Three-dimensional (3D) culture-primed placental mesenchymal stem cells decrease cellular heterogeneity, significantly enhancing osteogenesis via improving mitochondrial function.

Mesenchymal stem cells (MSCs) hold significant promise for bone tissue engineering, with adult bone marrow-derived MSCs (BMMSCs) widely used but limited by invasive harvesting and senescence-related osteogenic decline. Fetal-origin placental MSCs (PMSCs) offer a non-invasive and highly proliferative alternative, though their osteogenic capacity remains inconsistently reported. Because mitochondrial activity is closely linked to osteogenesis, and culture conditions strongly influence MSC function, we investigated how three-dimensional (3D) culture differentially modulates PMSCs and BMMSCs. Transcriptomic analyses revealed significant enrichment of cell adhesion and mitochondria-related pathways in PMSCs vs. BMMSCs; functional validation demonstrated larger viable spheroid formation and further augmentation of higher baseline mitochondrial activity in 3D-cultured PMSCs, along with upregulation of key osteogenic markers RUNX2 and osteoprotegerin. Surprisingly, even short-term 3D-priming led to elevated mitochondrial and osteogenic capacity in PMSCs but not BMMSCs. Single-cell RNA sequencing of PMSCs revealed that 3D culture promotes homogeneity, enriching for high mitochondrial- and osteogenic-expressing cell clusters. Functionally, inhibition of mitochondrial function suppressed osteogenic differentiation in 3D-primed PMSCs. Our findings reveal more robust baseline PMSC mitochondrial activity which can be further enhanced by even short-term, 1 day of 3D culture to significantly improve osteogenic commitment, demonstrating a practical strategy to improve MSC-based therapies for bone regeneration.