Isolation and Propagation of Neural Crest Stem Cells from Mouse Embryonic Stem Cells via Cranial Neurospheres

Abstract

The developmental fate of the multipotent neural crest (NC) is determined along with the neural axis in which NC cells are generated. Only the cranial NC can differentiate into mesectodermal derivatives such as osteoblasts, chondrocytes, and adipocytes in vivo. Here, we attempted to selectively differentiate mouse embryonic stem (ES) cells into cranial NC stem cells and propagate them to explore their developmental potential to differentiate into mesectodermal derivatives. Using aggregation cultures in feeder- and serum-free neural induction medium (NIM) without serum replacement and l-glutamine, we obtained NIM neurospheres composed of neuroepithelium. The NIM neurospheres expressed the rostral markers Otx1 and Otx2, but not nonrostral markers Hoxb4, Hoxb9, Lbx1, and TH, which characterize cranial neurospheres. Subsequently, AP2α, Sox9, p75, Snail, Slug, and Twist-positive NC cells were differentiated in 4-day adhesion cultures of cranial neurospheres. In addition, sphere clusters in adhesion cultures were differentiated into osteoblasts, while migrating cells were not. By taking advantage of the sphere-formation capability, we isolated and propagated NC stem cells from the sphere clusters and confirmed their multipotency. NC stem cells expressed NC and stem cell markers, and they maintained differentiation potency in the NC derivatives. These results show that cranial NC stem cells were obtained reproducibly and efficiently without special inducing factors, gene transfection, or fluorescence-activated cell sorting selection.

<b>Induction of neural crest cells from human dental pulp-derived induced pluripotent stem </b><b>cells </b>

“…Another study has found that a small neurosphere size is associated with efficient neuralization of ES cells (5). In contrast, large neurosphere size contributes to suppression of ES cell neuralization and promotes mesoderm differentiation (38). Further study is needed to determine whether larger neurosphere size would provide an advantage for NCC differentiation.…”

Stage-dependent differential gene expression profiles of cranial neural crest-like cells derived from mouse-induced pluripotent stem cells

“…In the present study, we derived cells from miPS which are closely migratory cNCCs genes. Previously, NCCs have been derived from ES or iPS cells using various approaches [91][92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107][108][109][110], and the protocol used in the present study was based on the methods outlined by Bajpai et al [23]; however, few studies have investigated changes in the properties of cNCCs at different time points after induction.…”

Mir-29b Mediates the Neural Tube versus Neural Crest Fate Decision during Embryonic Stem Cell Neural Differentiation

“…The NTE differentiation was performed according to published protocols (Wang et al., 2011) and the NCC differentiation was performed according to a published protocol (Minamino et al., 2015). For additional details, see Supplemental Experimental Procedures.…”

“…In vitro , ESCs can differentiate into NTE cells (Watanabe et al., 2005, Zhang, 2006) and NCCs (Lee et al., 2007, Liu et al., 2012, Minamino et al., 2015). Therefore, ESCs are an excellent model for the study of early development of the nervous system and the regulatory mechanisms that determine the differentiation of NTE cells and NCCs.…”