The LARGE Principle in AFiPSCs Figure 1. Senescence is bypassed by the derivation of human AFiPSCs from AFCs. (A-I) Primary human AFCs at passage 5, used for reprogramming. (A-II,-III) The same line at passage 17: Senescence is indicative by a striking morphological change (A-II) and beta-galactosidase staining (A-III). (A-IV) Colonies of AFiPSCs ten days post-transduction. (A-V) The morphology of a typical AFiPSC colony is indistinguishable from ESC colonies (scale bar = 200 mm). (B) Top panel: AFiPSC colonies stained for alkaline phosphatase (AP) at passage 1. 2nd to 7th panel: Immunocytochemistry for the human nuclear ESC pluripotency markers OCT4, SOX2 and NANOG and for ESC surface antigens SSEA4, TRA-1-60 and TRA-1-81 (scale bars = 200 mm). (C) AFiPSCs retain a normal karyotype after cellular reprogramming (mar = minimal altered region). (D) DNA fingerprinting verified the AFC origin of AFiPSC lines, excluding cross-contamination with ESC lines H1 and H9. doi:10.1371/journal.pone.0013703.g001 enlarged cells with typical cobblestone-like appearance (Figure 3A- III,-IV,-VII,-VIII) was observed. This is a characteristic feature of trophoblast differentiation of ESCs [41,42]. Gene expression profiling and qRT-PCR analyses revealed down-regulation of the key pluripotency markers POU5F1 and NANOG and up-regulation of the trophoblast markers CDX2, KRT7, HAND1, FOXF1, GATA3, and ID2 (Figure 3B). Both, BMP2 and BMP4, induced similar effects, however, BMP4 was more efficient. When we treated the AFiPSCs with a combination of 10 ng/ml BMP4 and 10 mM SB431542 over a period of seven days, the same morphological changes could be observed and human chorionic gonadotropin (hCG), a hormone secreted by trophoblastic cells of the placenta, was detected by immunofluorescence microscopy analysis (Figure 3C). PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13703