Figure 3

Neural differentiation of haploid ESCs carrying the Sox1-P2A-Venus reporter.

(a) Morphology and Venus fluorescence images of the differentiated cells in N2B27 culture from Hap-SV ESCs. Top shows Hap-SV ESCs before differentiation. BF, bright field; scale bar, 50 μm. (b) Venus fluorescence and DNA content analysis of the differentiated neural cells 9 days after the differentiation induction of Hap-SV cells. The Venus-negative population is shown in magenta and the Venus-positive population is shown in green. (c) The Venus-positive ratio in differentiated Hap-SV lines. FACS analysis was performed at day 9 after differentiation induction in N2B27 culture. Data from three Hap-SV lines are shown as the means ± SEM (n = 3). (d) Expression of differentiation marker genes in differentiated Hap-SV cells. RT-qPCR was performed 9 days after the differentiation induction in N2B27 culture. Venus-negative (magenta) and Venus-positive (green) sorted populations were analysed. Relative expression levels were normalised to the Gapdh expression level. Values in Venus-negative populations were set to 1. Data are shown as the means ± SEM (n = 3 for each line). Statistical significance was determined using Student’s t-test. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.0001. †, not detected. (e) Expression of stem cell marker genes in the undifferentiated and differentiated Hap-SV cells. Differentiated Hap-SV cells were collected at day 9 after differentiation in N2B27 culture. Values in undifferentiated Hap-SV ESCs were set to 1. Statistical significance was determined using Tukey’s multiple comparison analysis at the 5% (^*) and 1% (^**) significance level. Undiff., undifferentiated; ND, neurally differentiated.