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

Abstract

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 healingimpaired 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. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:584 -594

Characterization and Angiogenic Potential of Human Neonatal and Infant Thymus Mesenchymal Stromal Cells

“…We also evaluated the angiogenic characteristics of thymus MSCs using an established 3D sprouting assay [41][42][43] because 3D culture is known to enhance the regenerative abilities of MSCs and more closely approximates the in vivo setting [44][45][46][47]. In multicellular spheroids, HUVECs and thymus MSCs are in a 3D, highcell-density aggregate during initial formation, facilitating direct contact and paracrine cell interactions.…”

Physiologically Low Oxygen Enhances Biomolecule Production and Stemness of Mesenchymal Stem Cell Spheroids

“…43 VEGF had also been shown to be increased in spheroids formed on chitosan films for 7 days, with a 1.2-fold increase in VEGF, but with no significant change in bFGF. 44 This increase of growth factors due to low oxygen culture and 3D condition could in turn activate the collagen synthesis and migration of fibroblasts, leading to enhanced wound healing. 45 Well-maintained stemness is essential for clinical applications of hMSCs.…”

Three-Dimensional Aggregates of Mesenchymal Stem Cells: Cellular Mechanisms, Biological Properties, and Applications

“…5,6 Indeed, the formation of three-dimensional (3D) cellular aggregates, even short-term post-expansion, enhanced the regenerative capacity of MSCs by promoting the secretion of anti-inflammatory cytokines, proangiogenic, and chemotaxic factors. 5,18,19 The benefits of cell aggregation has also been shown in preclinical studies, in which intramyocardial transplantation of the 3D A-MSC and BM-MSC aggregates in a porcine model improved cell retention, survival, and integration. 20,21 Similarly, transplantation of synovial MSC aggregates promoted cartilage tissue regeneration in a rabbit model and enhanced meniscus tissue regeneration effectively in rats.…”