Establishment of bovine expanded potential stem cells

Significance Bovine embryonic stem cells and pluripotent stem cells hold the potential to substantially advance biotechnology and agriculture. We report the establishment of bovine expanded potential stem cells (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer (SCNT). EPSCs have broader developmental potential to generate embryonic and extraembryonic cell lineages. bEPSCs express high levels of pluripotency genes, propagate robustly in single cell passaging, are genetically stable, and permit efficient precise gene editing. They differentiate in vitro and in chimeras to both the embryonic and extraembryonic cell lineages. Importantly, genetically modified bEPSCs can be used as donors in SCNT or cloning.

All cell cultures in this paper were performed under conditions of 38.5°C and 5% CO2 unless stated otherwise. bEPSCs were frozen once they are~80% confluent using cryopreservation medium, which contains 90% FBS and 10% (vol/vol) DMSO. The bEPSCs should be stored with the density of 1.0-2.0 × 10 5 bEPSCs per cryotube. Before cryorecovery, BFFs feeder plates should be prepared at least 1 day in advance at a density of 1.2 × 10 4 cells per well (24 well plate), and 1.0-2.0 × 10 5 bEPSCs should be seeded to one well of feeder plates with bEPSCM.
Bovine Superovulation, Insemination and Embryo Recovery. Holsteins (approx. 14-18 months of age, 300-400 kg bodyweight) served as embryo donors. Bovine donors were received a CIDR-B device (DEC international NZ l, New Zealand) on day 0, and 1 mg estradiol benzoate (Ningbo Second Hormone Factory, China) was injected simultaneously. Then 200 mg FSH (Folltropin-V, Canada) was injected from day 5 to 8, twice injections daily, and with 0.6 mg PG (Shanghai Institute of Planned Parenthood Research, China) injection (twice daily) on Day 7. Then CIDR-B devices were removed on Day 8. Ovulation was induced by injection of Gonadorelin (Sansheng Biological Technology, China) 0.2 mg after onset of estrus on Day 9, and were inseminated 12 and 24 hours after onset of estrus. Morulae or blastocysts were recovered on Days 16 or 17, graded and cryopreservation for bEPSC derivation.

