title: Small-Molecule Induction of Canine Embryonic Stem Cells Toward Naïve Pluripotency.
abstract: Naïve and primed pluripotent stem cells (PSCs) reflect discrete pluripotent states that approximate the inner cell mass or the progressively lineage-restricted perigastrulation epiblast, respectively. Cells that occupy primed pluripotency have distinct epigenetic landscapes, transcriptional circuitry, and trophic requirements compared with their naïve counterparts. The existence of multiple pluripotent states has not been explored in dogs, which show promise as outbred biomedical models with more than 300 inherited diseases that also afflict humans. However, our understanding of canine embryogenesis and embryo-derived stem cells is limited. Herein, we converted leukemia inhibitory factor (LIF)-dependent and fibroblast growth factor 2 (FGF2)-dependent canine embryonic stem cells (cESCs) resembling primed PSCs toward a naïve pluripotent state using LIF and inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1/2 [called 2i and LIF (2iL)]. cESCs propagated in 2iL exhibited significant induction of genes associated with the naïve pluripotent state (eg, REX1, TBX3) and downregulation of primed pluripotency markers (eg, OTX2, FGF5) (P < 0.05). Differential phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell fate decisions on exposure to bone morphogenetic protein 4 (BMP4) suggested that a novel pluripotent identity has been established with 2iL. Accordingly, cESCs cultured with 2iL formed colonies at a greater efficiency than LIF-FGF2 cESCs following single-cell dissociation. Total genomic DNA methylation and histone H3 lysine 27 trimethylation signals were reduced in 2iL-treated cESCs. Our data suggest that 2iL culture conditions promote the conversion of cESCs toward an epigenetically distinct pluripotent state resembling naïve PSCs.
authors.affiliation: 1 Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario , London, Ontario, Canada .
authors.forename: Ian C
authors.initials: IC
authors.lastname: Tobias
authors.affiliation: 1 Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario , London, Ontario, Canada .
authors.forename: Courtney R
authors.initials: CR
authors.lastname: Brooks
authors.affiliation: 1 Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario , London, Ontario, Canada .
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Teichroeb
authors.affiliation: 2 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph , Guelph, Ontario, Canada .|3 Departamento de Producción Animal, Universidad de Guadalajara , Zapopan, Jalisco, Mexico .
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Villagómez
authors.affiliation: 1 Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario , London, Ontario, Canada .|4 Children's Health Research Institute, the University of Western Ontario , London, Ontario, Canada .|5 Molecular Medicine Research Group, Krembil Centre for Stem Cell Biology, Robarts Research Institute, the University of Western Ontario , London, Ontario Canada .
authors.forename: David A
authors.initials: DA
authors.lastname: Hess
authors.affiliation: 1 Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario , London, Ontario, Canada .|4 Children's Health Research Institute, the University of Western Ontario , London, Ontario, Canada .
authors.forename: Cheryle A
authors.initials: CA
authors.lastname: Séguin
authors.affiliation: 1 Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, the University of Western Ontario , London, Ontario, Canada .|4 Children's Health Research Institute, the University of Western Ontario , London, Ontario, Canada .
authors.forename: Dean H
authors.initials: DH
authors.lastname: Betts
mesh_terms: D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D019175:DNA Methylation / Q000187:drug effects; D004285:Dogs; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D055415:Bone Morphogenetic Protein 4; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D054852:Small Molecule Libraries; D014157:Transcription Factors