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

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.

Changes in testicular gene expression following reduced estradiol synthesis: A complex pathway to increased porcine Sertoli cell proliferation.

Porcine Sertoli cell number including number present at puberty is increased if testicular estradiol synthesis is reduced during the neonatal interval. Evaluating the changes in gene expression during the crucial interval of suppressed estradiol that leads to the increased Sertoli cell population will increase our understanding of Sertoli cell biology but this evaluation first required a more precise determination of the critical interval for treatment and timing of a detectable response. Previously, reduced testicular estrogens from 1 week of age were accompanied by increased Sertoli cell number at 6.5 weeks of age but the age at which Sertoli cell numbers were initially increased was unknown, one of the current objectives. Additional experiments were designed to further delineate the essential timing of treatment for the Sertoli cell response. Finally, changes in gene expression induced by the reduced estradiol synthesis were evaluated to elucidate molecular mechanisms. Experimental design typically consisted of one member of littermate pairs of boars treated with the aromatase inhibitor, letrozole, beginning at 1 week of age and the remaining member treated with canola oil vehicle. Weekly treatments continued through 5 weeks of age or tissue collection, whichever came first. Increases in Sertoli cell numbers were not detectable prior to 6.5 weeks of age and persistent treatment through 5 weeks of age was required to induce the increase in Sertoli cell numbers. This increase resulted from prolonging the first interval of Sertoli cell proliferation in the treated animals. Few genes exhibited dramatically altered transcription and similarities in pathway analysis or principal modified genes were quite limited in 2, 3, and 5-week-old boars. The critical timing and prolonged treatment required and the sequential changes in gene expression suggest a complex mechanism is involved in this model of increased proliferation of Sertoli cells.

Similarities between bovine and human germline development revealed by single-cell RNA sequencing.

The germ cell lineage ensures the creation of new individuals and perpetuates the genetic information across generations. Primordial germ cells are pioneers of gametes and exist transiently during development until they differentiate into oogonia in females, or spermatogonia in males. Little is known about the molecular characteristics of primordial germ cells in cattle. By performing single-cell RNA-sequencing, quantitative real-time PCR, and immunofluorescence analyses of fetal gonads between 40 and 90 days of fetal age, we evaluated the molecular signatures of bovine germ cells at the initial stages of gonadal development. Our results indicate that at 50 days of fetal age, bovine primordial germ cells were in the early stages of development, expressing genes of early primordial germ cells, including transcriptional regulators of human germline specification (e.g. SOX17, TFAP2C, and PRDM1). Bovine and human primordial germ cells also share expression of KIT, EPCAM, ITGA6, and PDPN genes coding for membrane-bound proteins, and an asynchronous pattern of differentiation. Additionally, the expression of members of Notch, Nodal/Activin, and BMP signaling cascades in the bovine fetal ovary, suggests that these pathways are involved in the interaction between germ cells and their niche. Results of this study provide insights into the mechanisms involved in the development of bovine primordial germ cells and put in evidence similarities between the bovine and human germline.

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

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.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

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.