1. ¹Neuroscience, Aging, and Stem Cell Research Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Rd, La Jolla, CA 92037, USA
2. ²Program in Neurogenetics, David Geffen School of Medicine, University of California at Los Angeles, 710 Westwood Plaza, Los Angeles, CA 90095, USA
3. ³Infectious and Inflammatory Disease Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Rd, La Jolla, CA 92037, USA

Correspondence: AV Terskikh, Neroscience, Aging, and Stem Cell Research Center, Burnham Institute for Medical Research, 10901 North Torrye Pines Road, La Jolla, CA 92037, USA. Tel: +858 795 5299; Fax: +858 795 5298; E-mail: terskikh@burnham.org

Received 11 June 2008; Revised 8 December 2008; Accepted 13 January 2009; Published online 13 March 2009.

Abstract

Insights into early human development are fundamental for our understanding of human biology. Efficient differentiation of human embryonic stem cells (hESCs) into neural precursor cells is critical for future cell-based therapies. Here, using defined conditions, we characterized a new method for rapid and uniform differentiation of hESCs into committed neural precursor cells (designated C-NPCs). Dynamic gene expression analysis identified several distinct stages of ESC neuralization and revealed functional modules of coregulated genes and pathways. The first wave of gene expression changes, likely corresponding to the transition through primitive ectoderm, started at day 3, preceding the formation of columnar neuroepithelial rosettes. The second wave started at day 5, coinciding with the formation of rosettes. The majority of C-NPCs were positive for both anterior and posterior markers of developing neuroepithelium. In culture, C-NPCs became electrophysiologically functional neurons; on transplantation into neonatal mouse brains, C-NPCs integrated into the cortex and olfactory bulb, acquiring appropriate neuronal morphologies and markers. Compared to rosette-NPCs,¹ C-NPCs exhibited limited in vitro expansion capacity and did not express potent oncogenes such as PLAG1 or RSPO3. Concordantly, we never detected tumors or excessive neural proliferation after transplantation of C-NPCs into mouse brains. In conclusion, our study provides a framework for future analysis of molecular signaling during ESC neuralization.