Stempro adipogenesis kit

The multi‐lineage potential of the cells was ascertained by inducing the samples post‐harvest with the StemPro Adipogenesis kit, StemPro Chondrogenesis kit and StemPro Osteogenesis kit (Life Technologies, UK). The media were prepared and used as per the manufacturer's instructions and the differentiation studies were performed as described in 30.

Multi‐parameter immunophenotypic analysis of the hMSCs was determined by flow cytometry and the antibodies were selected on the basis of the panel recommended by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006). This was performed using a Guava HT flow cytometer with excitation at 488 nM and is described in detail in Chan et al. (2014). Tri‐lineage differentiation potential of the cells was determined by using the StemPro Adipogenesis kit, StemPro Chondrogenesis kit and StemPro Osteogenesis kit (Life Technologies, ThermoFisher Scientific Company, Loughborough, UK) in accordance with the manufacturer's instructions on post‐harvest hMSC samples to induce differentiation. Staining of the samples to determine differentiation was performed as described in (Rafiq et al., 2013b). CFU‐f efficiency was determined by culturing the hMSCs in monolayer at a cell density of 10 cells/cm² and with a complete medium exchange every 4 days. After 15 days in culture, hMSCs were washed with PBS (Lonza, Visp, Switzerland) and fixed with 4% formaldehyde (v/v) (Sigma, UK) for 30 min. A 1% crystal violet (Sigma–Aldrich) in 100% methanol (w/v) was used for colony staining. The samples were incubated at room temperature for 30 min in the 1% crystal violet solution. All stained colonies that comprised of more than 25 cells were recorded as CFUs.