The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs.

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.

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.

The WNT signaling system plays an important but paradoxical role in the regulation of pluripotency. In the cow, IWR-1, which inhibits canonical WNT activation and has WNT-independent actions, promotes the derivation of primed pluripotent embryonic stem cells from the blastocyst. Here, we describe a series of experiments to determine whether derivation of embryonic stem cells could be generated by replacing IWR-1 with other inhibitors of WNT signaling. Results confirm the importance of inhibition of canonical WNT signaling for the establishment of pluripotent embryonic stem cells in cattle and indicate that the actions of IWR-1 can be mimicked by the WNT secretion inhibitor IWP2 but not by the tankyrase inhibitor XAV939 or WNT inhibitory protein dickkopf 1. The role of Janus kinase-mediated signaling pathways for the maintenance of pluripotency of embryonic stem cells was also evaluated. Maintenance of pluripotency of embryonic stem cells lines was blocked by a broad inhibitor of Janus kinase, even though the cells did not express phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Further studies with blastocysts indicated that IWR-1 blocks the activation of pSTAT3. A likely explanation is that IWR-1 blocks differentiation of embryonic stem cells into a pSTAT3+ lineage. In conclusion, results presented here indicate the importance of inhibition of WNT signaling for the derivation of pluripotent bovine embryonic stem cells, the role of Janus kinase signaling for maintenance of pluripotency, and the participation of IWR-1 in the inhibition of activation of STAT3.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.