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Osteogenic differentiation and adipogenic differentiation were evaluated by growing hAMSCs for 14 days in αMEM with 10% FBS supplemented with osteogenic and adipogenic supplements, respectively (R&D Systems, Minneapolis, MN, USA). For chondrogenic differentiation, we used DMEM/F12 containing both ITS and chondrogenic supplement (R&D Systems, Minneapolis, MN, USA). We used a specific antibody panel consisting of anti-hFABP4 (adipocyte marker), anti-hOC (osteocyte marker), and anti-hACAN (chondrocyte marker) (R&D Systems, Minneapolis, MN, USA) to analyze the respective mature phenotypes by immunofluorescence. Samples were analyzed using an EVOS™ FL Digital Inverted Fluorescence Microscope (Thermo Fisher Scientific, Waltham, MA, USA).

The differentiation analysis of hAMSCs was done in both 2D and 3D cultures. Spheroids were dissociated into a single-cell suspension and seeded to the culture plates for cell expansion before differentiation assay. To evaluate osteogenic and adipogenic differentiation, cells were grown for 14 days in α-minimum essential medium (α-MEM) supplemented with both 10% FBS and osteogenic and adipogenic supplements, respectively (R&D Systems, USA). Chondrogenic differentiation was analyzed by growing cells in DMEM/F12 medium containing both insulin-transferrin-selenium (ITS) supplement (R&D Systems, USA) and chondrogenic supplement (R&D Systems, USA). A panel of antibodies consisting of anti-hFABP4, anti-hOC, and anti-hACAN was analyzed by immunofluorescence to define the mature phenotypes of adipocytes, osteocytes, and chondrocytes, respectively (R&D Systems, USA). Fluorescence for each antibody was revealed using EVOS™ FL Digital Inverted Fluorescence Microscope (Fisher Scientific, UK), and signal intensities were calculated with ImageJ software.