In vitro Culture of Bovine Preimplantation Embryos and Derivation of Bovine EPSCs ES .
Bovine in vivo derived morulae or blastocysts from day 5-7 were used for the establishment of bovine EPSC lines. Zonae pellucidae of blastocysts were removed with Tyrode's solution, Acid (Sigma), then blastocysts were washed twice in SOF medium which includes MEM essential amino acids (GIBCO) and MEM non-essential amino acids (GIBCO) and 10 mg BSA ml -1 (SIGMA). Bovine blastocysts were cultured on a monolayer of Mitomycin C treated BFFs cells in bEPSC medium, supplemented with 10 μM Y27632 (Selleck) for 5 days, until initial outgrowths could be observed. Subsequently, bEPSCM medium without ROCKi was used for further culture. Medium was changed every other day. 12-20 days after plating, outgrowths were dissociated and passaged using TrypLE Select (Gibco) and were reseeded in the presence of the Rho kinase (ROCK) inhibitor Y-27632 (10 μM) into newly prepared wells with BFFs feeder cells. Growth of colonies was evaluated daily and approximately three days later cells began to form well-defined domed colonies. And then the bEPSCs ES were passaged every 2-3 days at a ratio of 1:4 as single cells by TrypLE Select without the Rho kinase (ROCK) inhibitor Y-27632.
Bovine EPSC Feeder Free Cultivation and Characterization. Bovine EPSCs iPS and EPSCs ES on feeder cells were switched to plates coated with 20 μg/ml fibronectin (Millipore, FC010) and maintained in bEPSCM. The bEPSCs were passaged every 2-3 days at a ratio of 1:4 with single cell suspension. The chemical defined bEPSC in different passages were collected for the expression of core pluripotent makers detection by RT-qPCR, AP staining and immunofluorescence staining.
Bovine Single EPS Cell Proliferation Ability Assay. Dissociation of bovine EPSCs and primed ES cells was performed in TrypLE™ Select for 2 min at 38.5°C, then resuspended with bEPSCM and CTFRM (3), carefully dissociated in a small drop of medium using blunted microcapillaries with an inner diameter large enough to accommodate approximately single cell. Dissociated cells were then individually plated into single wells of 96-well (Corning) plates with BFFs feeder cells. After continuous culturing for 5-20 days, single colony detection and assessment were carried out under a dissecting microscope (Leica), and AP staining was conducted for further verification the single cell source. More than one colony in one cell was excluded.
In vitro EB Formation Assay of Bovine EPSCs. In vitro differentiation, bovine EPSCs were detached from culture dishes using TrypLE Select, resuspended in 10% FBS DMEM without LIF and bFGF, and then seeded into ultra-low cell attachment U-bottom 96-well (Corning, 7007). After 4 days in suspension culture, EBs were transferred to gelatin-coated dishes and cultured for another 3 days prior to linage genes RT-qPCR and 7 days prior to immunostaining.
In vivo Chimera Assay. 6-12 tdTomato + bEPSCs iPS-Q36 were injected gently into the ICR mice 8cell stage embryo using a piezo-assisted micromanipulator attached to an inverted microscope (Zeiss, Eppendorf), the protocol was performed as previously described (7) . . The injected embryos were cultured in KSOM (Millipore) and bEPSCM mixture medium (1:1) at 37°C in a 5% CO2 atmosphere overnight and then transferred to the uteri of pseudopregnant ICR mice at 2.5 days post coitus (dpc). The embryos were isolated at embryonic stage E6.5 to check chimeric contribution. And also 5-10 bEPSCs ES-A15 (tdTomato + ) were injected into bovine morulae and blastocysts with the aid of a piezo-driven micromanipulator in SOF medium and bEPSCM mixture medium (1:1). After injection, bovine embryos were cultured in the same medium at 38.5°C in 5% CO2 and 5% O2 for 6-48 hours, part of blastocysts for the evaluation of chimerism, and the other embryos were transferred to the uteri of pseudopregnant bovine at 7 dpc. At day 23-30 after transplantation, pregnancy was diagnosed by ultrasonography and Rapid Visual Pregnancy Test Kit (IDEXX, 99-41369). The fetuses were isolated at embryonic stage day 38-72 to check chimeric contribution.

