| To isolate CD45+ haematopoietic cells, spleens were excised from 1-week-old Oct4-gfp mice (unless specified otherwise), minced by scissors and mechanically dissociated with pasture pipettes. Dissociated spleen cells were suspended with PBS and strained through a cell strainer (BD Biosciences). After centrifuge at 1,000 r.p.m. for 5 min, collected cells were re-suspended in DMEM medium and added to the same volume of lympholyte (Cedarlane), then centrifuged at 1,000g for 20 min. The lymphocyte layer was taken out and stained with CD45 antibody (ab25603, Abcam). CD45-positive cells were sorted by FACS Aria (BD Biosciences). After cell sorting, 1 × 106 CD45-positive cells were treated with 500 μl of low-pH HBSS solution (titrated to pH5.7 by HCl) for 25 min at 37 °C, and then centrifuged at 1,000 r.p.m. at room temperature for 5 min. After the supernatant (low-pH solution) was removed, precipitated cells were re-suspended and plated onto non-adhesive culture plates (typically, 1 × 105 cells ml−1) in DMEM/F12 medium supplemented with 1,000 U LIF (Sigma) and 2% B27 (Invitrogen). Cell cluster formation was more sensitive to the plating cell density than the percentage of Oct4-GFP+ cells. The number of surviving cells was sensitive to the age of donor mice and was low under the treatment conditions above when adult spleens were used. The addition of LIF during days 2–7 was essential for generating Oct4-GFP+ STAP cell clusters on day 7, as shown in Extended Data Fig. 1f. Even in the absence of LIF, Oct4-GFP+ cells (most of them were dim in signal) appeared transiently during days 2–5 in culture of low-pH-treated CD45+ cells, but subsequently disappeared, indicating that there is a LIF-independent early phase, whereas the subsequent phase is LIF-dependent. |
