Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines

Abstract

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cell nuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

Keywords: Bovine, single cell RNA seq; Cross-species comparisons; Embryo development; Epiblast stem cell; Formative pluripotency.

Fig. 1

Single-cell transcriptome sequencing deciphers lineage differentiation of bovine embryos. A The bovine embryo morphologies collected for scRNA-seq analyses encompass six distinct developmental stages: E5 (late morula), E6 (early blastula), E7 (late blastula), E10 (early bilaminar embryo), E12 (late bilaminar embryo), and E14 (primitive streak embryo). For the E5–E7 and E10 embryos, the scale bar is 100 μm, while for the E12 and E14 embryos, the scale bar is 1 mm. The E14 embryonic disc, scale bar is 200 μm. The dashed box indicates the position of embryonic disc in embryo at stages E10, E12, and E14. B UMAP plots showing the transcriptional similarity of all bovine embryonic cells. Different colored dots represent the indicated embryonic days, arrows represent known developmental trajectories. C The UMAP plots provide insights into cell typing at each embryonic stage, with distinct cell types represented by different colored dots. D–F Dot plots visualization depicting the expression patterns of characteristic genes across distinct cell types in E5–E7 embryos (D), E10 and E12 embryos (E), and E14 embryos (F). The size of the dot encodes the percentage of cells within a cell type, and the color encodes the average expression level. See also Figures S1, S2

Fig. 2

Tracing the dynamics of pluripotency of bovine embryonic lineages. A UMAP plots depicting the scRNA-seq data obtained from bovine embryonic lineages, encompassing E5–E7 pre-ICMs/ICMs, E10 and E12 EPIs, and E14 ECTOs. B Spearman’s correlation coefficients based on the mean expression levels of uniquely expressed genes within each embryonic lineage cells. C Enriched GO terms of pre-ICMs/ICMs, EPIs, and ECTOs specific genes. D Representative clusters of genes with similar expression trends showing the changes of naïve, formative, and primed pluripotency genes in pre-ICM, ICM, epiblast, and ectoderm cells during E5 to E14. E Heatmap showing the expression changes of genes associated with JAK/STAT3, TGFβ/SMADs, FGF/ERK, and Wnt/β-catenin signaling pathways in selected bovine embryonic lineages

Fig. 3

Comparative analyses of developmental and pluripotency changes of embryonic lineages in cattle and pigs. A PCA plot of embryonic single cell data for cattle and pigs, the round and triangular points represent cattle and pigs, respectively. With clusters color-coded based on distinct species and embryonic lineages. B Spearman’s correlation coefficients were calculated for cattle and pigs at various embryonic lineages. C Pseudotime analysis of embryonic lineages of cattle and pigs. D Schematic representation of distinct pluripotent states in the bovine and porcine embryonic lineages. E Heatmap showing representative marker genes for naïve, formative, and primed pluripotent states in selected embryonic lineages of cattle and pigs. F Heatmap depicting the expression of representative genes related to signaling pathways in selected embryonic lineages of cattle and pigs. G GO/KEGG enrichment terms of DEGs during pluripotent changes in selected embryonic lineages of cattle and pigs

Fig. 4

Generation and characteristics of bEpiSCs. A Strategies for establishment of bEpiSCs. B Efficiency of outgrowths derived from bovine embryos at different stages and cell lines established in 3i/LAF culture medium. C Morphological of outgrowths (up), and bEpiSCs (down), white arrows indicate the morphology of selected and passaged bEpiSC colonies. Scale bar, 200 μm. D Population doubling time of bEpiSCs. E Single cell cloning efficiency of bEpiSCs. F Alkaline phosphatase (AP) staining assay of bEpiSCs. Scale bar, 100 μm. G Karyotype analyses of bEpiSCs. For each cell line, 45 cells at metaphase were examined. H Immunostaining of pluripotency markers POU5F1, NANOG, and SOX2 in the bEpiSCs. DAPI was used to stain nuclei. Scale bar, 100 μm. I Immunostaining of pluripotency surface markers CDH1, SSEA1, and SSEA4 in the bEpiSCs. DAPI was used to stain nuclei. Scale bar, 50 μm. J In vitro EB differentiation assay. Immunostaining for ectodermal neuro-specific marker protein β-III-Tubulin, mesodermal muscle-specific marker protein α-SMA and endodermal specific marker protein SOX17. DAPI was used for staining nuclei. Scale bar, 100 μm. K In vivo teratoma formation assay. Haematoxylin and eosin (H&E) staining of teratomas derived from bEpiSCs. Scale bar, 50 μm. For D and E, error bars indicate ± SD (n = 3 independent experiments), ns., P ≥ 0.05. For C, and F–K, similar results were obtained in three independent experiments

