Abstract
Background and Aims Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. Results Using stromal-derived inducing activity (SDIA) of PA6 cells plus BMP4 and B27 supplements, the H9 hESC line was differentiated to neural crest stem-like cells and SN-like cells. After 7 days of PA6 cell coculture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker peripherin (PRPH) increased. Expression of the pluripotency marker OCT 4 decreased, whereas TP53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. On day 28 post p75 sorting, dual immunofluorescence identified sympathetic neurons by PRPH and TH copositivity cells in 20% of the cell population. Noradrenergic sympathetic neurons, identified by copositivity for both PHOX2B and DBH, were present in 9.4% ± 5.5% of cells. Conclusions We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma.
Highlights
The neural crest is a transient embryonic cell population which undergoes extensive migration and differentiation to give rise to a diverse range of cell populations in the embryo, ranging from the peripheral nervous system (sensory, enteric, and autonomic) to the craniofacial skeleton and pigment cells
The aim of the current study was to optimise a model of normal human sympathetic neuronal development using human embryonic stem cells (hESCs), which could be used to understand the normal development of the sympathetic nervous system (SNS) and in the future the pathogenesis of neuroblastoma and other neural crest-derived malignancies
We optimised Bone morphogenetic protein hESC (BMP) exposure and showed that BMP4 exposure alone was superior, in agreement with previous studies showing that early exposure to BMP4 can promote dorsal neural differentiation, when applied at later stages, BMP4 enhances the production of neural crest stem cell (NCSC) and autonomic neurons in primate and murine cells [9, 19]
Summary
The neural crest is a transient embryonic cell population which undergoes extensive migration and differentiation to give rise to a diverse range of cell populations in the embryo, ranging from the peripheral nervous system (sensory, enteric, and autonomic (sympathetic and parasympathetic)) to the craniofacial skeleton and pigment cells (reviewed by [1]). Neuroblastoma is an embryonal malignancy originating from neural crest cells which give rise to the sympathetic nervous system (SNS) [3]. Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma
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