Recombinant mouse alp protein

To evaluate the differentiation potential of MSCs in gels 1 d after encapsulation, they were cultured in medium supplemented with either an osteogenic chemical cocktail (No. CCM009) alone or both osteogenic and adipogenic (No. CCM011) cocktails for 7 or 10 d, respectively. All reagents for MSC differentiation were purchased from R&D Systems. One half of each sample was used to quantify an absolute number of viable cells by flow cytometry as described previously, while the other half was used to evaluate ALP activity. To quantify ALP activity, samples were lysed with 100 μL passive buffer (No. E1941, Promega) for at least 10 min at 4 °C. Each lysate was then added to a black 96‐well plate preloaded with 100 μL 4‐methylbelliferyl phosphate (4‐MUP) substrate (No. M3168, Sigma). Signals were acquired with excitation at 360 nm and emission at 450 nm using a plate reader. Recombinant mouse ALP protein (Novus Biologicals) was used to generate a standard curve for calibration. ALP activity of each sample was then normalized to the number of viable cells.

MSCs in gels were cultured for 1 day in a basal medium (No. CCM007), followed by the medium supplemented with both osteogenic (No. CCM007) and adipogenic (No. CCM11) cocktails for 10 days. The medium was refreshed on day 5. All reagents for MSC differentiation were purchased from R&D Systems. One‐half of each sample was used to quantify an absolute number of viable cells by calcein staining, while the other half was used to evaluate early osteogenesis or adipogenesis by ALP activity or lipid droplet staining, respectively. To quantify ALP activity, cells were lysed with 200 µl passive buffer (No. E1941, Promega) for 15 min at 4 °C. Each lysate was then added to a black 96‐well plate preloaded with 100 µl 4‐methylumbelliferyl phosphate (4‐MUP) substrate (No. M3168, Sigma). Signals were acquired with excitation at 360 nm and emission at 450 nm using a plate reader. Recombinant mouse ALP protein (Novus Biologicals) was used to generate a standard curve. To quantify lipid droplets in cells, cells were incubated with the LipidSpot™ 610 Lipid Droplet Stain (Biotium) at 37 °C for 30 min. Signals were acquired with excitation at 592 nm and emission at 638 nm using a plate reader.