Journal: Materials Today Bio

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

doi: 10.1016/j.mtbio.2026.103012

Figure Lengend Snippet: 3D culture decreases transcriptomic heterogeneity of PMSCs and enhances both mitochondrial and osteogenic processes. (A) Uniform Manifold Approximation and Projection (UMAP) analysis of cells from PMSC 2D and PMSC 3D as assessed using single-cell RNA sequencing data (National Center for Biotechnology Information-Gene Expression Omnibus or NCBI-GEO database: GSE268973 ). N = 6857 for 2D conditions; n = 10,705 for 3D conditions. (B) Cluster analysis of cells from PMSC 2D and PMSC 3D as visualized by UMAP. (C) Cluster analysis of cells from PMSC 2D as visualized by UMAP. (D) Cluster analysis of cells from PMSC 3D as visualized by UMAP. (E) Frequency analyses of clusters for PMSC 2D and PMSC 3D as assessed. (F) Mitochondrial transcription factor A ( TFM ), NADH:Ubiquinone oxidoreductase core subunit S1 ( NDUFS1 ), ubiquinol-cytochrome C reductase core protein 1 ( UQCRC1 ), cytochrome C oxidase subunit 6C ( COX6C ), and ATP synthase F1 subunit alpha ( ATP5F1A ) expression levels as visualized in cells from 2D- and 3D-preconditioned PMSCs by UMAP. (G) RUNX2, sirtuin1 ( SIRT1 ), alkaline phosphatase ( ALP ), bone sialoprotein ( IBSP ), and OPG expression levels as visualized in cells from PMSC 2D and PMSC 3D by UMAP. (H) Mitochondria- and bone-related GOBP pathways as enriched in cells from PMSC 3D vs. PMSC 2D by bubble plot analysis based on DEGs (fold change ≥1.5) and enrichment factor in Metascape. Bubble sizes represents the counts of genes associated with each GOBP pathway, while color gradient indicates p -values. (I) Mitochondria- and bone-related GOBP pathways as enriched in cells from PMSC 3D vs. PMSC 2D by bubble plot network analysis in Cytoscape. Bubble sizes represents the counts of genes associated with each GOBP pathway, while color gradient indicates NES. (J) Experimental design for the assessment of osteogenesis in 3D-preconditioned PMSCs: PMSCs were cultured under 2D or 3D conditions at a density of 1 × 10 5 cells/1 mL/cm 2 for one day. After preconditioning, both 2D- and 3D-cultured PMSCs were reseeded on 2D conditions with or without culture medium (CM) or osteogenic medium (OM). After 4 weeks, extracellular mineralization was assessed by staining calcium deposition with Alizarin Red (AR). (K) and (L) Representative and pooled data of extracellular mineralization in cells preconditioned from PMSC 2D and PMSC 3D as assessed with AR staining. Scale bar, 50 μm. N = 3 for each group. Data are shown as mean ± SD. One-way ANOVA: ∗∗, p < 0.01; ∗∗∗, p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 3D culture decreases transcriptomic heterogeneity of PMSCs and enhances both mitochondrial and osteogenic processes. (A) Uniform Manifold Approximation and Projection (UMAP) analysis of cells from PMSC 2D and PMSC 3D as assessed using single-cell RNA sequencing data (National Center for Biotechnology Information-Gene Expression Omnibus or NCBI-GEO database: GSE268973 ).

Techniques: Single Cell, RNA Sequencing, Gene Expression, Expressing, Cell Culture, Staining