title: Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen.
abstract: This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% (previously termed hypoxia) and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over a period of 2 weeks. This timescale is comparable to that observed at 20% O(2), but survival is enhanced. Sequential application of retinoic acid and purmorphamine (PM), from day 14 to day 28, directs differentiation toward spinal motor neurons. Alternatively, addition of fibroblast growth factor-8 and PM generates midbrain dopaminergic neurons. OLIG2 (encoding oligodendrocyte lineage transcription factor 2) induction in motor neuron precursors is twofold greater than that at 20% O(2), whereas EN1 (encoding engrailed homeobox 1) expression is enhanced fivefold. NPCs (at 3% O(2)) can be differentiated into all three neural lineages, and such cultures can be maintained long term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advancement for in vitro disease modeling and potentially for cell-based therapies.
authors.affiliation: Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. srls2@cam.ac.uk
authors.forename: S R L
authors.initials: SR
authors.lastname: Stacpoole
authors.forename: B
authors.initials: B
authors.lastname: Bilican
authors.forename: D J
authors.initials: DJ
authors.lastname: Webber
authors.forename: A
authors.initials: A
authors.lastname: Luzhynskaya
authors.forename: X L
authors.initials: XL
authors.lastname: He
authors.forename: A
authors.initials: A
authors.lastname: Compston
authors.forename: R
authors.initials: R
authors.lastname: Karadottir
authors.forename: R J M
authors.initials: RJ
authors.lastname: Franklin
authors.forename: S
authors.initials: S
authors.lastname: Chandran
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D004594:Electrophysiology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D051524:Fibroblast Growth Factor 8 / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D008636:Mesencephalon / Q000166:cytology*; D009025:Morpholines / Q000494:pharmacology; D009046:Motor Neurons / Q000166:cytology* / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects; D000076104:Oligodendrocyte Transcription Factor 2; D010100:Oxygen / Q000378:metabolism; D011687:Purines / Q000494:pharmacology; D013127:Spinal Nerves / Q000166:cytology; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D051524:Fibroblast Growth Factor 8; D018398:Homeodomain Proteins; D009025:Morpholines; D009419:Nerve Tissue Proteins; D000076104:Oligodendrocyte Transcription Factor 2; D010100:Oxygen; D011687:Purines; D014212:Tretinoin; C513076:EN1 protein, human; C405708:OLIG2 protein, human; C470893:purmorphamine