Establishing Primary Cultures of Trunk Neural Crest Cells

Abstract

Neural crest cells constitute a unique population of progenitor cells with extensive stem cell capacities able to navigate throughout various environments in the embryo and are a source of multiple cell types, including neurons, glia, melanocytes, smooth muscles, endocrine cells, cardiac cells, and also skeletal and supportive tissues in the head. Neural crest cells are not restricted to the embryo but persist as well in adult tissues where they provide a reservoir of stem cells with great therapeutic promise. Many fundamental questions in cell, developmental, and stem cell biology can be addressed using this system. During the last decades there has been an increased availability of elaborated techniques, animal models, and molecular tools to tackle neural crest cell development. However, these approaches are often very challenging and difficult to establish and they are not adapted for rapid functional investigations of mechanisms driving cell migration and differentiation. In addition, they are not adequate for collecting pure populations of neural crest cells usable in large scale analyses and for stem cell studies. Transferring and adapting the neural crest system in tissue culture may then represent an attractive alternative, opening up numerous prospects. Here we describe a simple method for establishing primary cultures of neural crest cells derived from trunk neural tubes using the avian embryo as a source of cells. This protocol is suited for producing pure populations of neural crest cells that can be processed for cytological, cellular, and functional approaches aimed at characterizing their phenotype, behavior, and potential. © 2020 Wiley Periodicals LLC. Basic Protocol: Primary cultures of avian trunk neural crest cells Support Protocol: Adaptations for immunofluorescence labeling and videomicroscopy

Non-canonical nuclear function of glutaminase cooperates with Wnt signaling to drive EMT during neural crest development

“…1A; Extended data Fig.1A). NCC delamination can be investigated experimentally in vivo [34] and can be mimicked in vitro under defined cell culture conditions [35]. Therefore, this system allows to analyze the process of delamination and its switch to migration and to decipher the individual and collective behaviors of NCCs, thereby providing a comprehensive spatiotemporal survey of the underlying metabolic events.…”

“…NCC cultures were produced from trunk NT obtained from quail embryos at different stages covering the delamination process: stage HH12-13 (17-18 somite pairs), stage 13 (19-21 somite pairs) and HH14 (23-25-somite stage) as described [24, 35]. After opening the eggshell, the yolk was transferred into phosphate-buffered saline (PBS).…”

“…(23-25-somite stage) as described [24,35]. After opening the eggshell, the yolk was transferred into phosphate-buffered saline (PBS).…”

Glucose oxidation and nutrients availability drive neural crest development

“…Generation of NCC primary cultures: NCC cultures were produced from NT explants obtained from quail embryos (Coturnix coturnix japonica) as described (Duband et al, 2020). Briefly, fertilized eggs purchased from a local farm (La Caille de Chanteloup, Corps Nuds, France) were incubated at 37-38°C for 54 h until the embryos reached stages corresponding to the chick stages 13-15 of Hamburger and Hamilton (HH) (1951), i.e.…”

“…Because investigating NCC metabolic features and their impact on NCC developmental program cannot be performed directly in intact embryos, we developed an alternative strategy based on ex vivo culture in defined conditions recapitulating the delamination, migration, and differentiation steps of NCC development, as well as their main features (timing and kinetics of cellular events, rate of proliferation, cellular interactions) (Duband et al, 2020). We characterized the metabolic traits of avian trunk NCC, and we modified the activity of some of the key metabolic enzymes or the nutrient supplies to decipher the role of carbon metabolism in NCC dispersion and differentiation.…”