Am J Stem Cell 2013;2(2):119-131

Original Article
A robust method to derive functional neural crest cells from human pluripotent stem
cells

Faith R Kreitzer, Nathan Salomonis, Alice Sheehan, Miller Huang, Jason S Park, Matthew J Spindler, Paweena Lizarraga, William A
Weiss, Po-Lin So, Bruce R Conklin

Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158, USA; California Pacific Medical Center
Research Institute, 475 Brannan Street, Suite 220, San Francisco, CA 94107, USA; Department of Neurology, University of California San
Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California San Francisco,
San Francisco, CA 94158, USA; Department of Medicine, University of California San Francisco, San Francisco, CA 94158, USA

Received April 2, 2013; Accepted June 3, 2013; Epub June 30, 2013; Published July 15, 2013

Abstract: Neural crest (NC) cells contribute to the development of many complex tissues of all three germ layers during embryogenesis,
and its abnormal development accounts for several congenital birth defects. Generating NC cells—including specific subpopulations
such as cranial, cardiac, and trunk NC cells—from human pluripotent stem cells will provide a valuable model system to study human
development and disease. Here, we describe a rapid and robust NC differentiation method called “LSB-short” that is based on dual
SMAD pathway inhibition. This protocol yields high percentages of NC cell populations from multiple human induced pluripotent stem
and human embryonic stem cell lines in 8 days. The resulting cells can be propagated easily, retain NC marker expression over multiple
passages, and can spontaneously differentiate into several NC-derived cell lineages, including smooth muscle cells, peripheral
neurons, and Schwann cells. NC cells generated by this method represent cranial, cardiac and trunk NC subpopulations based on
global gene expression analyses, are similar to in vivo analogues, and express a common set of NC alternative isoforms. Functionally,
they are also able to migrate appropriately in response to chemoattractants such as SDF-1, FGF8b, and Wnt3a. By yielding NC cells that
likely represent all NC subpopulations in a shorter time frame than other published methods, our LSB-short method provides an ideal
model system for further studies of human NC development and disease. (AJSC1304002).

Keywords: Neural crest, induced pluripotent stem cells, human, SMAD inhibition, differentiation, migration

Address correspondence to: Dr. Po-Lin So, Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA
94158, USA. E-mail: polin.so@gladstone.ucsf.edu
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