GSE211661
Human iPSC modeling recapitulates in vivo sympathoadrenal development and reveals an aberrant developmental subpopulation in familial neuroblastoma [scRNA-seq]
Pediatric malignancies, including neuroblastoma, are best understood as disorders of development. Neuroblastoma explicitly represents a failure of sympathoadrenal development. Yet, its molecular pathogenesis remains elusive. The application of an in vitro model of human sympathoadrenal development would allow for studying the normal developmental trajectory. Such a model could also interrogate the early steps toward neuroblastoma transformation utilizing genetic manipulation. However, in vitro models have thus far been unable to generate the cells of interest reliably. We developed and characterized a human in vitro pluripotent stem cell-based model via sequential single-cell RNA sequencing throughout sympathoadrenal development. We demonstrate the power of our model to study early events of the development of human neuroblastoma. We do so by identifying the differences between normal and patient-specific induced pluripotent stem cells, derived from a child with familial neuroblastoma, harboring a germline mutation in the Anaplastic Lymphoma Kinase (ALK) gene. Overall design: Induced pluripotent stem cells (iPSC) were differentiated along the sympathoadrenergic precursor (SAP) development during a 40 day long differentiation process. We performed single cell RNA sequencing (scRNAseq) on eight subsequent time-points to characterize the normal SAP development. We further investigated the effect of the ALK R1275Q mutation on the sympathoadrenergic developmental process by performing serial scRNAseq at four time points between days 25 and 32 using an ALKR1275Q patient-specific iPSC line. Prior to scRNAseq the cells of selected time-points were hashed and pooled together for scRNAseq analysis. The time-points D25-26, D27-28, D29-30 and D31-32 were hashed for the WT cells. For the ALK mutant cells time-points D25-27-29-32 were hashed. The single-cell RNA-Seq was performed on Chromium instrument (10X genomics).