Maintaining trunk neural crest cells as crestospheres

Neural crest cells have broad migratory and differentiative ability that differs according to their axial level of origin. However, their transient nature has limited understanding of their stem cell and self-renewal properties. While an in vitro culture method has made it possible to maintain cranial neural crest cells as self-renewing multipotent crestospheres (Kerosuo et al., 2015), these same conditions failed to preserve trunk neural crest in a stem-like state. Here we optimize culture conditions for maintenance of trunk crestospheres, comprised of both neural crest stem and progenitor cells. Trunk crestospheres display elevated expression of neural crest cell markers as compared to those characteristic of neural tube or mesodermal fates. Moreover, trunk crestospheres have increased expression of trunk-related markers as compared to cranial genes. Finally, we use lentiviral transduction as a tool to manipulate gene expression in trunk crestospheres. Taken together, this method enables long-term in vitro maintenance and manipulation of trunk neural crest cells in a premigratory stem or early progenitor state to probe the mechanisms underlying their stemness and lineage decisions.\n\nHighlightsO_LITrunk-derived multipotent neural crest stem cells can be cultured as crestospheres\nC_LIO_LITrunk-derived crestospheres require different conditions than cranial\nC_LIO_LITrunk crestospheres consist of neural crest stem and progenitor cells\nC_LIO_LITrunk crestospheres can be efficiently transduced using lentiviral vectors\nC_LI