Department of Animal Science, University of California, 450 Bioletti Way, Davis, CA, 95616, USA.
Instituto de Investigaciones Biotecnológicas 'Dr Rodolfo Ugalde', UNSAM-CONICET, Buenos Aires, Argentina.
Sci Rep. 2021 May 26;11(1):11045. doi: 10.1038/s41598-021-90422-0.
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.
牛胚胎干细胞（bESCs）可延长体外短暂多能性牛内细胞团的寿命。经过多年的研究，最近才成功报道了稳定的 bESC 的衍生。尽管成功了，但 bESC 的培养依赖于复杂的培养条件，需要定制的基础培养基和小鼠胚胎成纤维细胞（MEF）饲养层，限制了 bESC 的广泛应用。我们在此报告了简化的 bESC 培养条件，方法是用市售的替代物替代定制的基础培养基，并通过使用化学定义的基质消除对 MEF 饲养层的需求。bESC 系使用包含 N2B27 补充剂和 1%BSA（NBFR-bESCs）的基础培养基进行培养和衍生。新衍生的 bESC 系易于建立，易于繁殖，并且在长期培养后稳定。这些细胞表达多能性标记物，并在维持正常核型和分化为胚状体和畸胎瘤中所有三个生殖系衍生物的能力的情况下，可增殖超过 35 代。此外，NBFR-bESCs 在基于纤连蛋白和激活素 A 培养基补充的无饲养层培养系统中可传代多次，同时保持多能性。简化的条件将促进 bESC 在基因编辑应用和多能性及谱系分化研究中的应用。
