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TISSUE-SPECIFIC STEM CELLS

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

^aInstitute of Reconstructive Neurobiology, Life and Brain Center and Hertie Foundation,
^bInstitute of Molecular Medicine and Experimental Immunology, University of Bonn, Bonn, Germany;
^cDepartment of Neurology, University of Regensburg, Regensburg, Germany

Key Words. Human embryonic stem cells • Lineage selection • Human neurons • Cryopreservation

Correspondence: Prof. Oliver Brüstle, M.D., Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany. Telephone: +49-228-6885-500; Fax: +49-228-6885-501; e-mail: brustle{at}uni-bonn.de

Received January 4, 2008; accepted for publication April 6, 2008.
First published online in STEM CELLS EXPRESS   April 17, 2008.

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.