Figure 1.

Validation of naive and primed states at derivation, and at early and late passages

(A) Study design – four hESC lines, namely Lis38, Lis39, Lis45, and Lis46, were derived and cultured in NHSM conditions. At passage 20 (p20, derivation), half of the cells from each line were transferred to primed conditions (conversion), and half continued to be cultured in NHSM conditions. After 10 passages cells in the NHSM (p30, Early NHSM) and primed (p30, Early primed) cultures were analyzed. Cells were analyzed again at after an additional 20 passages (p50, Late NHSM and Late primed).

(B) Colony morphology of NHSM-derived cells and their isogenic primed counterparts (scale bar – 200 mm) and immunofluorescence staining for the pluripotent markers OCT4, TRA-1-60, and SSEA4 and for the naive marker TFE3 (scale bar – 100 mm).

(C) Relative mRNA expression levels of the naive markers KLF17, TFCP2L1, and STELLA as determined by qRT-PCR in NHSM (red column) and their primed counterparts (blue column) lines, relative to primed control (black column). All data are expressed as mean ± SEM, ∗∗∗p < 0.005 by a paired t-test.

(D) In vivo differentiation was demonstrated by teratoma formation at p40. Tissues representing the three germ layers were identified using hematoxylin and eosin staining and marked by white arrows (scale bar – 50 μm).