Single colony expansion of hESCs.

(A) Schematic diagram of the procedure. Parental hESCs were gamma-irradiated and used as metabolically active but replication defective feeder cells. Lentivirally-transduced hESCs expressing a fluorescent reporter protein were enriched by FACS and seeded at low density on to the feeders, expanded as single colonies, and manually selected for serial passage as indicated (Methods). (B) FACS-isolated hESCs plated onto irradiated hESC feeder layer at low dilution after FACS. (C�CF) Fluorescence micrographs showing examples of the sorted cells at different times after plating. Individual cells seen one day after sorting (C) grow into small colonies as imaged at days 7, 9 and 16 (D�CF) (different colonies). (G) H2BmCherry-positive differentiated cells did not proliferate under the expansion conditions. (H,I) Phase contrast (H) and fluorescence (I) micrographs of clonal H2BmCherry hESCs originated from a single cell at day 7 following passage 1.