Published: 27 March 2023| Version 1 | DOI: 10.17632/76prtz548d.1


Studies defining normal and disrupted human neural crest cell development have been challenging given its early timing and intricacy of development. Consequently, insight into the early disruptive events causing neural crest related disease such as pediatric cancer neuroblastoma is limited. To overcome this problem, we developed an in vitro differentiation model to recapitulate the normal in vivo developmental process of the sympathoadrenal lineage which gives rise to neuroblastoma. We used human in vitro pluripotent stem cells and single-cell RNA sequencing to recapitulate the molecular events during sympathoadrenal development. We provide a detailed map of dynamically regulated transcriptomes during sympathoblast formation and illustrate the power of this model to study early events of the development of human neuroblastoma, identifying a distinct subpopulation of cell marked by SOX2 expression in developing sympathoblast obtained from patient derived iPSC cells harboring a germline activating mutation in the Anaplastic Lymphoma Kinase (ALK) gene. Bulk RNA-seq: Induced pluripotent stem cells (iPSC) were differentiated along the sympathoadrenergic precursor (SAP) development during a 40 day long differentiation process. We performed bulk RNA sequencing on selected time-points (D0/Start, D3, D7, D11, D16, D21, D23, D25, D27, D29, D31, D35, D40) covering the entire differentiation process. The experiments were repeated four times, twice with a human induced pluripotent stem cell line (J2) and twice with a human embryonic stem cell line (H9) for a total of four biological replicates. Single cell RNA-seq: 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).


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Bulk RNA-seq: Samples were barcoded and run on a HiSeq 4000 in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina Cat nr. TG-410-1002). FASTQ files were mapped using STAR (v2.7.3a). The counts per gene were normalized using DESeq2 (Love et al., 2014) The counts for each gene in each sample is divided by the geometric mean of the gene across all samples. The median of the ratios for the genes in a sample is the estimated size “scaling” size factor used to adjust the total mapped reads count from each sample. Single cell RNA-seq: The sequenced libraries were quantified with Cell Ranger software (version 6.0.0) (Zheng et al., 2017) with default parameters. The standard reference provided with Cell Ranger (version 2020-A) was used. This version was build on a GRCh38 human genome (bundle 3.0.0) and a filtered version of Gencode v32. In addition, HTO surface antibodies were quantified with the Cell Ranger count feature reference option. The feature reference file is provided on GitHub (


RNA Sequencing, Single-Cell RNA Sequencing