Figure 5. TGFβR2i allows efficient expansion of ER^pos cells.

(a) Population doublings as a function of passage number calculated by continuous cell number recordings in triplicate cultures before confluency and plating at a fixed number of 4,000 cells per cm² per flask at each split. TGFβR2i allows proliferation for up to six passages, corresponding to 15 population doublings (open diamond). If RepSox is omitted, the cells cannot be expanded beyond fourth passage (cross). Initial plating on 3T3 feeders with quantification starting in passage three extends proliferation to more than 10 passages, corresponding to more than 25 population doublings (open circle). Cells from a different sorting (albeit followed for a shorter period) exhibit similar extended proliferative capacity (closed circle). hTERT/shp16-transduced CD166^high/CD117^low cells subsequently passaged at a fixed number of 6,000 cells per cm² at each split extended the proliferative capacity even further (closed triangle), and the cells have now been growing for more than 12 passages. hTERT/shp16-transduced CD166^high/CD117^low cells derived from a different biopsy, split at a ratio of up to 1:4, has so far been growing up to passage 9 (open square). (b) Even beyond 20 population doublings (passage 9), ER^pos cells with definitive lifespan maintain ER and PR expression as shown by immunoperoxidase and haematoxylin staining (cells in ninth passage seeded at 4,000 cells per cm² and stained at day 5 with SP1 prediluted and SAN27, respectively). Scale bar, 50 μm.