title: Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.
abstract: The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.
authors.affiliation: NURTURE, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Priddle
authors.forename: Cinzia
authors.initials: C
authors.lastname: Allegrucci
authors.forename: Paul
authors.initials: P
authors.lastname: Burridge
authors.forename: Maria
authors.initials: M
authors.lastname: Munoz
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Smith
authors.forename: Lyndsey
authors.initials: L
authors.lastname: Devlin
authors.forename: Cecilia
authors.initials: C
authors.lastname: Sjoblom
authors.forename: Sarah
authors.initials: S
authors.lastname: Chamberlain
authors.forename: Sue
authors.initials: S
authors.lastname: Watson
authors.forename: Lorraine E
authors.initials: LE
authors.lastname: Young
authors.forename: Chris
authors.initials: C
authors.lastname: Denning
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D014162:Transfection