title: Lineage selection of functional and cryopreservable human embryonic stem cell-derived neurons.
abstract: A major prerequisite for the biomedical application of human embryonic stem cells (hESC) is the derivation of defined and homogeneous somatic cell types. Here we present a human doublecortin (DCX) promoter-based lineage-selection strategy for the generation of purified hESC-derived immature neurons. After transfection of hESC-derived neural precursors with a DCX-enhanced green fluorescent protein construct, fluorescence-activated cell sorting enables the enrichment of immature human neurons at purities of up to 95%. Selected neurons undergo functional maturation and are able to establish synaptic connections. Considering that the applicability of purified hESC-derived neurons would largely benefit from an efficient cryopreservation technique, we set out to devise defined freezing conditions involving caspase inhibition, which yield post-thaw recovery rates of up to 83%. Combined with our lineage-selection procedure this cryopreservation technique enables the generation of human neurons in a ready-to-use format for a large variety of biomedical applications.
authors.affiliation: Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Ladewig
authors.forename: Philipp
authors.initials: P
authors.lastname: Koch
authors.forename: Elmar
authors.initials: E
authors.lastname: Endl
authors.forename: Banu
authors.initials: B
authors.lastname: Meiners
authors.forename: Thoralf
authors.initials: T
authors.lastname: Opitz
authors.forename: Sebastien
authors.initials: S
authors.lastname: Couillard-Despres
authors.forename: Ludwig
authors.initials: L
authors.lastname: Aigner
authors.forename: Oliver
authors.initials: O
authors.lastname: Brüstle
mesh_terms: D000818:Animals; D019070:Cell Lineage; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D004594:Electrophysiology; D053595:Embryonic Stem Cells / Q000166:cytology*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000196:embryology; D006801:Humans; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D009474:Neurons / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics*; D011401:Promoter Regions, Genetic
chemical_list: C000714328:DCX protein, human; C000714847:Dcx protein, mouse; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; D049452:Green Fluorescent Proteins