Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells

Abstract

Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.

Figure 1

Derivation and characterization of bESCs in NBFR conditions. (a) Bright field images at different stages of bESC derivation from whole blastocysts. Images represent day-7 embryos selected for ESC derivation (arrows), outgrowth after 3 days in culture, and typical colony morphology at passages 1 and 7. (b) Immunofluorescence staining of OCT4 and SOX2 pluripotency factors, and SSEA4 surface marker. Detection of alkaline phosphatase activity, and chromosome number (2n = 60) of bESCs at different passages. D: day; P: passage. Scale bars 100 μm.

Figure 2

Epigenomic landscape of NBFR-bESCs. (a) Functional enrichment of genes containing H3K4me3, H3K27me3 or bivalent domains. The top 10 GO terms are shown. (b) Track view of genes containing H3K4me3, H3K27me3 or bivalent H3K4me3/H3K27me3 domains. (c) Track view of H3K4me3 and H3K27me3 profiles in primed and naïve pluripotency markers. Two independent NBFR lines (NBFR1 and NBFR2) were analyzed by CUT&RUN for H3K4me3 (K4) and H3K27me3 (K27) and compared with Chip-Seq data from CTFR-bESC7 (CTFR1 and CTFR2).

Figure 3

In vitro and in vivo differentiation of NBFR-bESCs. (a) Gene expression levels of pluripotency (OCT4, SOX2, NANOG), trophectoderm (CDX2), endoderm (FOXA2), ectoderm (PAX6), and mesoderm (MEOX1) markers in representative tissues and two independent NBFR-bESCs lines before (NBFR#A and NBFR#D at passage 10) and after adaptation to feeder-free culture (NBFR-FF #A and #D, passage 10 and 20 after adaptation, respectively). Relative expression was calculated using the comparative CT method (ΔΔCT), normalizing values to HMBS. (b) Bright field images of NBFR-bESCs and embryoid bodies obtained at different times of differentiation. RT-PCR for genes representative of different lineages (OCT4, CDX2, SOX17, PAX6, and FOXA2). HMBS: housekeeping gene. Scale bar 100 μm. (c) Teratomas obtained 12 weeks after injection of two independent NBFR-bESC lines (NBFR #D and #Y) into immunodeficient mouse. Representative histological images showing derivates of the 3 germ lineages. BFF: bovine fetal fibroblasts; Bls: blastocysts, d: days.

Figure 4

Characteristics of NBFR-bESCs grown under feeder-free conditions. (a) Immunostaining of OCT4 and SOX2, and normal chromosome number (2n = 60) of NBFR-bESCs grown for multiple passages on vitronectin supplemented with Activin A (b) Flow cytometry analysis of bovine fibroblasts and NBFR-bESCs cultured in feeder-free conditions (NBFR-FF-bESCs) indicating the DNA content and the percentage of cells in each phase of the cell cycle. (c) Quantification of OCT4 expression in NBFR-FF-bESCs (blue peak) and secondary only control (unshaded peak). (d) Expression levels of genes (OCT4, FOXA2, SOX17, CDX2, MEOX1, PAX6) representative of different lineages after 3D (3 weeks) and 2D (4 weeks) in vitro differentiation in NBFR-bESCs lines before (NBFR#B at passage 10) and after adaptation to feeder-free culture (NBFR-FF#B at passage 11). Relative expression was calculated using the comparative CT method (ΔΔCT), normalizing values to HMBS. P: passage. Scale bar 100 μm.

Figure 5

IWR-1 supplementation is required for pluripotency of NBFR-bESCs cultured on MEF feeders. Immunostaining of pluripotency factors OCT4 and SOX2 in NBFR-bESCs grown on MEF feeders for 5 passages after IWR-1 withdrawal. Scale bar 100 μm.

Figure 6

Feeder-free NBFR-bESCs depend on FGF2 and Activin A for pluripotency maintenance. Immunostaining of pluripotency factors OCT4 and SOX2 in NBFR-bESCs grown in feeder-free conditions supplemented with different combinations of Activin A, CHIR2291, FGF2, IWR-1 and TGFβ1 for 4 passages. Scale bar 100 μm.