Fig. 1.

Derivation of naïve mouse ESC-like induced pluripotent stem cells. (A) Strategy and representative images of C1 cultures and subcloned cell line C1.2 observed at different stages during reprogramming. p, passage number. NOD mESCs and C1.2 hiPSCs after DOX withdrawal are also shown. (B) C1 hiPSC line maintained in conventional bFGF/serum-supplemented human ES growth conditions (hESM) was transferred into N2B27 PD/CH/LIF + DOX, and emerging colonies were subcloned. Representative C1.10 hiPSC clone is shown. (C) Signaling dependence of pluripotent cell lines. Pluripotent cells were equally divided and plated on feeders in the indicated growth medium in which these cell lines are normally maintained, and 36 h later the wells were supplemented with the indicated inhibitors or growth factors. After 6 days, wells were fixed and stained for Nanog to determine the relative percentage of pluripotent colonies. Colony formation is normalized to an internal control growth medium only without inhibitors. (D) C1.2 hiPSC line was electroporated with mammalian expression vectors expressing the indicated reprogramming factors and cells were subjected to puromycin selection and passaged in PD/CH/LIF without DOX. Values indicate relative percentage of SSEA4+ colonies obtained in comparison with control cells that were transfected with an Oct4/Klf4/Sox2-encoding polycistronic construct. (E) Screening of factors that allow propagation of transgene-independent (i.e., DOX-independent) C1 hiPSCs in PD/CH/LIF-supplemented media. Effect of the removal of individual factors from the pool of 13 indicated small molecules or cytokines on the stabilization of pluripotent C1 hiPSCs independent of DOX. C1 cells were plated on feeders in N2B27 media with the indicated factors. P values using Student's t test indicate significant change in comparison with cells grown in DOX/PD/CH/LIF conditions, which were defined as a control (100% survival).