Fig. 5

3i/LAF are essential for the long-term subcultured of bEpiSCs. A Colonies morphologies and AP staining were observed in bEpiSCs treated with subtractive factors, with the 3i/LAF group serving as the control. Scale bar, 200 μm. B Colonies size analyses of the bEpiSCs culture system following one-by-one subtractive factor treatment. More than 30 colonies were counted for each treatment group. C Quantitative real-time PCR analyses of bEpiSCs gene expression differences between 3i/LAF and I/F groups. D, E The mRNA expression levels of representative pluripotent (D) and lineage (E) marker genes involved in the CHIR, IWR, and WH-depleted groups were compared with those in the 3i/LAF group using qRT–PCR. F The mRNA expression levels of representative pluripotent and lineage marker genes involved in the FGF2-depleted group were compared with those in the 3i/LAF group using qRT-PCR. G The mRNA expression levels of representative pluripotent and BMPs signaling pathway marker genes involved in the Activin A-depleted group were compared with those in the 3i/LAF group using qRT-PCR. H The mRNA expression levels of representative pluripotent and JAK/STAT3 signaling pathway marker genes involved in the LIF-depleted group were compared with those in the 3i/LAF group using qRT-PCR. For B–H, error bars indicate ± SD (n = 3 independent experiments), ns., P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For (A–H), similar results were obtained in three independent experiments. See also Figure S3

Fig. 6

Transcriptomic profiling of bEpiSCs. A The PCA plot illustrating the distribution of scRNA-seq data obtained from bovine embryonic lineage cells and bEpiSCs, with clusters color-coded based on distinct embryonic lineages and bEpiSC lines. B Spearman’s correlation analysis was performed on bovine embryonic lineage cells and bEpiSCs. A color gradient ranging from blue to red represents an increasing correlation from low to high. C The violin plots illustrating the expression levels of naïve, formative, and primed pluripotency representative genes in bovine embryonic lineages as well as bEpiSCs derived from different embryonic stages. D The PCA plot were generated to compare bEpiSCs with publicly established Primed bESCs [15], bEDSCs [16], bEPSCs, and biPSCs [18], where each data point represents a distinct cell line. E Differentially expressed genes (DEGs) were identified between bEpiSCs and published bPSCs. The red bar represents genes that are upregulated in bEpiSCs, while the green bar represents genes that are downregulated in bEpiSCs, compared to the published bPSCs through pairwise comparisons. F Scatter plots illustrating the average gene expression levels comparison between bEpiSCs and published bPSCs, with upregulated genes highlighted in orange and downregulated genes in blue. Key genes are appropriately annotated. See also Figure S4

Fig. 7

Potential applications of bEpiSCs. A Prospects for the application of bEpiSCs in a model diagram. B Colonial morphologies at different stages of myogenic differentiation of bEpiSCs are shown. Scale bar, 200 μm. C Quantification of mRNA expression for myogenic differentiation associated genes by qRT-PCR. D Immunostaining of muscle cell associated proteins in myogenic differentiated bEpiSCs. Scale bar, 200 μm. E Morphology of day 4 PGCLCs embryonic body induction from bEpiSCs. Scale bar, 200 μm. F Relative expression levels of pluripotency and PGCLCs related marker genes in PGCLCs versus bEpiSCs. G Colony morphology of GFP-bEpiSCs, scale bar, 50 μm. H Morphologies of cloned embryos derived from GFP-bEpiSCs, scale bar, 100 μm. I Statistics on cloning efficiency using bEpiSCs as donor cells for nuclear transfer. J Colony morphology of de novo derived GFP-bEpiSCs from bovine cloned embryos, scale bar, 200 μm. For C and F, error bars indicate ± SD (n = 3 independent experiments), ns., P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For B–H, similar results were obtained in three independent experiments