Data supporting the article "3D MICROENVIRONMENT DIFFERENTIALLY REGULATES ADIPOGENIC AND ANGIOGENIC POTENTIAL OF ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS"

The data set includes the following files: Figures described in the article Fig. 1. Contraction dynamics and cytoskeletal organization of AT-MSCs in 3D constructs.(A, B) Time-dependent changes in the diameter of collagen I (A) and Hplasma (B) hydrogels containing AT-MSCs (cell-seeded) or acellular controls. Statistical comparisons were performed between cell-seeded hydrogels 20 minutes after embedding and subsequent time points (***p < 0.001, ****p < 0.0001), and between acellular and cell-seeded hydrogels at corresponding time points (# p < 0.05, #### p < 0.0001). Data are presented as mean ± SD (N = 6).(C) Time-dependent changes in spheroid diameter (****p < 0.0001). Data are presented as mean ± SD (N = 8).(D) Representative immunocytochemical staining of F-actin in collagen I, Hplasma, and spheroid cultures at days 3 and 7. Arrows indicate merging of two individual Hplasma constructs at day 7 (N = 4 for each construct type). Fig. 2. Metabolic activity and viability of AT-MSCs in 3D culture systems.(A–C) Metabolic activity of AT-MSCs cultured in collagen I hydrogels (A), Hplasma hydrogels (B), and spheroids (C), measured using the Alamar Blue assay at days 1, 3, 5, and 7. Data are presented as mean ± SD (N = 3-4, technical triplicates for each donor, *p < 0.05, **p < 0.01, ***p < 0.001).(D) Representative LIVE/DEAD™ staining of AT-MSCs in 3D constructs at days 3 and 7 (N = 5, three constructs per donor). Fig. 3. Hypoxia- and autophagy-related responses of AT-MSCs in 3D culture systems.(A) Immunocytochemical staining of HIF-1α in collagen I, Hplasma, and spheroid constructs at days 3 and 7, showing differences in expression levels and spatial distribution within the constructs (N = 4). (B) Heat map of relative gene expression (normalized to 2D monolayer controls) of hypoxia-related (HIF1A, EPAS1) and autophagy-related (BECN1, ATG5) genes, as well as microenvironment-responsive factor CXCL12 (N = 5). Fig. 4. Secretion of angiogenic growth factors by AT-MSCs in 3D culture systems after 3 days.Quantification of growth factors secreted by AT-MSCs cultured in collagen I, Hplasma, and spheroid systems after 3 days, assessed by Luminex-based analysis (N = 3, performed in duplicate). Secretion levels are shown relative to 2D monolayer controls. Statistical significance between 3D cultures and 2D control is indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are presented as median ± interquartile ranges. Fig. 5. Secretion of angiogenic growth factors by AT-MSCs in 3D culture systems after 7 days.Quantification of growth factors secreted by AT-MSCs cultured in collagen I, Hplasma, and spheroid systems after 7 days, assessed by Luminex-based analysis (N = 3, measured in duplicate). Secretion levels are shown relative to 2D monolayer controls. Statistical significance between 3D cultures and 2D control is indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are presented as median ± interquartile ranges. Fig. 6. Adipogenic differentiation of AT-MSCs in 3D culture systems (A) Representative fluorescence images of lipid droplets stained with Nile Red in collagen I, Hplasma, and spheroid cultures at days 5, 10, and 15 of differentiation.(B) Quantification of lipid droplet area per cell, analyzed using ImageJ. Data are presented as mean ± SD (N=3; three constructs per donor per condition; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).(C) Relative expression of PPARG, LPL, FABP4, and CEBPA in collagen I, Hplasma, and spheroid cultures, determined by qPCR and normalized to non-differentiated control cultures (at day 5 of culture). Data are presented as log₂ fold-change values (median with interquartile range, N=3 per condition, technical duplicates, *p < 0.05, ***p < 0.001, ****p < 0.0001). 2. The individual raw microscopy images underlying the figures presented in the manuscript. Each TIFF file corresponds to a representative field/image used for figure preparation or quantitative analysis. File names indicate experimental condition (3D culture system), time point (day 3 or day 7), and staining/assay type (live and dead, actin staining, Nile Red staining, HIF staining). 3. The Excel file (Raw data_figures.xlsx) contains the underlying quantitative data used for graph preparation and statistical analysis.