Abstract 1670: A human pluripotent stem cell derived differentiation model for the study of normal and transformed sympatho-adrenal progenitors

Abstract Background: Neuroblastoma (NB) is a clinically and genetically heterogeneous embryonal cancer thought to arise due to a disruption of normal sympatho-adrenergic progenitors (SAPs) development. Defining the unknown molecular pathways of normal SAP differentiation is essential to improve our limited understanding of NB pathogenesis, and devise more effective and safer therapies. Currently, accurate human model systems for normal and aberrant SAP development are lacking. Using human pluripotent stem cells (hPSCs), we optimised and characterised an in vitro differentiation model to generate SAPs to dissect normal SAP development and NB oncogenesis. Aims: Using hPSCs, we optimised and validated an in vitro differentiation model to investigate normal and aberrant SAP development. Methods/materials: Utilizing a differentiation protocol developed by the Studer laboratory at Memorial Sloan Kettering Cancer Center, we performed in vitro differentiations of hPSCs into SAPs. Throughout a 40-day differentiation process, we harvested cells for bulk RNA-sequencing to analyse time-course expression profiles of specific neurodevelopmental markers. Next, utilizing 10XGenomics, we further mapped molecular phenotypes at the single-cell level. Additionally, we combined our data with the publicly available human SAP single cell-data set by the Adamayko laboratory to identify our cells of interest and pinpoint the distinct cell populations. Results: During differentiation, we could confirm the development of truncal neural crest cells (NCCs) based on the expression of SOX10 and truncal HOX genes, including B7 and B9. Further differentiation of truncal NCCs into SAPs confirmed SOX10 downregulation and upregulation of neurodevelopmental transcription factors such as ASCL1, PHOX2B and STMN2. By comparing our in vitro results with in vivo data generated from human embryos, we discovered that we could generate the in vivo SAP developmental subpopulations. We further characterised all the subpopulations using sets of tissue-specific makers (i.e., PLP1, CHGA, and ISL1). These results confirm that our model can accurately generate SAPs that are comparable to their in vivo counterparts. Conclusion: We have developed a human in vitro differentiation model to generate SAPs resembling their in vivo counterparts. This model allows for mechanistic studies that are unfeasible using in vivo systems, with human cells that faithfully recapitulate the NB cell of origin. We are now using our model to explore NB transformation in vitro using genetically modified and patient-specific hPSCs. Our system will provide critical insights into early NB oncogenesis and will serve as a platform for pre-clinical studies in the context of NB. Citation Format: Stephane Van Haver, Yujie Fan, Wouter Van Loocke, Katleen De Preter, Alex Kentsis, Lorenz Studer, Stephen Roberts, Frank Speleman. A human pluripotent stem cell derived differentiation model for the study of normal and transformed sympatho-adrenal progenitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1670.