Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts

Abstract

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

Keywords: bovine; embryonic stem cell; inner cell mass; pluripotency.

Fig. 1.

Derivation and characterization of CTFR-bESCs. (A) Bright-field images and AP staining showing typical colony morphologies of CTFR-bESCs (note that the feeder layer is negative for AP). P3, passage 3; P24, passage 24. (Scale bars, 50 μm.) (B) IF staining for SOX2, POU5F1, GATA6, and CDX2 in bovine blastocysts [Top Row (magnification: 20× objective)] and CTFR-bESCs (Middle and Bottom Rows). (C) Expression analysis of ICM, TE, and PE lineage-specific markers in CTFR-bESCs, whole blastocysts, and fibroblasts. Transcriptome analysis was done using RNA-seq. Samples include two independent CTFR-bESC lines (P10), two independent pools of whole blastocysts (10 each), and two lines of bovine fibroblasts. The color scale goes from red (high expression) to green (low/no expression). (D) Representative images showing H&E staining of histological sections derived from teratomas generated by CTFR-bESCs. CTFR-bESC–derived teratomas contained tissues of all three germ lineages: ectoderm, endoderm, and mesoderm. (Magnification: 10×.)

Fig. 2.

Histone methylation landscape of CTFR-bESCs. (A) Transcriptional status of genes containing H3K4me3, H3K27me3, or bivalent domains. Genes with RPKM ≥0.4 were considered expressed, and genes with RPKM <0.4 were considered nonexpressed. Mean RPKM ± SEM values of expressed genes are shown inside the bar plot, and mean RPKM ± SEM values for all genes (expressed and nonexpressed) are shown on the x axis. (B) Functional characterization of genes containing H3K4me3 (n = 8,816), H3K27me3 (n = 2,553), or bivalent domains (n = 3,886). The top-10 GO terms are shown. The bar plot shows the −log₁₀ of the P value for selected GO term biological processes from DAVID. (C) Genome browser snapshot of genes containing H3K4me3 (TGFBR1, FGF8, SALL4, TRIM8, SBDS, and TAF8), H3K27me3 (OOEP, REC8, SLITRK4, LRRC4B, ARRX, and CSNB1), or bivalent domains (WNT2, WNT7A, MATN2, CHL1, MSX2, and ETV4). H3K4me3-, H3K27me3-, and bivalent-selected genes were associated with three different GO terms. The start of the gene is denoted by a black arrow.

Fig. 3.

CTFR-bESCs show molecular signatures characteristic of primed pluripotency. (A) Transcriptome analysis of selected naive and primed pluripotency markers in CTFR-bESCs. RNA-seq was performed, and RPKM values were used to define expressed (RPKM ≥0.4; red) and nonexpressed (RPKM <0.4; green) genes. The means of two biological replicates are shown. (B) Genome browser snapshots of histone methylation profiles of primed and naive pluripotency markers in CTFR-bESCs. (C) Genome browser snapshots of H3K4me3 and H3K27me3 marks in core pluripotency genes (POU5F1, SOX2, NANOG, SALL4) in CTFR-bESCs.

Fig. 4.

Potential applications of CTFR-bESCs for genomic selection. (A) CTFR-bESC derivation efficiency using three different plating methods (whole blastocyst, mechanical isolation of ICM, and immunosurgery-derived ICM) and different embryo sources (IVM-IVF, OPU-IVF, SCNT, and Holstein and Jersey breeds). Derivation efficiency was measured as the percentage of blastocysts that gave rise to a stable CTFR-bESC line at P3 over the total number of embryos seeded per method. (B) A schematic diagram showing the strategy of using CTFR-bESCs for genomic selection to produce animals of superior genetic value through highly efficient CTFR-bESC derivation and NT. (C) CTFR-bESCs generated from different sources can be used as nuclear donors for cloning.