Extended Data Fig. 4: Further characterization of ruminant haSCs.
From: Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells
a. DNA copy number variation (CNV) analysis of ruminant haSCs at passage 25. Note that no significant genomic alternations. The results were displayed on a log2 scale. Chromosomes are arranged in numerical order and in different colours. b. Summary of karyotyping analysis of ruminant haSCs at different passages. Total, the total number of examined cells in which chromosome was successfully spread. c. Determination of the sex chromosome of ruminant haSCs by PCR assay. The primers are specific for X chromosome-specific PHEX and Y chromosome-specific ZFY genes. Normal XX and XY diploid SCs were used as controls. M, DNA marker. n = 3 independent experiments with similar results. d. Scatter-plots showing the reproducibility of buRNA-seq between different biological replicates of ruminant haSCs. The Pearson’s correlation coefficients (Cor.) are shown. e. Principal-component analysis (PCA) showing the separation of b-haSCs and bovine pre-implantation embryos. f. Scatter plot based on PCA of b-haSCs, diploid primed b-SCs, and diploid expanded b-SCs, showing that the projection position of b-haSCs is located between expanded and primed b-SCs. g. Correlation matrices showing coefficients among b-haSCs, diploid primed b-SCs, diploid expanded b-SCs, and pre-implantation bovine embryos. h. Heatmap displaying the differentially expressed genes (DEGs) among b-haSCs, primed b-SCs, and expanded b-SCs (P < 0.05, 2-fold change; left). Gene Ontology (GO) terms about the biological processes enriched in DEGs are listed (right). i. Developmental progression of pluripotency in embryos during peri-implantation. Formative pluripotency may be present at the early stages of gastrulation, according to developmental time and lineage segregation of mouse, monkey, pig, and human embryos10,71,72,73,74,75,76. E, embryonic day; ICM, inner cell mass; EPI, epiblast; HYPO, hypoblast; TE, trophectoderm. j. A uniform manifold approximation and projection representation (UMAP) of bovine peri-implantation embryos (E8 ~ E14) and b-haSCs sequenced by 10x Genomics. RNA-seq data of b-haSCs generated in this study, previously published data for bovine diploid pre-implantation embryos are from ref. 11 PMID: 24591639 (GSE52415), diploid primed b-SCs are from ref. 13 (GSE110040), diploid expanded b-SCs are from ref. 14 (GSE129760).
