Recovery and stability of the TNGA ES cell population following puromycin selection. (A) Profile of two consecutive passages from a steady-state population of TNGA ES cells grown for several weeks without puromycin treatment. Notice that under these conditions, cells accumulate in the LN. Compare with Figure 1A. (B) Cells from the culture shown in (A) were treated with puromycin (left column) for three successive passages. The treatment selects for cells expressing high levels of Nanog-GFP and results in the elimination of cells that do not express Nanog-GFP. After three passages, puromycin selection was removed, and Nanog-GFP expression was monitored in the population over eight consecutive passages by flow cytometry. Notice that following the puromycin treatment, the majority of the cells in the culture are in the HN peak (99.96%) and that the relative ratio of LN cells progressively increases until the third/fourth passage, when the distribution stabilizes with a LN population between 15%–25% of the total. This distribution is comparable to the one noted in Chambers et al. [25] (and see Figure 1) and persists for four more passages (P5–P8) thereafter, suggesting that this is a steady-state distribution. For comparison, we show the culture from (A) grown in parallel, without selection in the same medium the cells were before the selection (right column). Long-term culture without selection leads to the increase of cells in the LN peak. (0.95 MB TIF)