Human Neural Crest Cells Research Articles

Analysis of early human neural crest development

The outstanding migration and differentiation capacities of neural crest cells (NCCs) have fascinated scientists since Wilhelm His described this cell population in 1868. Today, after intense research using vertebrate model organisms, we have gained considerable knowledge regarding the origin, migration and differentiation of NCCs. However, our understanding of NCC development in human embryos remains largely uncharacterized, despite the role the neural crest plays in several human pathologies. Here, we report for the first time the expression of a battery of molecular markers before, during, or following NCC migration in human embryos from Carnegie Stages (CS) 12 to 18. Our work demonstrates the expression of Sox9, Sox10 and Pax3 transcription factors in premigratory NCCs, while actively migrating NCCs display the additional transcription factors Pax7 and AP-2α. Importantly, while HNK-1 labels few migrating NCCs, p75 NTR labels a large proportion of this population. However, the broad expression of p75 NTR – and other markers – beyond the neural crest stresses the need for the identification of additional markers to improve our capacity to investigate human NCC development, and to enable the generation of better diagnostic and therapeutic tools.

Generation of neural crest progenitors from human embryonic stem cells

The neural crest (NC) is a transient population of multipotent progenitors arising at the lateral edge of the neural plate in vertebrate embryos, which then migrate throughout the body to generate diverse array of tissues such as the peripheral nervous system, skin melanocytes, and craniofacial cartilage, bone and teeth. The transient nature of neural crest stem cells make extremely challenging to study the biology of these important cells. In humans induction and differentiation of embryonic NC occurs very early, within a few weeks of fertilization giving rise to technical and ethical issues surrounding isolation of early embryonic tissues and therefore severely limiting the study of human NC cells. For that reason our current knowledge of the biology of NC mostly derives from the studies of lower organisms. Recent progress in human embryonic stem cell research provides a unique opportunity for generation of a useful source of cells for basic developmental studies. The development of cost-effective, time and labor efficient improved differentiation protocols for the production of human NC cells is a critical step toward a better understanding of NC biology.

CHD7 cooperates with PBAF to control multipotent neural crest formation

Heterozygous mutations in the gene encoding the CHD (chromodomain helicase DNA-binding domain) member CHD7, an ATP-dependent chromatin remodeller homologous to the Drosophila trithorax-group protein Kismet, result in a complex constellation of congenital anomalies called CHARGE syndrome, which is a sporadic, autosomal dominant disorder characterized by malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. Although it was postulated 25 years ago that CHARGE syndrome results from the abnormal development of the neural crest, this hypothesis remained untested. Here we show that, in both humans and Xenopus, CHD7 is essential for the formation of multipotent migratory neural crest (NC), a transient cell population that is ectodermal in origin but undergoes a major transcriptional reprogramming event to acquire a remarkably broad differentiation potential and ability to migrate throughout the body, giving rise to craniofacial bones and cartilages, the peripheral nervous system, pigmentation and cardiac structures. We demonstrate that CHD7 is essential for activation of the NC transcriptional circuitry, including Sox9, Twist and Slug. In Xenopus embryos, knockdown of Chd7 or overexpression of its catalytically inactive form recapitulates all major features of CHARGE syndrome. In human NC cells CHD7 associates with PBAF (polybromo- and BRG1-associated factor-containing complex) and both remodellers occupy a NC-specific distal SOX9 enhancer and a conserved genomic element located upstream of the TWIST1 gene. Consistently, during embryogenesis CHD7 and PBAF cooperate to promote NC gene expression and cell migration. Our work identifies an evolutionarily conserved role for CHD7 in orchestrating NC gene expression programs, provides insights into the synergistic control of distal elements by chromatin remodellers, illuminates the patho-embryology of CHARGE syndrome, and suggests a broader function for CHD7 in the regulation of cell motility.

