Supporting data (after revision) for the article "METABOLIC REPROGRAMMING OF MULTIPOTENT MESENCHYMAL STROMAL CELLS IN THREE-DIMENSIONAL SPHEROIDS"

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The data set for the study https://doi.org/10.1186/s13287-026-05268-y The data set includes the following files: Figure 1. Formation of spheroids from AT MSCs and size characterization over time. (A) Representative images of spheroid formation in BiofloatTM plates from the seeding point to day 7. Scale bar = 100 μm. (B) Quantitative evaluation of spheroid diameter. Data are presented as mean ± SD, N=10 spheroids per time point. Note: **** - p<0.0001 compared to day 1 of culture. (C) Comparison of cell size following isolation from spheroids versus cells cultured in monolayer conditions and subsequently trypsinized. The values are expressed as mean ± SD, n=32 corresponding to 8 dissociated spheroids or 8 trypsinized wells of monolayer cultured cells from 4 independent donors. Note: **** - p<0.0001 compared to monolayer-cultured cells. (D) Immunofluorescence staining of F-actin (left) and N-cadherin (right) at days 3 and 7 of spheroid culture. Figure 2. Spheroid culture-driven changes in secretory protein expression and release (N=4 independent donor-derived MSCs in duplicates). (A) Expression of TSG6, HGF, and VEGFA in monolayer-cultured and spheroid-cultured cells. Box plots show median, interquartile range, 5th and 95th percentiles. The boxplots represent PPIA-normalized relative mRNA expression (2⁻ΔΔCt) to the monolayer shown as 1. Note: TSG6, HGF, and VEGFA mRNA expression was significantly increased in spheroids compared with their respective time-matched monolayer controls (p<0.05); # - p<0.05 day 7 vs. day 3 of spheroid culture. (B) Alteration in the release of cytokines and growth factors during spheroid culture compared to monolayer conditions. Box plots show median, interquartile range, 5th and 95th percentiles. Note: The secretion levels of all analyzed factors were significantly higher in spheroids than in the corresponding monolayer cultures (p<0.05); # - p<0.05 day 7 vs. day 3 of spheroid culture. (C) Representative immunofluorescence images of spheroids at day 3 and day 7 of culture, stained with anti-HIF-1α antibody. Figure 3. Metabolic activity of AT MSCs in monolayer and spheroids (N=4 independent donor-derived MSCs per condition). (A) Resazurin reduction by cells cultured in spheroids and monolayers. Box plots show median, interquartile range, 5th and 95th percentiles. Note: * - p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001 compared to day 1 of culture in corresponding conditions. (B) The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of monolayer-cultured AT MSCs, spheroid-derived cells, and cells in spheroids. AT MSCs in each group were assessed using the Seahorse assay, and results were normalized to equal cell number. Note: X and Y axes scales and measurement conditions for spheroids differ from other panels because of protocol adjustments for 3D structures. (C) AMP and ADP levels in monolayer and spheroid cultures. Note: * - p<0.05, ** - p<0.01, *** - p<0.001. Figure 4. Viability, apoptosis, and proliferation markers of AT-MSC spheroids during 3D culture. Representative fluorescence images of day 3 and day 7 spheroids stained with Live/Dead, cleaved caspase-3, p53, Ki-67, and EdU. In Live/Dead staining, viable cells are shown in green and dead cells in red. For cleaved caspase-3, a positive signal is shown in red; for p53, Ki-67, and EdU staining, a positive signal is shown in green. Figure 5. Metabolomic reprogramming of cells in spheroids cultured for 3 and 7 days (N=4 individual donors per condition). (A) Principal component analysis (PCA) of metabolomic profiles from monolayer-cultured cells (white circles) and spheroid-cultured cells (blue – 3 days; violet – 7 days of 3D culture). (B) Volcano plots showing changes in key metabolite and lipid classes/subclasses in spheroid-cultured MSCs compared with monolayer cultures. Separate plots are shown for day 3 spheroids versus monolayer cultures (left) and day 7 spheroids versus monolayer cultures (right). Annotated classes include amino acids, purine/pyrimidine metabolites, phospholipids, glycerides, fatty acids, and lysophospholipids. (C) KEGG pathway enrichment analysis of significantly altered polar metabolites performed in MetaboAnalyst. Each dot represents a metabolic pathway, with color indicating p-value, and the dot size reflecting the pathway’s impact values. Figure 6. Lipid profile of cells cultured in monolayer and spheroids for 3 and 7 days. A doughnut chart illustrates the distribution of 664 identified lipid species grouped into 12 most abundant lipid classes. Lipid classes representing less than 3% of the total lipid profile were combined in the category “Others”. The proportion of upregulated (white segments) and downregulated (grey segments) lipid species within each class at days 3 and 7 of spheroid culture, relative to monolayer conditions, is presented as horizontal bar charts (N=4 individual donors per condition). Figure 7. Changes in phospholipid and fatty acid selected classes and expression of corresponding metabolic enzymes between day 3 and day 7 of spheroid culture (N=4 individual donors per condition). (A) Total TIC-normalized abundances of main phospholipid subclasses in spheroids and monolayer cultures. Box plots show median, interquartile range, 5th and 95th percentiles. Note: * - p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001. (B) Heatmap illustrating the changes in enzymes related to phospholipid and triacylglycerol synthesis, fatty acid synthesis, and desaturation occurring during spheroid culture. (C) Comparison of the fatty acid saturation profiles between monolayer-cultured and spheroid-cultured cells. Figure 8. Spheroid culture is associated with the release of the 10,000 × g large extracellular particle fraction. (A) Scanning electron microscopy image showing round extracellular particles on spheroid surface; arrows indicate representative particle-like structures. Scale bar, 20 µm. (B) Confocal microscopy and (C) Spinning disk super-resolution microscopy images showing discrete labeled particles and particle clusters in the LEP fraction of the secretome released by spheroids formed from PKH26-labeled AT-MSCs (cyan LUT pseudocolor is applied for enhanced contrast and overall presentation quality). Scale bars, 15 µm in B and 2 µm in C. (D) Nanoparticle tracking analysis illustrating a broad, polydisperse particle-size distribution (average for 4 donor preparations). (E–H) Cryo-EM analysis of the LEP fraction. (E) Representative round-to-ovoid vesicular particle bordered by an apparent limiting membrane. (F) Smaller and non-spherical/elongated vesicular profiles. (G) Vesicles of different diameters within the same field, illustrating size heterogeneity. (H) Larger irregular membrane-bound particle with heterogeneous granular contents and internal vesicle-like/membranous features. Cryo-EM scale bars, 50 nm. Figure 9. Comparison of lipid composition between spheroids and the large extracellular particle (LEP) fraction released by spheroids. (A) Overlap of lipid species identified in spheroids (day 3 of culture) and their LEPs. (B) Pearson correlation between absolute abundances of individual lipid species in spheroids and LEPs. (C) Pearson correlation between absolute abundances of key lipid classes in spheroids and LEPs. (D) Pearson correlation between fold changes in individual lipid species in spheroids relative to monolayer cultures and their corresponding absolute levels in LEPs. (E) Pearson correlation between fold changes in lipid classes in spheroids relative to monolayer cultures and their corresponding absolute levels in LEPs. Supplemental materials - file includes: Figure S1. Total TIC-normalized abundances of glycerols in spheroids and monolayer cultures. Box plots display the median, interquartile range, and the 5th and 95th percentiles. Note: * - p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001.Figure S2. Total TIC-normalized abundances of main lysophospholipid subclasses in spheroids and

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