Isolation and transcriptional profiling of embryonic human neural crest cells
Creators: name: Thomas, Sophie
name: Thomas, Marie
name: Wincker, Patrick
name: Babarit, Candice
name: Pu-Ting Xu
name: Speer, Marcy C.
name: Munnich, Arnold
name: Lyonnet, Stanislas
name: Vekemans, Michel
name: Etchevers, Heather
Publisher: name: F1000Research
Year: 2016

description: <i>Introduction</i><br>During development, neural crest cells (NCC) emerge from the dorsal part of the neural tube (NT) and migrate to various locations within the embryo to generate most structures of the PNS (peripheral nervous system) and non-neural tissues such as melanocytes, cells with endocrine function, and craniofacial bones, cartilage, smooth muscle, dermis and more (mesectoderm, Fig. 1).<br> <br><i>Isolation of human neural crest</i><br>A. NT of a human embryo at C13 (ca 31 days). B-D: After 16 hours, hNCC have left the dorsal NT (B), which is then detached from the culture dish (C) leaving an enriched hNCC population (D). E. Evaluation of hNCC migration from 31 explanted NT. Peaks occur during C11 (ca. 24 days) at cephalic levels, and during late C12 (ca. 29 days) at rostral trunk levels.<br> <br><i>In vitro</i>, hNCC synthesized (A) α-smooth muscle actin and/or neural cell adhesion molecule (NCAM), (B) neuron-specific beta III tubulin; (C) nuclear SOX9; (D) nuclear and cytoplasmic SOX2 proteins.<br> <br><i>Serial Analysis of Gene Expression (SAGE)</i><br>We derived a SAGE bank from hNCC of a female C13 embryo. 51,374 counted tags led to the discovery of 14,120 annotated / 8,832 unannotated transcripts. (GEO accession GSM207304). <i>In situ </i>hybridization (not shown) of 13 genes confirms <i>in vivo </i>relevance of SAGE results.<br> <br>A. Presence in different hNCC lines (N1- N5) of "typical" neural crest genes identified in SAGE bank made from N5 RNA. Most are also expressed by the human neural tube at C12 and C13 (T12, T13) and by the C13 liver bud (L). B. As assessed by quantitative RT-PCR, both <i>PAX3 </i>and <i>GJA1 </i>are more abundant in hNCC lines relative to the C13 liver.<br> <br><i>hNCC and ES cell whole transcriptomes are highly similar</i><br> A. Hierarchical cluster dendrogram of hNCC-expressed transcript list compared to fourteen normal human tissue SAGE banks (uncentered correlation, average linkage). The global transcriptome of hNCC is most similar to human ES cell lines hES3 and hES4. B. Expression in five hNCC lines of highly characteristic human ES cell genes. C. Heat map representing expression levels in hNCC, hES3, hES4, BM-MSC, UC-MSC, adult liver and adult lung cells of lists of genes attributed by IP software to various signaling pathways. Growth factor pathways are similarly activated in hNCC and hES cells, relative to other cell types.<br> <br><i>Interspecies NCC transcriptome comparison </i><br>Comparison of human and mouse NCC transcriptomes. A. Venn diagram with common and specific genes to hNCC, mouse neural crest stem cells (mNCSC, 2d after isolation) and neural crest progenitors (mNCP, 7d after isolation), as described by Hu et al. 2006. B. Schematic view of part of the Notch pathway expressed specifically in hNCC (pink) in contrast to mNCSC/mNCP (green). Human NCC express many Notch ligands, receptors, co-activators, effectors and transcriptional targets. Phenotypic differences <i>in vitro </i>also exist between species.<br> <br><i>hNCC express a conserved epithelio-mesenchymal transition network</i><br>Protein interaction network around <i>pebble </i>(<i>pbl</i>) using the <i>Drosophila </i>BioGrid (Breitkreutz et al., 2008)<br> <br><i>hNCC-specific marker discovery </i><br>A. Scatter plot showing the distribution of each hNCC-expressed gene relative to its preferential abundance and expression range among 15 tissues including those in Fig 5A. A black box highlights ubiquitous genes. Dots in the blue box represent the 119 most hNCC-specific transcripts, of which 48 are also highly specific to hES cells. B. Across 16 human cell types, 43 transcripts were most specific to hNCC.<br> <br><i>Conclusion </i><br>The transcriptional profile of early human neural crest cells at a local scale is unique, yet shares the identifying qualities of uncommitted stem cells.<br>