title: Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells.
abstract: Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression.
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, UK.
authors.forename: Tariq
authors.initials: T
authors.lastname: Enver
authors.forename: Shamit
authors.initials: S
authors.lastname: Soneji
authors.forename: Chirag
authors.initials: C
authors.lastname: Joshi
authors.forename: John
authors.initials: J
authors.lastname: Brown
authors.forename: Francisco
authors.initials: F
authors.lastname: Iborra
authors.forename: Torben
authors.initials: T
authors.lastname: Orntoft
authors.forename: Thomas
authors.initials: T
authors.lastname: Thykjaer
authors.forename: Edna
authors.initials: E
authors.lastname: Maltby
authors.forename: Kath
authors.initials: K
authors.lastname: Smith
authors.forename: Raed
authors.initials: R
authors.lastname: Abu Dawud
authors.forename: Mark
authors.initials: M
authors.lastname: Jones
authors.forename: Maryam
authors.initials: M
authors.lastname: Matin
authors.forename: Paul
authors.initials: P
authors.lastname: Gokhale
authors.forename: Jonathan
authors.initials: J
authors.lastname: Draper
authors.forename: Peter W
authors.initials: PW
authors.lastname: Andrews
mesh_terms: D000222:Adaptation, Physiological / Q000502:physiology*; D015295:Antigens, Tumor-Associated, Carbohydrate; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002877:Chromosomes, Human / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004622:Embryo, Mammalian / Q000166:cytology*; D044127:Epigenesis, Genetic / Q000235:genetics*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006028:Glycosphingolipids / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055746:Stage-Specific Embryonic Antigens; D013234:Stem Cells / Q000166:cytology*; D049951:X Chromosome Inactivation / Q000235:genetics
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D017931:DNA Primers; D006028:Glycosphingolipids; D055746:Stage-Specific Embryonic Antigens; C052924:stage-specific embryonic antigen-3