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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.

The brainstem is a posterior region of the brain, composed of three parts, midbrain, pons, and medulla oblongata. It is critical in controlling heartbeat, blood pressure, and respiration, all of which are life-sustaining functions, and therefore, damages to or disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells (hPSCs) recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models are limited in the extent hPSCs recapitulate human brain development and hence are not able to fully elucidate the diseases affecting various components of the brain such as brainstem. Here, we developed a method to generate human brainstem organoids (hBSOs), containing midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, dopaminergic neurons, and neural crest lineage cells. Single-cell RNA sequence (scRNA-seq) analysis, together with evidence from proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem, which raises the possibility of making use of hBSOs in investigating central nervous system disorders affecting brainstem and in efficient drug screenings.