Publication
Differentiation of Human Embryonic Stem Cells to Sympathetic Neurons: A Potential Model for Understanding Neuroblastoma Pathogenesis
Publisher:
Hindawi Limited
Date:
11-2018
DOI:
10.1155/2018/4391641
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.