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APPLICATION OF ZEBRAFISH NEURAL CREST CULTURE ASSAYS TO ELUCIDATE STEM CELL PROPERTIES
Presenter: Beste Kinikoglu, PhD
Co-Authors: Kong Y; Liao EC
Massachusetts General Hospital and Harvard Medical School

Purpose: Vertebrate neural crest development depends on pluripotent, migratory neural crest (NC) cells. Isolation and culture of zebrafish NC cells has not been previously reported. In vitro culture of NC cells allows evaluation of in vivo findings in a more controlled environment. Here we report for the first time the isolation, in vitro culture and characterization of NC cells from zebrafish embryos. We apply the NC culture to determine if these cells possess stem cell or progenitor cell properties of multi-lineage differentiation, maintenance and renewal.

Methods: NC cells were isolated from transgenic sox10::egfp embryos using FACS and cultured in a complex proliferation medium, on substrates coated with extra cellular matrix proteins, with growth factors to induce differentiation into various lineages. NC multi-lineage differentiation was determined by immunocytochemistry and RT-PCR, cell migration was assessed by wound healing assay, and the proliferation index of the NC cells was calculated by immunostaining against the mitosis marker phosphohistone H3.

Results: NC cells isolated from sox10::egfp embryos expressed early markers of NC cells, HNK1 and p75. We showed that these NC cells give rise to multiple NC lineages. They could be propagated in vitro, passaged, and directed towards neuronal lineage (neurons), glial lineage (Schwann cells), and mesenchymal lineage (smooth muscle cells). Retinoic acid, a morphogenetic signaling molecule in vertebrate embryos, had a profound effect on NC cell morphology, significantly inhibited proliferation and enhanced cell migration.

Conclusions: In this study we established a novel in vitro neural crest system and methods for isolating and culturing these unique cells from zebrafish embryos. We demonstrate that we are able to identify NC progenitors and characterize them with reproducible assays of cell differentiation, proliferation, and migration. The availability of high numbers of NC cells offers new opportunities for studies of NC development with complementary in vivo and in vitro approaches.


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