Using human stem cell derived in vitro modeling to characterize the effect of MYCN overexpression on early hSAP development and tumor formation

In this study we use human stem cell derived in vitro modelling to characterize the effect of MYCN overexpression on early hSAP development and tumor formation. We developed and characterized a human in vitro pluripotent stem cell-based model via sequential single-cell RNA sequencing throughout sympathoadrenal development. We differentiated an induced pluripotent stem cell (iPSC) line with a doxycycline inducible MYCN construct along the hSAP developmental trajectory in vitro. We induced MYCN overexpression at multiple timepoints during the critical time frame for the hSAP developmental window during differentiation, as defined by expression changes of key genes including PLP1, ASCL1 and STMN2, covering both early and late hSAP development. During this window we induced MYCN overexpression for 48hrs at four time-points (day 24, 26, 28 and 30). We then collected cells for single cell RNA-seq.

本研究采用人源干细胞体外建模技术，表征MYCN过表达（MYCN overexpression）对早期人源交感肾上腺祖细胞（human sympathoadrenal progenitor, hSAP）发育与肿瘤发生的影响。我们通过覆盖交感肾上腺发育全程的时序性单细胞RNA测序（single-cell RNA sequencing），构建并表征了基于人源多能干细胞的体外模型。我们在体外沿hSAP发育轨迹，对携带多西环素诱导型MYCN表达构建体的诱导多能干细胞（induced pluripotent stem cell, iPSC）株进行定向分化。我们在分化过程中hSAP发育的关键窗口期内的多个时间点诱导MYCN过表达；该关键窗口期由PLP1、ASCL1与STMN2等关键基因的表达变化界定，涵盖hSAP发育的早、晚两个阶段。在此窗口期内，我们分别在第24、26、28、30天四个时间点诱导MYCN过表达，诱导时长为48小时。随后收集细胞用于单细胞RNA测序。