Extended Data Fig. 4: Gene expression analysis of chicken ESCs and cells isolated from different developmental stages of chicken embryos.

a, Immunostaining of chicken ESCs cultured in OT/3i/chLIF. n = 3 independent experiments. b, RT-qPCR analysis illustrating the relative expression levels of lineage marker genes in embryoid bodies derived from chicken ESCs. Data represent the mean ± s.d. of three biological replicates. c, Oil-red staining of chicken ESC-derived adipocytes. n = 3 independent experiments. d, Heatmap analysis comparing the differential gene expression profiles of chicken embryonic stem cells (ESCs) and chicken primary cells isolated from EGK.I to HH4 stage embryos. EGK.I to VIII data sourced from accession number GSE86592; HH4 data obtained from GenBank: SRX893876. e, Scatter plots showing FPKM values with Pearson correlations between chicken ESCs/EGK.X, chicken ESCs/HH4, and EGK.X/HH4. Statistical significance was shown by a two-sided p-value. f, Gene set enrichment analysis showing enrichment score of EGK.X and HH4 signature in chicken ESCs respectively. NES, normalized enrichment score. Enrichment score was calculated using KS-like running sum statistic. g, Heatmap analysis showing the differential gene expression profiles between chicken ESCs, EGK.X and HH4. h, Gene Ontology analysis of Group B upregulated genes (g) in chicken ESCs. Group B upregulated genes represent genes expressed at higher levels in chicken ESCs and were not differentially expressed in EGK.X and HH4 stages. The significance was determined using Hypergeometric test. Scale bars, 50 μm.