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

¹ Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation; Germany.
² Institute of Molecular Medicine and Experimental Immunology, University of Bonn; Germany.
³ Department of Neurology, University of Regensburg, Germany.

^* To whom correspondence should be addressed. E-mail: brustle{at}uni-bonn.de.

	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. Following transfection of hESC-derived neural precursors with a DCX-EGFP construct, fluorescence-activated cell sorting (FACS) 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.