Generating trunk neural crest from human pluripotent stem cells

Abstract

Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior “cranial” NCC form craniofacial bone, whereas solely posterior “trunk” NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages.

Transcriptional Profiling Reveals a Common Metabolic Program in High-Risk Human Neuroblastoma and Mouse Neuroblastoma Sphere-Forming Cells

“…This observation indicates that TH-MYCN neuroblastoma sphere-forming cells underwent spontaneous differentiation in culture. We further investigated the responsiveness of these sphere-forming cells to retinoic acid (RA), a differentiation inducer of neuroblastoma cells (Sidell, 1982) and neural crest cells (Dupin and Le Douarin, 1995; Huang et al, 2016). Treatment of mouse neuroblastoma spheres with RA induced growth arrest as evidenced by the marked reduction in sphere size and numbers, as well as the total number of sphere cells (Figures 2A-2C).…”

WNT/β-CATENIN modulates the axial identity of ES derived human neural crest

“…Human NC formation models have reported mixed and even contradictory information regarding the contribution of BMP signaling. Some groups require BMP inhibition (Chambers et al, 2012; Mica et al, 2013), but others found BMP inhibition dispensable for NC formation from hPSCs (Fukuta et al, 2014; Huang et al, 2016; Menendez et al, 2011). By contrast, our model of anterior NC formation requires BMP signaling (Leung et al, 2016).…”

“…Retinoic Acid (RA) signaling has been linked with posteriorization of embryonic tissues and neural crest (Fattahi et al, 2016; Fukuta et al, 2014; Huang et al, 2016; Mica et al, 2013); specifically, addition of RA promotes the expression of vagal identity HOX genes in NC cultures. It has been proposed that RA and FGF exert antagonistic effects to modulate the anterior-posterior character of the neural tube and NCCs (Cunningham et al, 2015; Diez del Corral et al, 2003; Olivera-Martinez and Storey, 2007).…”

“…Initial efforts relied on co-cultures, the use of serum or serum replacement cocktails, and either embryoid bodies or neural rosettes (Bajpai et al, 2010; Brokhman et al, 2008; Curchoe et al, 2010; Jiang et al, 2009; Lee et al, 2007; Liu et al, 2012; Rada-Iglesias et al, 2012; Sparks et al, 2018). Simpler models where cell density was controlled along with defined media components have enabled a better analysis of contributing factors during human NC development from hESCs (Fukuta et al, 2014; Hackland et al, 2017; Huang et al, 2016; Leung et al, 2016; Menendez et al, 2011). Common to these and other models of NC development is the activation of the WNT/β-CATENIN signaling pathway (Chambers et al, 2012; Fukuta et al, 2014; Hackland et al, 2017; Leung et al, 2016; Menendez et al, 2011; Mica et al, 2013), which recapitulates WNT’s known role in NC induction in model organisms (García-Castro et al, 2002; LaBonne and Bronner-Fraser, 1998; Lewis et al, 2004).…”

Development of Human Neuroblastomas in Mouse-Human Neural Crest Chimeras

“…Neural crest (NC) related deficiencies are the cause of multiple human diseases and the term "neurocristopathies" has been proposed to denote syndromes or tumors involving NCCs. Neural crest derived malignancies include cancers such as melanoma, neuroblastoma and neurofibromatosis (Vega-Lopez et al, 2018) and the use of hPSCs-derived NCCs for modeling human NC diseases is an attractive in vitro experimental approach (Fattahi et al, 2016, Huang et al, 2016. We showed that hPSCs-derived NCC, when injected into the gastrulating mouse embryo, migrated along the dorso-lateral migration route contributing to the pigment system of the mouse, suggesting that this platform may be used to model neurocristopathies in vivo (Cohen et al, 2016).…”