Fig. 2

SP5 is required for normal hPSC differentiation. a Schematic of the SP5 gene. A deletion of the sequence encoding the C2H2 Zinc-finger DNA binding domain was generated using CRISPR-Cas9 with two guide RNAs (designated by red arrowheads). Gene structure: thin lines for intron and intergenic regions, medium lines for non-coding exonic sequence, and thick lines for open reading frame. The position of genotyping primers is indicated by arrows. b Wild-type and mutant SP5 protein detection. Lysates from unstimulated (−) and CHIR-stimulated (+) WT and SP5 mutant (dZF1 and 2) hESCs were analyzed by immunoblotting. Short (i) and long (ii) exposures of the same immunoblot are shown. CHIR = CHIR98014; kD kilo Daltons. c Altered EB formation from SP5 mutant cell lines. SP5 mutant EBs acquire abnormal morphologies at later time points. Scale bar = 100 μm. d Altered gene expression in SP5 mutant EBs. RNA was isolated from EBs at the indicated days after initiation of differentiation and analyzed by qPCR for expression of markers indicative of endoderm (FOXA2), mesoderm (MIXL1), and ectoderm (PAX6). RQ relative quantity. (Error bars are SEM for four technical replicates; Student’s t test: **p < 0.01; ***p < 0.001; ****p < 0.0001). e Altered gene expression in monolayer differentiation of SP5 mutant cells. WT and SP5 mutant (dZF 1 and 2) hESCs were differentiated through removal of FGF2 and addition of fetal bovine serum. RNA was extracted at 3 and 10 days after initiation of differentiation and analyzed by RNA-Seq. Correlation matrices representing 5253 differentially expressed genes indicate increased divergence of WT (two independent differentiations) and dZF cells at days 3 and 10 of differentiation