Extended Data Fig. 1: Medium optimization for generating human naïve PSC.
From: Rolling back human pluripotent stem cells to an eight-cell embryo-like stage
a. Schematic of the compound screening workflow. b. Representative phase contrast images of primed H9 ESC cultured in different basal recipes for three passages. hLIF, human LIF; CHIR, CHIR99021; PD, PD0325901; 2i, PD+CHIR. Scale, 40 μm. Representative of three independent experiments. c. RT-qPCR for the indicated pluripotency genes in primed H9 ESC cultured in different basal recipes for three passages. Data are the mean values ± standard error of the mean (SEM) of the fold-change compared to primed ESC. n = 3 biological replicates. d. Table of tested compounds and their known targets. e and f. RT-qPCR for the indicated pluripotency genes in primed H9 ESC cultured in different modified basal recipes for three passages. Data are presented as mean values ± SD of fold-change compared to primed ESC in basal medium with PD, IWR1 and human LIF. n = 3 biological replicates. g. RT-qPCR for the indicated pluripotency genes in human primed HUES1 and HUES7 ESC, and human primed iPSC-1, iPSC-2 and iPSC-3 clones cultured in 4CL for three passages (day 12). h. Representative images of G-banding karyotype of primed H9 ESC and primed iPSC-4 clone cultured in 4CL for 15 passages. Twenty metaphases were counted for each. All 20 metaphase spreads for H9 ESC are shown in Supplementary Figure 1. i. Representative phase contrast images of primed H9 ESC cultured in 4CL on feeders, ECM-coated surface (feeder-free), or in suspension, for three passages (day 12). Scale, 40 μm. Representative of three independent experiments. j. RT-qPCR for the indicated pluripotency genes in H9 ESC cultured in 4CL on either feeders or ECM-coated surface for three passages (day 12). Data are presented as mean values ± SD of fold-change compared to primed ESC. n = 3 biological replicates. k. RT-qPCR for the indicated pluripotency genes in primed H9 ESC, HUES1 ESC and iPSC-4 clone converted by 4CL in suspension for three passages (day 12). l. RT-qPCR for the indicated pluripotency genes in primed H9 ESC cultured in 4CL with or without (w/o) Vc for three passages (day 12). Data are presented as mean values ± SD of fold-change compared to 4CL with Vc. n = 3 biological replicates. P value was calculated using two-tailed unpaired Student’s t-test. m. RT-qPCR for the indicated genes in H9 ESC cultured in 4CL with different TSA or DZNep substitutes for three passages (day 12). Data are presented as mean values ± SD of fold-change compared to 4CL with TSA and DZNep. n = 3 biological replicates.
