Human mesencult adipogenic differentiation kit

Adipogenic differentiation was induced using Human MesenCult™ Adipogenic Differentiation Kit (STEMCELL Technologies #05412). According to manufacturer’s protocol, DMEM media was replaced with differentiation media once the ASC culture reached 90%–100% confluency. ASC culture was differentiated for 14 days with media changes every 3 days before they were used for downstream applications.

According to the manufacturer's protocols, direct differentiation of human UC‐MSCs toward adipocytes, osteoblasts, and chondrocytes was initiated with a Human MesenCult Adipogenic Differentiation Kit (05412, STEMCELL Technologies Inc., Canada), Human MesenCult Osteogenic Differentiation Kit (05465, STEMCELL Technologies Inc., Canada), and MesenCult‐ACF Chondrogenic Differentiation Kit (05455, STEMCELL Technologies Inc., Canada), respectively. Two to four weeks later, the cells were either fixed for Oil Red (O1391, Sigma–Aldrich, USA) staining and immunostaining or exacted by using RNA isolate Total RNA Extraction Reagent (R401‐01, Vazyme, China) for RNAs. To calculate the differentiation efficiency of adipocytes, photos of three fields at random were taken with a 10× objective lens with NIS‐Elements F software by a microscope (Nikon Ts2‐FL, Japan) for the UC‐MSCs after Oil Red O staining. Then the cells were counted with the ImageJ 1.52v software for further analysis. For quantification of lipids from Oil Red‐stained cells, 100% isopropanol was used to extract Oil Red for 5 min with gentle rocking. The absorbance at 492 nm was read by a Synergy HT Multi Detection Reader (BioTek Instruments, Winooski, VT) for the extracted liquid.