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 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.

Keywords: Adult stem cells; Angiogenesis; Cell migration; Cell transplantation; Tissue regeneration.

Figure 1.

Phenotypic characterization of ASCs derived from spheroid and monolayer cultures. (A): The ASCs were plated in monolayer or spheroid cultures for 7 days, followed by replating in monolayer culture for an additional 7 days. The expression level of ASC surface antigens was determined by flow cytometry and is shown as the proportion of positively stained cells relative to an isotype control. (B): Microscopic images of monolayer and spheroid-derived ASCs cultured in adipogenic and osteogenic induction media for 14 days. Cell cultures were stained with Oil Red O for detection of adipogenesis and alizarin red for detection of osteogenesis. Scale bars = 100 μm. (C): Real-time polymerase chain reaction measurements for adipogenic and osteogenic marker genes (PPAR-γ and Runx2, respectively) in monolayer and spheroid-derived ASCs at day 7 of induction culture. No significant difference between the monolayer and spheroid-derived cells was noted. Abbreviation: ASC, adipose-derived stem cell.

Figure 2.

Spheroid-derived ASCs exhibited higher growth rate and lower senescence. (A): Cumulative population doubling curve of monolayer and spheroid-derived ASCs. Cells were plated at low density and passaged every 7 days. (B): LDH efflux assay of monolayer and spheroid-derived ASCs after serum starvation for 1, 3, 5, and 7 days. At all indicated times during serum starvation, significantly less LDH activity was observed in medium overlying the spheroid-derived ASCs. (C): SA-β-gal staining of monolayer and spheroid-derived ASCs after 7 and 21 days of culture revealed significantly more SA-β-gal-positive cells in the monolayer group. Scale bars = 100 μm. (D): Real-time polymerase chain reaction and Western blot analysis for senescence marker p21 showed less p21 expression in spheroid-derived ASCs on day 7. *, p < .05 versus the monolayer condition at the same time point. Abbreviations: ASC, adipose-derived stem cell; LDH, lactate dehydrogenase; SA-β-gal, senescence-associated β-galactosidase; RQ, relative quantity.

Figure 3.

Expression of stemness markers and angiogenic growth factors in spheroid-derived ASCs. (A): Real-time polymerase chain reaction measurements for pluripotency marker genes (Nanog, Sox-2, and Oct-4) and angiogenic growth factors (HGF, VEGF, and FGF-2) in monolayer and spheroid-derived ASCs. (B): Western blot analysis of the expression of pluripotency markers and angiogenic growth factors in monolayer and spheroid-derived ASCs. (C): Release of HGF from monolayer and spheroid-derived ASCs into the media as determined by enzyme-linked immunosorbent assay. *, p < .05 versus the monolayer condition. Abbreviations: ASC, adipose-derived stem cell; FGF-2, fibroblast growth factor-2; HGF, hepatocyte growth factor; RQ, relative quantity; VEGF, vascular endothelial growth factor.

Figure 4.

Enhanced CXCR4 expression in spheroid-derived ASCs. (A): Flow cytometric analysis of a stem cell homing marker, CXCR4, revealed significantly more CXCR4-positive cells in ASCs that had experienced spheroid cultures. (B): Real-time polymerase chain reaction and Western blot analysis showed higher CXCR4 expression in spheroid-derived ASCs compared with monolayer cells. (C): Monolayer and spheroid-derived ASCs were incorporated with BrdU for 24 hours and then detected by enzyme-linked immunosorbent assay, which indicated proliferative activity. The significant increase of BrdU incorporation in spheroid-derived ASCs could be reversed by adding a CXCR4 antagonist, AMD3100. (D): The percentages of viable monolayer and spheroid-derived ASCs after exposure to H₂O₂ were determined by flow cytometry measuring propidium iodide uptake and annexin V-fluorescein isothiocyanate labeling. The ratio of survival cells was significantly higher in the group that had undergone short-term spheroid culture, and the ratio decreased after addition of AMD3100. *, p < .05 versus the monolayer condition; #, p < .05 versus the spheroid + AMD3100 condition. Abbreviations: ASC, adipose-derived stem cell; BrdU, 5-bromo-2′-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RQ, relative quantity.

Figure 5.

Enhanced MMP expression and chemotaxis in spheroid-derived ASCs were associated with CXCR4 upregulation. (A, B): Real-time-polymerase chain reaction (A) and Western blot analysis (B) for MMP-9 and MMP-13 expression revealed significantly higher MMP-9 and MMP-13 expression in spheroid-derived ASCs compared with monolayer cells, and the enhanced expression was inhibited by adding a CXCR4 antagonist, AMD3100. *, p < .05 versus other conditions for each MMP. (C): In vitro cell migration was evaluated by scratching confluent ASCs with a pipette tip. After 10 hours, the area of the cell-free zone covered by the migrated cells was measured. *, p < .05 versus the monolayer condition. (D): Chemotactic assay evaluated ASC migration through 8 μm pore size Transwell inserts toward SDF-1. Cells were stained with DAPI (green). Enhanced migration and chemotaxis of spheroid-derived ASCs compared with monolayer cells could be inhibited by supplementing with AMD3100. Scale bars = 50 μm. *, p < .05 versus all other conditions. Abbreviations: ASC, adipose-derived stem cell; DAPI, 4′,6-diamidino-2-phenylindole; MMP, matrix metalloproteinase; RQ, relative quantity; SDF, stromal cell-derived factor; w, with; w/o, without.

Figure 6.

Delivery of spheroid-derived ASCs enhanced cutaneous wound closure. (A): Gross pictures and wound area curves show accelerated healing in wounds treated with spheroid-derived ASCs. (B): Hematoxylin and eosin staining of wound sections at day 7. A significantly larger area of granulation tissue was observed in wounds treated with spheroid-derived ASCs. Scale bars = 200 μm. *, p < .05 versus the other two groups. Abbreviations: ASC, adipose-derived stem cell; G, granulation tissue; PBS, phosphate-buffered saline.

Figure 7.

Enhanced cell engraftment and angiogenesis in wounds treated with spheroid-derived ASCs. (A): Double immunofluorescent staining of HNA and an endothelial marker, CD31. White arrows indicate human ASCs incorporated into vasculature. (B): The ratio of HNA-positive cells was significantly higher in the spheroid group, suggesting a higher ASC retention rate at both day 7 and day 14. Quantification of capillary density per high-power field also revealed significantly enhanced angiogenesis in wounds treated with spheroid-derived ASCs. The numbers of capillary and HNA-positive cells were calculated from 10 randomly selected high-power fields per sample. (C): Double immunofluorescent staining of HNA and an epithelial marker, pancytokeratin. White arrows indicate human ASCs incorporated into the epidermal structure. Cells were counterstained with DAPI (blue). Scale bars = 50 μm. *, p < .05 versus other groups at the same time point. Abbreviations: ASC, adipose-derived stem cell; DAPI, 4′,6-diamidino-2-phenylindole; HNA, human nuclear antigen; PBS, phosphate-buffered saline.