title: Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency.
abstract: Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state.
authors.affiliation: Instituto de Medicina Molecular and Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa, Avenida Prof. Egas Moniz, Lisboa 1649-028, Portugal Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Avenida Brasilia - Doca de Pedrouços, Lisboa 1400-038, Portugal elsa.abranches@gmail.com henrique@fm.ul.pt.
authors.forename: Elsa
authors.initials: E
authors.lastname: Abranches
authors.affiliation: Instituto de Medicina Molecular and Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa, Avenida Prof. Egas Moniz, Lisboa 1649-028, Portugal Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Avenida Brasilia - Doca de Pedrouços, Lisboa 1400-038, Portugal.
authors.forename: Ana M V
authors.initials: AM
authors.lastname: Guedes
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Moravec
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street, Philadelphia, PA 19104, USA.
authors.forename: Hedia
authors.initials: H
authors.lastname: Maamar
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Svoboda
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street, Philadelphia, PA 19104, USA.
authors.forename: Arjun
authors.initials: A
authors.lastname: Raj
authors.affiliation: Instituto de Medicina Molecular and Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa, Avenida Prof. Egas Moniz, Lisboa 1649-028, Portugal Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Avenida Brasilia - Doca de Pedrouços, Lisboa 1400-038, Portugal elsa.abranches@gmail.com henrique@fm.ul.pt.
authors.forename: Domingos
authors.initials: D
authors.lastname: Henrique
mesh_terms: D000818:Animals; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics; D049109:Cell Proliferation; D002999:Clone Cells; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D000071317:Nanog Homeobox Protein; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D025341:Principal Component Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013269:Stochastic Processes; D059008:Time-Lapse Imaging; D014158:Transcription, Genetic
chemical_list: D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D012333:RNA, Messenger
keywords: Gene expression heterogeneity; Lineage priming; Nanog; Pluripotency; Stem cells