In vitro gametogenesis from embryonic stem cells in livestock species: recent advances, opportunities, and challenges to overcome.

Pluripotent stem cells (PSC) can be stabilized in vitro from pre-implantation stage embryos (embryonic stem cells, ESC) or by reprogramming adult somatic cells (induced pluripotent stem cells, iPSC). The last decade has seen significant advances in the livestock PSC field, particularly the development of robust methods for long-term culture of PSC from several livestock species. Along with this, considerable progress has been made in understanding the states of cellular pluripotency and what they mean for cell differentiation capacity, and significant efforts are ongoing to dissect the critical signaling pathways required for the maintenance of PSC in different species and distinct states of pluripotency. Among the cell types that can be generated from PSC, the germline holds special importance as they are the genetic link between generations; and devising methods to enable in vitro gametogenesis (IVG) and produce viable gametes could revolutionize animal agriculture, wildlife conservation, and human assisted reproduction alike. Within the last decade, many pivotal studies about IVG were published using rodent models, filling some critical knowledge gaps in the field. Most importantly, the entire female reproductive cycle was reproduced in vitro from mouse ESC. Although complete male gametogenesis in vitro has not yet been reported, significant advances were made showing the capacity of germline stem cell-like cells to generate healthy offspring. In this review, we provide an overview of PSC and advances in the establishment of livestock PSC; we present the breakthroughs made in rodents regarding IVG and the current progress towards livestock IVG, including the importance of a detailed understanding of fetal germline development. Finally, we discuss some key advances that will be critical to enable this technology at scale. Given the potential impact of IVG for animal agriculture, major efforts will likely continue to be employed by research institutions and industry towards the development of methods to achieve efficient generation of gametes in vitro.

In this review, we summarize the current state of livestock embryonic stem cell establishment and the advances in production of sperm and eggs in vitro in rodents and livestock. We also discuss the potential and challenges of developing systems that support in vitro gametogenesis in livestock and the opportunities for this new technology in the reproductive field.

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.

Dietary Calcium and Phosphorus Amounts Affect Development and Tissue-Specific Stem Cell Characteristics in Neonatal Pigs.

BACKGROUND: Dietary calcium and phosphorus are required for bone and muscle development. Deficiencies of these macrominerals reduce bone mineral and muscle accretion potentially via alterations of mesenchymal stem cell (MSC) and satellite cell (SC) activities. OBJECTIVES: With increasing interest in the role of early-life events on lifetime health outcomes, we aimed to elucidate the impact of dietary calcium and phosphorus, from deficiency through excess, on MSC and SC characteristics during neonatal development. METHODS: Neonatal pigs [30 females, 1-d-old, 1.46 ± 0.04 kg body weight (BW)] were fed milk replacers for 16 d that were isonitrogenous and isocaloric with a consistent ratio of calcium to phosphorus, but either 25% deficient (calcium: 0.78%; phosphorus: 0.60%; CaPD), adequate (calcium: 1.08%; phosphorus: 0.84%; CaPA), or 25% in excess (calcium: 1.38%; phosphorus: 1.08%; CaPE) of calcium and phosphorus requirements based on sow-milk composition and extrapolation from NRC requirements for older pigs. BW and feed intake were recorded daily. Blood was collected for serum phosphorus, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23) determination. Humeri were collected for MSC isolation and radii/ulnae bone were collected for analysis. Longissimus dorsi muscle was collected for SC isolation and analysis. RESULTS: There was 4.6% increase in bone ash percentage in CaPE- versus CaPD-fed pigs (P < 0.05). In vivo proliferation indicated a 41.3% increase in MSCs in CaPA compared with CaPD and a 19% increase in SCs in CaPA compared with both CaPE and CaPD. MSCs from CaPD had 2- to 5-fold greater expression of peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), and lipoprotein lipase (LPL) but lower osteocalcin (BGLAP) and fibronectin (FN1) expression than CaPA (P < 0.05). SCs from CaPD-fed pigs had 19% lower in vivo proliferation than in CaPA-fed pigs. CONCLUSIONS: These findings demonstrated that feeding a diet marginally deficient in calcium and phosphorus to neonatal pigs had a great impact on bone development, MSC, and SC characteristics. These dietary deficiencies may program future bone health and muscle development by altering MSC and SC activities.