title: WNT/β-catenin signaling affects cell lineage and pluripotency-specific gene expression in bovine blastocysts: prospects for bovine embryonic stem cell derivation.
abstract: Despite many attempts, true bovine embryonic stem cells (bESC) still remain elusive. The WNT pathway has been associated with stem cell control in vertebrates and its role in pluripotency maintenance has been proven for several mammalian species, including rodents and primates. Thus, we have aimed to investigate the effect of WNT activation on pluripotency marker gene expression in the inner cell mass (ICM) and the trophectoderm (TE) and to study the derivation potential of primary bESC lines from blastocysts obtained in the presence of the glycogen synthase kinase 3 inhibitor (GSK3i). WNT activity clearly exerted a positive effect on pluripotency gene expression in developing bovine embryos, manifested by upregulation of OCT4, NANOG, REX1, SOX2, c-MYC, and KLF4 in the ICM and downregulation of CDX2 in the TE. However, the prolonged exposition of preimplantation embryos to the GSK3i resulted in reduced potential to form primary ESC-like colonies. The results of bESC derivation experiments allowed us to speculate that the derived cell lines may share features of both naïve and primed ESCs. Similar to mouse epiblast stem cells and human ESCs, the derived lines grew as flat monolayer colonies intolerant to passaging as single cells. JAK/STAT signaling was indispensable for proper colony formation and proliferation, yet LIF alone was inefficient to support self-renewal. Concomitant with the naïve state of mouse ESCs, WNT activity supported by LIF had beneficial effects on cell culture propagation, survival after passage, morphology, and pluripotency-related marker gene expression. Moreover, colonies derived in the presence of LIF and GSK3i maintained KLF4 transcription over several passages, whereas EpiSCs virtually do not express KLF4.
authors.affiliation: Department of Genetics and Animal Breeding, Poznan University of Life Sciences , Poznan, Poland .
authors.forename: Zofia Eliza
authors.initials: ZE
authors.lastname: Madeja
authors.affiliation: Department of Genetics and Animal Breeding, Poznan University of Life Sciences , Poznan, Poland .
authors.forename: Kamila
authors.initials: K
authors.lastname: Hryniewicz
authors.affiliation: Department of Genetics and Animal Breeding, Poznan University of Life Sciences , Poznan, Poland .
authors.forename: Maciej
authors.initials: M
authors.lastname: Orsztynowicz
authors.affiliation: Department of Genetics and Animal Breeding, Poznan University of Life Sciences , Poznan, Poland .
authors.forename: Piotr
authors.initials: P
authors.lastname: Pawlak
authors.affiliation: Department of Genetics and Animal Breeding, Poznan University of Life Sciences , Poznan, Poland .
authors.forename: Anna
authors.initials: A
authors.lastname: Perkowska
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D002417:Cattle; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental; D000090062:Kruppel-Like Factor 4; D039904:Pluripotent Stem Cells / Q000166:cytology; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism*
chemical_list: C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051176:beta Catenin