Figure 1.

Derivation and maintenance of spheres. A, Growth kinetics of USSCs cultured in stem cell proliferation medium, HEScGRO and PSM and USSC growth medium^ACF. Cells were seeded at low density and total cell number was determined every two days. B, Time course of the formation of spheres. i, USSCs in stem cell proliferation medium; ii-iv, USSCs in USSC growth medium^ACF; ii, after three passages USSCs begin to cluster; iii, within 24 hours of formation, clusters detach from the surface; iv, with continued culturing, clusters increase in size and have defined boundaries. (Magnification, ×200; scale bar is 100 μm)C, Size of spheres on day seven. (Magnification, ×100; scale bar is 100 μm)D, Growth of spheres after mechanical dissociation and re-plating on extracellular matrix-coated and non-ECM-coated surfaces; i, clusters of cells continue to grow in USSC growth medium^ACF; ii, dissociated spheres can grow as an adherent monolayer on fibronectin and collagen-coated surfaces in USSC growth medium^ACF; iii, dissociated spheres can grown as an adherent monolayer on gelatin-coated surfaces in USSC growth medium^ACF; iv, in the absence of mechanical dissociation, spheres cultured in stem cell proliferation media grow as an adherent monolayer. (Magnification, ×200; scale bar is 100 μm).