Neural crest (NC) cells are multipotent cells that are specified at the prospective neural plate border, undergo an epithelial-to-mesenchymal transition to form NC cells, and migrate from the dorsal neural tube to their target tissues where they differentiate into various cell types [1
]. The abnormal development of NC cells can result in severe congenital birth defects that affect craniofacial, cardiac, and peripheral nervous system structure and function. These defects include some of the most frequently observed birth defects such as cleft palate, conotruncal heart malformations, and Hirschprung disease [2
]. In addition, NC-derived tumors such as neuroblastomas represent 8% of all childhood tumors and account for 15% of cancer-related deaths in children. While many studies have begun to elucidate the mechanisms governing NC development, many questions remain unanswered, particularly with respect to how alterations in NC development can cause disease.
It is known that the regulation of specific developmental pathways, including the bone morphogenetic protein (BMP)/Activin and Wnt signaling axes, are required for proper NC development during embryogenesis [3
]. To better understand the precise role of these signaling pathways, much effort has been devoted to developing methods to induce NC cell formation in vitro
. However, the first methods to do this were complex, multi-step procedures that relied on the formation of embryoid bodies and the use of stromal feeder co-cultures [4
]. The first embryoid body–independent methods to generate NC cells were focused on producing neural stem cells [6
], whereby NC cells were observed as a by-product of neural differentiation. However, it was soon realized that these neural precursor methods could be used to obtain larger numbers of NC cells. These newer methods included the use of Noggin, an endogenous secreted peptide that antagonizes BMP signaling by blocking BMP interaction with its receptors, and SB435142, a small molecule that inhibits the Activin type–I receptor ALK4 and the nodal type–I receptor ALK7, in human induced pluripotent stem (iPS) cells and human embryonic stem (ES) cells [6
]. More recently, it was demonstrated that LDN-193189 [7
], a potent, synthetic small-molecule inhibitor of BMP type–I receptors ALK2 and ALK3, could substitute for Noggin during peripheral nerve differentiation of iPS cells [8
]. Blocking BMP and Activin A/Nodal signaling resulted in the inhibition of downstream SMAD signaling, thereby facilitating differentiation into neural and NC lineages [6
]. Concurrent activation of Wnt signaling can also induce NC cell formation from pluripotent stem cells [9
In spite of this progress, most existing methods for in vitro NC cell formation remain relatively inefficient. They yield highly heterogeneous populations and require cell-sorting techniques to enrich for NC cells, making it difficult to study NC-specific phenotypes in culture. Also, the functionality of these derived NC cells, such as their migratory potential in response to known chemoattractants, has not been extensively evaluated in vitro. Understanding this intrinsic migratory potential will be important for improving our understanding of NC cell migratory defects in various NC disorders. Furthermore, NC cells are comprised of diverse subpopulations, including cranial, cardiac, and trunk NC cells, and it is unclear whether the existing methods can generate all of these subpopulations. This consideration is critical when studying specific NC disorders that result in craniofacial, cardiac, or enteric developmental abnormalities. Therefore, more robust and efficient methods for NC derivation will be required for applications in regenerative medicine and drug discovery.
The advent of human iPS cell technology provides an invaluable tool for studying both normal NC development as well as NC disease mechanisms [11
]. In this report, we applied this technology to develop a rapid, robust, and reproducible method, known as “LSB-short”, to derive NC cells from human pluripotent stem cells. The LSB-short method is similar to the previously published LSB method, which uses a combination of LDN-193189 and SB435142 to induce NC formation in approximately two weeks [8
]. Our method generates high yields of NC cells in a shorter time frame compared to other published methods. The NC cells could be cultured over multiple passages while maintaining NC marker expression, and could spontaneously differentiate into different NC lineages. Global gene-expression analyses revealed the multipotency potential of NC cells, while migration assays confirmed their migratory potential. The LSB-short method is therefore well suited for regenerative medicine and drug-screening applications, and could be used to advance our understanding of NC biology in development and disease.