Production of Nuclear Transfer Embryos Reconstructed with bEPSCs ES .
The bEPSCs ES within passages 15-25 were dispersed to a single cell suspension by TrypLE select (Invitrogen) and recovered in bEPSCM. They were used as donor cells for NT. The NT protocol was performed as previously described (8). Briefly, matured oocytes were denuded of cumulus cells, and oocytes with the first polar body were transferred into TCM199-Hepes medium containing 7.5 μg/ml cytochalasin B. Enucleation was performed with a 20-mm (internal diameter) glass pipette by aspirating the first polar body and approximately 5% of the adjacent surrounding cytoplasm. Single bEPSCs were individually transferred to the perivitelline space of the recipient cytoplasts. Cell fusion was performed using two direct current pulses of 1.0 kV/cm for 10 μs by an ECM 830 Electroporation System (BTX, San Diego, CA, USA) in 0.30 M mannitol, 0.05 mM CaCl2, 0.1 mM MgCl2, and 0.05% BSA. Successfully reconstructed embryos were kept in modified synthetic oviductal fluid (mSOF) (containing 5 mg/mL cytochalasin B) for 2 hours until activation. All fused embryos were further activated in 5-mM ionomycin for 5 minutes, followed by exposure to 2 mM 6-dimethylaminopurine in SOF for 4 hours. After the activation, NT embryos were washed and transferred into 500 μl of SOF media covered with mineral oil in 4well plate, under an atmosphere of 5% CO2, 5% O2, 90% N2. The cleavage rates were determined 48h after culturing, and the blastocyst rates were determined 7 days after culturing.  Table. KAPA SYBR FAST Universal qPCR Kit (Roche-KAPA, KK4601) were used for RT-qPCR assays. All RT-qPCR reactions were performed on Veriti 96 cell Thermal Cycler (Applied Biosystems, USA). Gene expression was determined relative to GAPDH using the ΔΔCt method. Data are shown as the mean and SD.
Cryosectioning and Immunofluorescence Staining. Immunofluorescence staining of bEPSCs was performed as described previously (9). The slides were imaged with a confocal microscope FLUOVIEW FV1000 (Olympus). For immunofluorescence staining of cryo-sections of chimeric fetus, all fetus were half separated along vertical axis, one half part for genotyping, and the other half part was immersed in 4% PFA immediately after collection overnight at 4°C, then dehydration 1 hour in 5% sucrose, then 10% sucrose 1 hour, 20% sucrose 1 hour. Next, fetuses were embedded in mixture of Tissue-Tek O.C.T. compound (SAKURA, 4583) and 20% sucrose (Sigma) overnight at 4°C, at last for liquid nitrogen quick freezing. The frozen OCT blocks were sectioned by CRYOSTAR NX50 (Thermo), at 10 µm each section. Sections were first permeabilized with 0.1% Triton and blocked with 5 % donkey serum plus 1% BSA followed by incubations with primary antibodies overnight at 4°C. The primary antibodies were used as follows: OCT4 (Santa Cruz Biotechnology), SOX2 (Millipore), NANOG (Thermo Fisher Scientific), AFP (R&D Systems), SMA (R&D Systems), PL-1 (SantaCruz), KRT7 (SantaCruz), SOX17 (R&D Systems), CDX2 (BioGenex), GATA6 (R&D Systems), hCGß (Abcam), SDC1 (Abcam), GATA3 (R&D Systems), Beta-III Tubulin (R&D Systems) and E-CADHERIN (BD Biosciences). Fluorescence-conjugated secondary antibodies were used to incubate the slides at room temperature for 1 hour. The antibodies are listed in Table S3. Whole Genome Bisulfite Sequencing (WGBS) Library Construction and Sequencing. The WGBS libraries were prepared according to the standard protocol (10,11). Shortly, genomic DNA was isolated from cells using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's protocol. Then, approximately 2 µg of genomic DNA spiked with 10ng (0.5%) unmethylated lambda DNA (Fermentas) was sheared into an average size of 200-300 bp using Covaris S2 system and purified with DNA Clean & Concentrator™-5 Kit (Zymo research). The fragmented DNA was end-repaired, dA-tailed and ligated with cytosine-methylated adapters (NEB), followed by cleanup of adaptor-ligated DNA using AMPure XP beads. Subsequently, bisulfite conversion was performed with EZ-96 DNA Methylation-Direct™ MagPrep (Zymo research) using the manufacture's standard manual. After bisulfite conversion, the converted templates were PCR amplified followed by purification and quantified using Qubit ds DNA high sensitivity dye (Invitrogen). The final quality-insured libraries were sequenced on Illumina HiSeq2000 platforms with 150-bp paired-end mode (sequenced by Novogene).

RNA-seq Library Preparation and
Processing RNA-Seq Data. RNA-seq raw sequencing data were firstly removed reads with adapters and low-quality bases using the customer script. Clean reads were then aligned into the bovine genome bosTau9 (UCSC version) with software TopHat (version: 2.0.12) using default parameters (12). Uniquely mapped reads were then counted with software HTSeq (13), and gene expression levels were quantified with the RPKM (reads per kilobase million).

Batch Correction in Cross-Species Comparisons.
Batch effects in RNA-seq data were obvious among different species and different studies which can be detected in principal component analysis (PCA), and therefore, batch correction was essential in cross-species comparisons. We set source studies corresponding to RNA-seq data as batch labels, and then removed batch effects with function ComBat in R package sva (16). The corrected RNA-seq data were used to perform PCA with R function prcomp and unsupervised clustering with R function hclust.