a, Optimization of pH conditions for Oct4-GFP induction. Five days after CD45-positive cells were exposed to acidic solution treatment at different pH, Oct4-GFP expression was analysed by FACS (n = 3, average?±?s.d.). b, Gating strategy for Oct4-GFP+ cell sorting. Top: representative results 7?days after the stress treatment. Bottom: non-treated control. P3 populations were sorted and counted as Oct4-GFP+ cells for all experiments. c, Controls for FACS analysis. In Oct4-GFP+ cell analysis, the grey and white histograms indicate the negative control (non-stress-treated Oct4-gfp haematopoietic cells) and the positive control (Oct4-gfp ES cells), respectively. Also, the green histograms indicate non-treated cells (left) and stress-treated cells at day?7 (right). In CD45+ cell analysis, the grey and white histograms indicate the negative (isotype) and positive controls, respectively. The red histograms indicate non-stress-treated cells (left) and stress-treated cells at day?7 (right). d, Oct4-GFP+ cell generation from various subpopulations of CD45+ cells. Seven days after the stress treatment, Oct4-GFP expression was analysed by FACS (n = 3, average?±?s.d.). Among total CD45+ fraction and its subfractions of CD19+, CD90+, CD34+ and CD34− cells, the efficacy of CD34+ cells was significantly lower than the others. P?<?0.05 by the Newman–Keuls test and P?<?0.01 by one-way ANOVA. e, Comparison of culture conditions for low-pH-induced conversion. Stress-treated cells were cultured in various media. The number of Oct4-GFP-expressing clusters was counted at day 14 (n = 3, average?±?s.d.). ***P?<?0.001 (B27+LIF versus all other groups); Tukey’s test. In the case of 3i medium, although the clusters appeared at a moderate efficiency, they appeared late and grew slowly. ACTH, ACTH-containing ES medium; ES+LIF+FBS, 20% FBS+LIF-containing ES culture medium; B27, DMEM/F12 medium containing 2% B27; B27+LIF, DMEM/F12 medium containing 2% B27+LIF; EpiSC, EpiSC culture medium containing Fgf2+activin. f, Signalling factor dependency of STAP cell generation. Growth factors that are conventionally used for pluripotent cell culture such as LIF, activin, Bmp4 and Fgf2 were added to basal culture medium (B27-supplemented DMEM/F12) in different culture phases (days 0–7, 2–7 and 4–7), and Oct4-GFP expression was analysed by FACS at day 7 (n = 3, average?±?s.d.). g, h, Time course of apoptosis after the low-pH exposure. Stress-treated cells and non-stress-treated control cells were stained with CD45, annexin-V and propidium iodide at day 0 (immediately after stress treatment), day 3 and day 7. g, Blue bars, GFP+CD45−; orange bars, GFP−CD45+. Percentages in total cells included propidium-iodide-positive cells. h, Annexin-V-positive cells in these cell populations were analysed by FACS.