Ectopic expression of Ink4a in young ETPs causes reduced cell production. (A) Representative FACS plots showing that young ETPs transduced with Ink4a/GFP did not generate thymocytes in fetal thymic cultures compared with young ETPs transduced with GFP alone. Plots are representative of 3 independent experiments. (B) Number of GFP- and p16^Ink4a/GFP-expressing thymocytes recovered from the fetal thymic cultures shown in panel A. (C) Frequencies of 7-amino-actinomycin D⁺ (dead) and annexin V⁺ (apoptotic) cells in cell suspensions harvested from cultures established by seeding Ink4a/GFP- or GFP-only–transduced young ETPs on OP9-Δ1 stromal cells 2 weeks after seeding. Average frequencies ± SEM are based on data pooled from 3 independent experiments. (D) GFP⁺ cell recovery from cultures established by seeding young (4-6 weeks) and old (18 months) ETPs transduced with shInk4a/GFP- or GFP-only–containing retroviral vectors onto OP9-Δ1 stromal cell layers. Cell recovery was determined 7 and 14 days after seeding. The mean ± SEM based on 3 independent experiments is shown.