Lineage selection of functional and cryopreservable human embryonic stem cell-derived neurons.
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.
Ladewig
Julia
J
Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany.
Koch
Philipp
P
Endl
Elmar
E
Meiners
Banu
B
Opitz
Thoralf
T
Couillard-Despres
Sebastien
S
Aigner
Ludwig
L
Brüstle
Oliver
O
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
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