utility of bone marrow mesenchymal stem cells (BMSCs). We sought intrinsic regulators that preserve stemness and mapped their regulatory relationships. An integrated discovery framework combined a single-cell atlas of murine bone (GSE128423; 11,976 cells), weighted gene co-expression network analysis, and cross-dataset validation in two microarray cohorts that contrasted BMSCs with neural crest-derived stem cells (GSE30419) and adipose-derived stem cells (GSE43781). We inferred cell–cell communication to identify pathway context and built an mRNA–miRNA interaction network to prioritize upstream control. Validation in primary rat BMSCs used gain- and loss-of-function perturbations of the prioritized gene and its upstream and downstream factors, with readouts that included dual-luciferase reporter assays, qPCR, Western blotting, immunoprecipitation, colony formation, and osteogenic and adipogenic differentiation. Integrated analyses prioritized Tet2 as a central regulator of BMSC stemness. miR-29b-3p directly targeted Tet2 and reduced its mRNA and protein, while Tet2 negatively regulated E-cadherin. Increasing miR-29b-3p or E-cadherin, or reducing Tet2, elevated stemness-associated transcription factors and strengthened colony formation and lineage differentiation. These findings define a miR-29b-3p–Tet2–E-cadherin axis that provides a molecular basis to help preserve BMSC stemness during expansion and motivates in vivo evaluation.