Fig. 9.

Clonogenic growth of rabbit limbal G0-G1 and SP cells on 3T3 feeder cells. G0-G1 cell collection was restricted to cells having an FSC range comparable to that of the SP cells. Colonies from G0-G1 (A-C) and SP cells (D-F) were generated from 2000 plated cells after 11 days (A,B) or after 14 days (C,D) of culture. PMA (100 μM) was included in these cultures for 4–72 hours post-plating interval. Note that essentially all colony formation by the G0-G1 cells is arrested whereas a substantial number of colonies grow from the SP cells, and the SP culture contains a substantial number of microcolonies (mc). Microscopic examination showed that most of these colonies consist of small, morphologically undifferentiated cells. (C,F) Fate of microcolonies. Dishes were complemented with 400 limbal epithelial cells and treated with PMA as described for B and E. After 14 days, colonies were present only in the SP dish. These large fast-growing colonies were removed by swiping (clear cell-free areas marked by asterisks on the grayish background generated by the 3T3 cell mat) and the culture was continued for two more weeks. The microcolonies continued to grow to form new large colonies.