title: Dual kinase inhibition promotes pluripotency in finite bovine embryonic cell lines.
abstract: Embryonic pluripotent stem cells (ePSCs) can generate all somatic cell types, as well as functional gametes. In mouse and rat, derivation of ePSCs from the early epiblast is promoted by the double inhibition ("2i") of mitogen-activated protein kinase kinase (MAP2K), antagonizing fibroblast growth factor signaling (FGF), and glycogen synthase kinase 3 (GSK3), stimulating the WNT pathway. However, it has remained unclear whether this culture regime is applicable to nonrodent livestock species. Here we report the generation of bovine ePSCs under minimal conditions. Inner cell masses (ICMs) were immunosurgically isolated from in vitro fertilized bovine blastocysts and cultured feeder-free in 2i medium. Dual kinase inhibition primed bovine ICMs for stem cell derivation and sustained expression of epiblast-specific pluripotency markers SOX2 and NANOG, while repressing the hypoblast marker GATA4. Following mechanical passage, 2i supported limited proliferation for several weeks. Continuously cultured ePSC lines expressed discriminatory markers of naïve pluripotency and primordial germ cells, but not of primed epiblast stem cells. In female ePSCs, most OCT4-positive cells lacked epigenetically silenced X-chromosomes, displaying a diagnostic feature of naïve pluripotency. Bovine ePSCs maintained a normal karyotype and differentiated into derivatives of all three germ layers in suspension culture. This culture system provides a screening platform for factors that maintain long-term proliferation of pluripotent embryonic cattle cells without genetic intervention.
authors.affiliation: AgResearch, Ruakura Research Centre, Hamilton 3214, New Zealand.
authors.forename: Vinod
authors.initials: V
authors.lastname: Verma
authors.forename: Ben
authors.initials: B
authors.lastname: Huang
authors.forename: Prasanna Kumar
authors.initials: PK
authors.lastname: Kallingappa
authors.forename: Björn
authors.initials: B
authors.lastname: Oback
mesh_terms: D000818:Animals; D017209:Apoptosis; D015415:Biomarkers; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000502:physiology*; D005346:Fibroblast Growth Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050991:GATA4 Transcription Factor / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000502:physiology*; D055748:SOXB1 Transcription Factors / Q000378:metabolism
chemical_list: D015415:Biomarkers; D050991:GATA4 Transcription Factor; D018398:Homeodomain Proteins; D055748:SOXB1 Transcription Factors; D005346:Fibroblast Growth Factors; D038362:Glycogen Synthase Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases