## 3 Results
### Differential Gene Expression Patterns in MSC Spheroids and C-MSCs Cultured in 3D Aggregates  
To uncover the distinctions in biological properties between MSC spheroids and C-MSCs, we performed whole transcriptome sequencing. Specifically, the experimental setup involved collecting RNA from 2D-cultured MSCs (200,000 cells) on a 24-well plate, eight MSC spheroids (25,000 cells each) in an ultra-low-binding 24-well plate, and a single C-MSC aggregate (200,000 cells) in a similar plate, which was then subjected to RNA-seq (Figure 4A). We first assessed the typical biological capacities of MSC, including immunomodulatory and cell protective factor expressions, as well as osteogenic, adipogenic, and chondrogenic differentiation potential. Both MSC spheroids and C-MSCs demonstrated elevated expressions of immunomodulatory and cell protective factors compared to their 2D counterparts. Interestingly, C-MSCs had a more pronounced increase in these genes than MSC spheroids (Figures 4B and S1). However, the expression of trilineage differentiation genes remained unchanged, suggesting that 3D aggregation alone does not promote differentiation. In addition, qPCR analysis of PTGS2, TSG-6, and IL-1 RN gene expression revealed consistent mRNA expression patterns with those observed in the RNA-seq heatmap data (Supplementary Figure S2), validating the results of our RNA-seq  
Figure 2: (**A**,**B**) MSC spheroids (**A**) and C-MSCs (**B**) were generated from the indicated number of cells (within a range from 25,000 (25K) to 200,000 (200K) cells) as described in the Section 2. Subsequently, semi-serial sections were prepared, and H&E and TUNEL staining were performed. The left panels of H&E staining show lower magnification (bar = 500 \(\upmu\)m), whereas magnified photos from the boxed regions are displayed in the right panels (bar = 50 \(\upmu\)m). A similar magnification (bar = 50 \(\upmu\)m) applies to the TUNEL staining images. (**C**) Graphs express the mean \(\pm\) S.D. of the cell number derived from four views of magnified images for each cell aggregate. (**D**) Results display the percentage of TUNEL-positive apoptotic cells. Values represent mean \(\pm\) S.D. of four tested samples. \({}^{*}\)\(p\)\(<\) 0.05, \({}^{**}\)\(p\)\(<\) 0.01 (ANOVA). All images and graphs are representative of three independent experiments. analysis. The volcano plot analysis revealed that 2043 mRNAs were differentially expressed between MSC spheroids and C-MSCs (Figure 4C). Then, gene ontology (GO) analysis on 1035 genes highly expressed in MSC spheroids and 1008 genes elevated in C-MSCs was conducted to assess the biological characteristics in each cell aggregate. The results indicated that genes predominantly expressed in MSC spheroids were mainly associated with \"cell adhesion\", whereas \"cytokine-mediated signaling pathway\" was the most enriched category for C-MSCs in the GO analysis pertaining to cellular processes (Figure 4D). To further elucidate the characteristics of MSC spheroids and C-MSCs in a 3D environment, we included 2D-cultured MSCs for a comparative gene expression study. Venn diagram analysis highlighted a set of 623 genes that showed significant differences among 2D MSCs, MSC spheroids, and C-MSCs, representing the intersection in the diagram (Figure 4E). The clustergram analysis clearly showed differences in gene expression among 2D MSCs, MSC spheroids, and C-MSCs (Figure 4F). For the identified 623 genes, we performed a gene set enrichment analysis (GSEA) for the KEGG pathway, comparing C-MSCs with MSC spheroids. In line with the GO analysis in Figure 4D, the \"cytokine-cytokine receptor interaction\" was notably upregulated in C-MSCs (Figure 4G). Categories such as \"TNF signaling pathway (NES = 2.42, Q-value \(<\) 0.001)\", \"NF-kappa B signaling pathway (NES = 2.14, Q-value \(<\) 0.001)\", and \"JAK-STAT signaling pathway (NES = 2.19, Q-value \(<\) 0.001)\", which are associated with the immunomodulatory and cytoprotective properties of MSCs, were also enriched in C-MSCs.