Isolation and Characterization of Porcine Stem/Progenitor Cells

STCs were isolated from fresh pig kidneys, as previously shown [7 (link),12 (link)]. Briefly, pig cortical and medullary sections were washed with phosphate-buffered saline, diced, and digested with 2 mg/mL collagenase for 1 h. Samples were then forced through a 60-mesh (250 μm) steel sieve to remove the fibrous component [34 (link)]. The cellular fraction was passed through 100 μm cell strainer followed by the addition of Medium 199 containing 3% FBS (Gibco BRL, Waltham, MA, USA) [35 (link)] at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was replaced every 2 days to remove non-adherent cells. Two weeks later, the adherent cells were harvested with TrypLE™ Express (Gibco) treatment and sub-cultured. Cultured pig STCs were characterized using flow cytometry and immunofluorescence staining, which confirmed their positivity for CD133 (Novus Biologicals, Centennial, CO, USA) and CD24 (Abcam, San Francisco, CA, USA).