An in vitro cell lineage model to identify alterations in the differentiation program of neuroblastoma

Neuroblastoma (NB) is a pediatric solid tumor that arises from trunk neural crest cells (tNCCs), a transient cell population involved in the development of the sympathetic nervous system. The tNCCs are migrating cells that differentiate into sympathoadrenal precursors (SAPs) and Schwann cell precursors (SCPs). The SAPs are the direct progenitors of sympathetic ganglia neurons (SGNs), and of chromaffin cells (ChCs), the major cell population of the adrenal medulla (AM). The SCPs are known to be precursors of the mature Schwann cells but recently a new role has been highlighted for them in forming the ChCs. Furthermore, according to a new hypothesis of differentiation, the SCPs trans-differentiate in a SAP-like intermediate cells, generating a connecting population able to terminally differentiate in ChCs. During tNCCs differentiation, oncogenic insults can occur leading to the malignant transformation giving origin to neuroblastoma. The precise steps along the differentiation path in which the tumorigenic alterations bring to a differentiation block is still unclear. In the attempt to elucidate this point, we successfully established an efficient differentiation protocol to obtain from human embryonic stem cells (hESCs) the tNCCs, SAPs, SCPs, SCP/SAP intermediate cells, SGNs and ChCs cells. Marker analysis by real-time qPCR, immunofluorescence, ELISA assays and functional characterization by multi- electrode array recording (MEA), confirmed the effectiveness of our differentiation protocols. The resulting differentiated cells will be used to perform bulk RNAseq to compare the gene expression profile of our in vitro models with those reported in the available single-cell RNA sequencing (scRNAseq) datasets of both normal and tumoral human AM. These new cellular differentiation systems will provide us a unique tool to study in depth the role of the genomic alterations in NB pathogenesis.