Transcriptomic Profiling Reveals Mechanisms of Anoikis Resistance in Spheroid-Cultured Human Umbilical Cord Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem (stromal) cells are a promising cell source for regenerative medicine, but their therapeutic efficacy is often limited by poor engraftment and survival post-transplantation. One major contributing factor is anoikis, a form of apoptosis triggered by cell detachment from the extracellular matrix. Spheroid culture systems have shown potential to enhance cell survival and stemness, yet the mechanisms by which they confer resistance to anoikis remain unclear. METHODS: We established spontaneous spheroids from human umbilical cord-derived mesenchymal stem (stromal) cells (UC-MSCs) and performed RNA-sequencing analysis to compare gene expression profiles between spheroid and monolayer cultures. Differentially expressed genes were identified and subjected to GO and pathway enrichment analyses. Functional assays included the use of PI3K/Akt and HIF-1 pathway inhibitors to dissect their role in anoikis regulation. Expression levels of apoptosis-related genes were validated by qRT-PCR. RESULTS: Spheroid UC-MSCs exhibited significantly enhanced resistance to anoikis. Transcriptomic analysis revealed upregulation of both pro-apoptotic and anti-apoptotic genes, suggesting a balanced but regulated apoptotic threshold. Downregulation of executioner genes such as BAX, BAK1, and FADD, along with activation of PI3K/Akt and HIF-1α pathways, suggested effective suppression of apoptotic execution. Inhibitor experiments confirmed these pathways as key contributors to anoikis resistance. CONCLUSION: Our findings demonstrate that spheroid formation promotes a survival-permissive gene expression profile in UC-MSCs, driven in part by PI3K/Akt and hypoxia signaling. These insights advance the understanding of spheroid-mediated anoikis resistance and may inform strategies to enhance stem cell-based therapies.