RESUMO
The neural crest (NC) is a transient embryonic cell population with remarkable characteristics. After delaminating from the neural tube, NC cells (NCCs) migrate extensively, populate nearly every tissue of the body and differentiate into highly diverse cell types such as peripheral neurons and glia, but also mesenchymal cells including chondrocytes, osteocytes, and adipocytes. While the NC has been extensively studied in several animal models, little is known about human NC development. A number of methods have been established to derive NCCs in vitro from human pluripotent stem cells (hPSC). Typically, these protocols comprise several cell culture steps to enrich for NCCs in the neural derivatives of the differentiating hPSCs. Here we report on a remarkable and hitherto unnoticed in vitro segregation phenomenon that enables direct extraction of virtually pure NCCs during the earliest stages of hPSC differentiation. Upon aggregation to embryoid bodies (EB) and replating, differentiating hPSCs give rise to a population of NCCs, which spontaneously segregate from the EB outgrowth to form conspicuous, macroscopically visible atoll-shaped clusters in the periphery of the EB outgrowth. Isolation of these NC clusters yields p75NTR(+)/SOXE(+) NCCs, which differentiate to peripheral neurons and glia as well as mesenchymal derivatives. Our data indicate that differentiating hPSC cultures recapitulate, in a simplified manner, the physical segregation of central nervous system (CNS) tissue and NCCs. This phenomenon may be exploited for NCC purification and for studying segregation and differentiation processes observed during early human NC development in vitro.
