Inhibition of FGF signaling accelerates neural crest cell differentiation of human pluripotent stem cells

https://doi.org/10.1016/j.bbrc.2016.10.147Get rights and content

Abstract

Neural crest (NC) is a transient population, arising during embryonic development and capable of differentiating into various somatic cells. The defects of neural crest development leads to neurocristopathy. Several signaling pathways were revealed their significance in NC cell specification. Fibroblast growth factor (FGF) is recognized as an important signaling during NC development, for instance Xenopus and avian; however, its contributions in human species are remained elusive. Here we used human pluripotent stem cells (hPSCs) to investigate the consequences of FGF inhibition during NC cell differentiation. The specific-FGF receptor inhibitor, SU5402, was used in this investigation. The inhibition of FGF did not found to affect the proliferation or death of hPSC-derived NC cells, but promoted hPSCs to commit NC cell fate. NC-specific genes, including PAX3, SLUG, and TWIST1, were highly upregulated, while hPSC genes, such as OCT4, and E-CAD, rapidly reduced upon FGF signaling blockage. Noteworthy, TFAP-2α, a marker of migratory NC cells, abundantly presented in SU5402-induced cells. This accelerated NC cell differentiation could be due to the activation of Notch signaling upon the blockage of ERK1/2 phosphorylation, since NICD was increased by SU5402. Altogether, this study proposed the contributions of FGF signaling in controlling human NC cell differentiation from hPSCs, the crosstalk between FGF and Notch, and might imply to the influences of FGF signaling in neurocristophatic diseases.

Introduction

Neural crest (NC) cells arise at the border between the neural plate and the adjacent non-neural ectoderm [1], [2]. They are transient and multipotent that can migrate extensively and differentiate vastly to form NC derivatives, such as peripheral neurons, glia, melanocytes, and mesenchymal cells [3], [4]. NC cell is marked by the expression of neural plate border-specific genes, like ZIC1, DLX5 and MSX1/2 [5]. NC progenitor cells remain within the neural folds and subsequently localize within the dorsal portion of the neural tube during the neural tube folding process, marked by the expression of NC-specifier genes, SNAIL, SLUG and TWIST1 [6], [7]. Following neural tube formation, NC progenitor cells undergo epithelial to mesenchymal transition, delaminating from neuroepithelium, and migrating from the neural tube to various embryonic tissues. Disruption of genes involved in the ontogeny of NC development can lead to congenital disorders, such as albinism and neurocristopathy, while disruption at later stages contributes to cancers, such as melanoma and neuroblastoma [8], [9].

Specification of NC cells from embryonic ectoderm is directed by multiple signaling pathways, including WNT, BMP, Notch and FGF [10], [11], [12]. FGF is proposed to act indirectly during the inductive phase by activating WNT ligand in the mesoderm. Several FGFs are expressed in a spatiotemporal manner and associated with the induction of NC cells. In Xenopus embryos, Fgf ligands, in particular Fgf8, can induce neural crest cell fate through the activation of STAT pathway [13]. FGF/ERK pathway is demanded in the gastrula epiblast for avian NC commitment, and impedes the ectopic expression of lateral ectoderm markers [11]. Although the previous evidence of various species proposed the instructive roles of FGF signaling in promoting NC cell fate, the actual contributions of FGF signaling in controlling human NC cell differentiation has not yet been investigated.

Human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are extensively used to explore the mechanisms of human development and diseases [14], [15]. The differentiation of hPSCs is follow the hierarchical sets of signals that regulate embryonic development [16], [17]. Specific cell types could efficiently be obtained from hPSCs when appropriate signallings are manipulated [16], [18]. Besides, the roles of numerous cell signallings in controlling human development are established by using hPSCs [19], [20]. In this paper, we reported the contributions of FGF signaling involving in the differentiation of NC cells of hPSCs. The specific FGF receptor inhibitor, SU5402, was used to block FGF/ERK during NC differentiation, and it was found that hPSCs accelerated their fate commitment into NC lineage. This finding might imply the significance of FGF signaling in neurocristopathy, and guide the potential therapeutic approaches.

Section snippets

Culture and maintenance of human pluripotent stem cells, transfections

FES29 hESCs and HEL11.4 iPSCs (both from Biomedicum Helsinki) were cultured in Geltrex-coated plates with StemPro complete medium (Invitrogen) and propagated with combination of collagenase IV treatment and mechanic disassociation.

Neural crest, neural lineage and mesenchymal derivative differentiation

NC differentiation protocol was based on the previous report [19]. Briefly, when confluent, hESCs where passaged with 0.02%EDTA (Sigma) into a Geltrex-coated dishes. NC lineage was induced by N2B27 medium, supplemented with 2 μM Dorsomorphin (Sigma) and 5 μM

Human embryonic stem cells expressed pluripotent markers and retain pluripotency

To validate that hESCs used in this study, immunocytochemistry was performed to investigate the expression of pluripotent stem cell markers and differentiation potential. hESC colony exhibited typical shape with large nuclei (Fig. 1A). When spontaneously differentiated, cells became flat with various cellular structures. Antibody staining against hPSC markers showed that the cells homogeneously expressed key pluripotent transcription factors, OCT4 and NANOG (Fig. 1A). After 2 weeks of

Discussion

NC cells are specified at a region called the neural plate border. The specification of NC cell fate from hPSCs involves the modulation of multiple signaling pathways, for instance Wnt, BMPs and Notch signaling [19], [25]. FGF signaling is reportedly demanded for the maintenance and differentiation of hPSCs, and ERK1/2 activation was responsible for the FGF-mediated regulation of neural differentiation [26]. The defects in FGF signaling is one of the causatives that lead to NC developmental

Disclosures

There is no conflict of interest in this study.

Acknowledgments

This work was supported by Suranaree University of Technology (SUT) Research and Development Fund.

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