Journal: bioRxiv

Article Title: A Modular Bioinstructive Platform for Additive-Free, Topography-Driven Stem Cell Differentiation and Patterning

doi: 10.1101/2025.07.11.664383

Figure Lengend Snippet: A) Heatmap showing expression of selected genes associated with skeletogenesis across experimental conditions at days 3 and 14 post-seeding. B, C) Volcano plots displaying key differentially expressed genes associated with cytoskeleton, osteogenesis, and chondrogenesis in dimpled versus smooth microparticle cultures at day 3 (B) and day 14 (C) post-seeding (log 2 fold change > 1 and p adj < 0.05, N= 3 donors). D, E) Relative quantitative real-time PCR (qPCR) analysis of gene expression at specific time-points in serum-reduced medium, relative to 2D controls: RUNX2 (D; N= 5 donors) after 3 days, and SP7 (E; N= 5 donors) after 10 days. 2D positive control treated with 2 μM purmorphamine is shown as a reference point for RUNX2 expression. (F) Relative qPCR analysis of BGLAP expression after 10 days of either 300 nM KAAD-cyclopamine treatment or 0.06% ( v/v ) DMSO in serum-reduced medium, and relative to 2D vehicle-only controls (N= 5 donors). Statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons was applied. Values are presented as mean ± SD (** p < 0.01, *** p < 0.001, **** p < 0.0001). G, H) Representative maximum intensity projection confocal images of hMSCs stained for COL1A1 (green; G), and OCN (green; H) with nuclei counter-stained in blue (DAPI) after 10 days of culture (Scale bars: 50 μm). A single optical slice is used to represent brightfield for clarity. Abbreviations: SOX9, SRY-box transcription factor 9; COL2A1, collagen type II alpha 1 chain; COL10A1, collagen type x alpha 1 chain; COL1A1, collagen type I alpha 1; COL6A1, collagen type VI alpha 1 chain; ACAN, aggrecan; RUNX2, runt-related transcription factor 2; SP7, specificity protein 7, also known as osterix; MSX2, Msh homeobox 2; SPP1, secreted phosphoprotein 1, also known as osteopontin; POSTN, periostin; PAX9, paired box 9; BGLAP, bone gamma-carboxyglutamate protein, also known as osteocalcin; ALPL, alkaline phosphatase; TNC, tenascin-c; SCUBE3, signal peptide-CUB-EGF domain-containing protein 3 ; SERPINE2, serine protease inhibitor 2; GALNT1, N-acetylgalactosaminyltransferase 1; LEPR, leptin receptor; SOSTDC1, sclerostin domain-containing protein 1; CNMD , chondromodulin; BMP, bone morphogenetic protein; IGF-II, insulin-like growth factor 2; IGFBP, insulin-like growth factor binding protein; DAPI, 4′,6-Diamidino-2-phenylindole; OI media, Osteoinductive media; Pur, Purmorphamine; DIMP, Dimpled; SM, Smooth; DIMP_KD, Dimpled cultures treated with KAAD-cyclopamine .

Article Snippet: The following primary antibodies were used, goat anti-GLI-1 affinity purified polyclonal antibody (1:100; AF3455, R&D systems, RRID: AB_2247710), rabbit anti-osteocalcin (OCN) polyclonal antibody (1:75; AB10911, Millipore, RRID: AB_1587337), goat anti-type I collagen polyclonal antibody (1:200; #1310-01, SouthernBiotech, RRID: AB_2753206) and monoclonal anti-Vinculin (1:70; V4505, Sigma-Aldrich, RRID: AB_477617).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Positive Control, Staining, Protease Inhibitor, Binding Assay