Human neural crest cells display molecular and phenotypic hallmarks of stem cells

The fields of both developmental and stem cell biology explore how functionally distinct cell types arise from a self-renewing founder population. Multipotent, proliferative human neural crest cells (hNCC) develop toward the end of the first month of pregnancy. It is assumed that most differentiate after migrating throughout the organism, although in animal models neural crest stem cells reportedly persist in postnatal tissues. Molecular pathways leading over time from an invasive mesenchyme to differentiated progeny such as the dorsal root ganglion, the maxillary bone or the adrenal medulla are altered in many congenital diseases. To identify additional components of such pathways, we derived and maintained self-renewing hNCC lines from pharyngulas. We show that, unlike their animal counterparts, hNCC are able to self-renew ex vivo under feeder-free conditions. While cross species comparisons showed extensive overlap between human, mouse and avian NCC transcriptomes, some molecular cascades are only active in the human cells, correlating with phenotypic differences. Furthermore, we found that the global hNCC molecular profile is highly similar to that of pluripotent embryonic stem cells when compared with other stem cell populations or hNCC derivatives. The pluripotency markers NANOG, POU5F1 and SOX2 are also expressed by hNCC, and a small subset of transcripts can unambiguously identify hNCC among other cell types. The hNCC molecular profile is thus both unique and globally characteristic of uncommitted stem cells.

PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells

Human embryonic stem cells (hESC) have been directed to differentiate into CNS cells with clinical importance. However, for study of development and regeneration of the human PNS, and peripheral neuropathies, it would be useful to have a source of human PNS derivatives. We have demonstrated that peripheral sensory neuron-like cells (PSN) can also be derived from hESC via neural crest-like (NC) intermediates, and from neural progenitors induced from hESC using noggin. Here we report the generation of higher purity PSN from passagable neurospheres (NSP) induced by murine PA6 stromal cells. hESC were cultured with PA6, and colonies that developed a specific morphology were cut from the plates. Culture of these colonies under non-adhesive conditions yielded NSPs. Several NC marker genes were expressed in the NSP, and these were also detected in 3–5week gestation human embryos containing migrating NC. These NSPs passaged for 2–8weeks and re-plated on PA6 gave rise to many Brn3a+/peripherin+ cells, characteristic of early sensory-like neurons. Re-culturing PA6-induced NSP cells with PA6 resulted in about 25% of the human cells in the co-cultures differentiating to PSN after 1week, compared to only about 10% PSN obtained after 3 weeks when noggin-induced NSP were used. Two month adherent cultures of PA6-induced NSP cells contained neurons expressing several PSN neuropeptides, and voltage-dependent currents and action potentials were obtained from a molecularly identified PSN. hESC-derived PA6-induced NSP cells are therefore an excellent potential source of human PSN for study of differentiation and modeling of PNS disease.

Expression of an epithelial membrane glycoprotein by neurons arising from the human olfactory plate through development

Human terminal-vomeronasal neural crest cells began to express a 34,000 molecular weight epithelial membrane glycoprotein, which was detected by the mouse monoclonal antibody Ber-EP4 soon after their migration into the olfactory plate. Expression of this antigen continued in neurons arising from these cells, which were olfactory sensory neurons and luteinizing hormone-releasing hormone-producing neurons migrating into the brains of the embryos of approximately 44-52 postovulatory days. Ber-EP4 immunoreactivity appeared over the entire surface membranes of these neurons, including their processes, not only in their extracerebral portions, but also within the brain parenchyma from 48 to 53 postovulatory days. Ber-EP4 immunoreactivity of these migrating neurons became weaker as they matured into luteinizing hormone-releasing hormone-producing neurons and disappeared from the postnatal hypothalamic neurons; however, it remained in the primary sensory nerve fibers throughout postnatal life. Except for the neurons arising from the olfactory plate, no other human neurons express this epithelial antigen during the course of development. The mechanism and significance of the expression of this antigen in mature sensory neurons remains unclear, but the intramucosal location and morphological kinship of these neurons to epithelial cells may be related to this phenomenon. The results of the present study indicate that neurons arising from the olfactory plate are distinct from other neurons by having a 34,000 molecular weight epithelial membrane glycoprotein that can be detected by the mouse monoclonal antibody, Ber-EP4, during the embryonic stage. This antigen disappears from the luteinizing hormone-releasing hormone-producing neurons concomitant with maturation, but is preserved in the olfactory primary sensory nerve throughout postnatal life.