Lineage Selection of Functional and Cryopreservable Human Embryonic Stem Cell‐Derived Neurons

• Published in: Stem cells (Dayton, Ohio) Vol. 26; no. 7; pp. 1705 - 1712
• Main Authors: Ladewig, Julia, Koch, Philipp, Endl, Elmar, Meiners, Banu, Opitz, Thoralf, Couillard‐Despres, Sebastien, Aigner, Ludwig, Brüstle, Oliver
• Format: Journal Article
• Language: English
• Published: Bristol John Wiley & Sons, Ltd 01.07.2008
• Subjects: Animals; Cell Lineage; Cells, Cultured; Cryopreservation; Cryopreservation - methods; Electrophysiology; Embryonic Stem Cells - cytology; Flow Cytometry; Green Fluorescent Proteins - metabolism; Hippocampus - embryology; Human embryonic stem cells; Human neurons; Humans; Lineage selection; Mice; Microtubule-Associated Proteins - genetics; Neurons - metabolism; Neuropeptides - genetics; Promoter Regions, Genetic
• ISSN: 1066-5099; 1549-4918; 1549-4918
• DOI: 10.1634/stemcells.2008-0007

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. Disclosure of potential conflicts of interest is found at the end of this article.