Short-Term Spheroid Formation Enhances the Regenerative Capacity of Adipose-Derived Stem Cells by Promoting Stemness, Angiogenesis, and Chemotaxis

<jats:title>Abstract</jats:title> <jats:p>Adipose-derived stem cells (ASCs) represent an important source of mesenchymal stem cells for clinical application. During in vitro culture, ASCs quickly lose the expression of transcription factors associated with pluripotency and self-renewal (Sox-2, Oct-4, and Nanog) and CXCR4, the key receptor responsible for stem cell homing. To enhance their therapeutic potential despite in vitro passages, we examined whether ASCs exhibit superior regenerative capacity by expanding them in monolayers following short-term spheroid formation. Spheroid-derived ASCs retained the expression pattern of cell surface markers and adipogenic/osteogenic differentiation capabilities of ASCs constantly cultured in monolayers. However, spheroid-derived ASCs exhibited higher expansion efficiency with less senescence. Moreover, spheroid-derived ASCs expressed significantly higher levels of pluripotency markers, CXCR4, and angiogenic growth factors. Enhanced in vitro migration, associated with the increased expression of matrix metalloproteinases (MMP-9 and MMP-13), was also observed in spheroid-derived ASCs. The enhanced migration and MMP expression could be inhibited by a CXCR4-specific peptide antagonist, AMD3100. Using a murine model with healing-impaired cutaneous wounds, we observed faster healing and enhanced angiogenesis in the wounds treated with spheroid-derived ASCs. Significantly more cellular engraftment of spheroid-derived ASCs in the cutaneous wound tissue was also noted, with evidence of ASC differentiation toward endothelial and epidermal lineages. These findings suggest that short-term spheroid formation of ASCs before monolayer culture enhances their properties of stemness, angiogenesis, and chemotaxis and thereby increases their regenerative potential for therapeutic use.</jats:p>