Abstract A22: Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis

Background and Aims: Neuroblastoma is an embryonal malignancy derived from neural crest cells which give rise to the sympathetic nervous system (SNS). One of the main therapeutic challenges in neuroblastoma continues to be the emergence of relapsed disease, which occurs in 50% of patients with high risk disease and is associated with resistance to therapy. Our aim was to differentiate human embryonic stem cells (hESCs) to sympathetic neurons (SN) to model normal human SNS development. Results: Using the stromal-derived inducing activity (SDIA) of murine PA6 cells in combination with BMP4 and B27 neuronal supplement, H9 and a newly derived hESC line Ncl(R)14, were induced to differentiate to neural crest stem cells and SN. After 7 days of PA6 cell co-culture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker Peripherin increased. Q-RT-PCR showed that expression of the pluripotency marker OCT 4 decreased, whereas p53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A marked increase in expression of the catecholaminergic marker Tyrosine Hydroxylase (TH) and the noradrenergic marker Dopamine Beta Hydroxylase (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 Peripherin. SN were identified by immunofluorescence by co-expression of TH u0026 Peripherin or DBH u0026 PHOX2B in p75+ cells. Live cell analysis and imaging showed increased migration in p75+ cells compared with p75- cells, consistent with a migratory neural crest phenotype in-vitro. Conclusions: We have established a model of nor-adrenergic SNS development using two hESC lines to improve our understanding of normal human SNS development and in future studies the pathogenesis of neuroblastoma. Citation Format: Jane Carr-Wilkinson, Nilendran Prathalingam, Deepali Pal, Helen Forgham, Majlinda Lako, Mary Herbert, Deborah A. Tweddle. Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A22.