Figure 1. 2a2iL induces conversion of primed hPSCs into a naïve‐like state.

• A
  Determination of a time window for 2a2iL treatment needed to induce a naïve‐like pluripotency state in primed hPSCs. One day of treatment with 2a followed by the addition of 2a2iL for up to 14 days. Scale bars: 200 μm.
• B
  Schematic illustration that outlines induction of the naïve‐like state from primed hPSCs. Primed hPSCs were first cultured as single cells in conventional hPSC medium in the presence of ROCK inhibitor (ROCKi) on day 0. The medium was replaced by serum‐free medium plus 2a2iL on day 1 without ROCKi. Compact dome‐shaped colonies began to appear on day 3. The resultant naïve‐like cells were passaged as single cells in the absence of ROCKi on day 5 in 2iL medium. 2a2iL‐hPSCs were passaged every 3–4 days in 2iL, and kept typical naïve‐like morphology for over 50 passages.
• C
  Characteristics of 2a2iL‐RH6 cells represented by dome‐shaped colonies, ALP activity, and a normal male karyotype (46, XY). 2a2iL‐RH6 expressed the common pluripotency markers OCT4, NANOG, SOX2, and TRA‐1-81, but not SSEA1. Nuclei were stained with 4′, 6‐diamidino‐2-phenylindole (DAPI) and propidium iodide (PI). Scale bars: 200, 100, and 50 μm in the inset.