Integration of central and peripheral neurons in human elongating multilineage organized (EMLO) gastruloids

Human stem cell technologies including self-assembling 3D tissue models provide unprecedented access to early neurodevelopment and are enabling fundamental insights into neuropathologies. Gastruloid models have yet to be used to investigate developing neuronal systems. Here we generate elongating multi-lineage-organized (EMLO) gastruloids with trunk identity that co-develop central and peripheral nervous system (CNS, PNS) correlates. We identify neural crest cells that differentiate to form peripheral neurons integrated with an upstream spinal cord region. This follows initial EMLO polarization events and is coordinated with primitive gut tube elongation and multicellular spatial reorganization. We evaluate EMLOs over a forty-day period, applying immunofluorescence of multi-lineage and functional biomarkers, including day 16 single-cell RNA-Seq, and use them to investigate the impact of mu opioid receptor modulation on neuronal activity. This comprehensive study demonstrates the first combined human CNS-PNS model of early organogenesis in the trunk to benefit biomedical research. Overall design: 10x scRNA-Seq of day16 EMLO human hiPSC line H3.3.1

包括自组装三维组织模型在内的人类干细胞技术，为早期神经发育研究开辟了前所未有的路径，也助力学界对神经病理学获得基础性认知。类原肠胚（gastruloid）模型尚未被用于探究发育中的神经元系统。本研究构建了具备躯干特性的伸长型多谱系组装（elongating multi-lineage-organized，EMLO）类原肠胚，其可协同发育出与中枢神经系统（central nervous system，CNS）和外周神经系统（peripheral nervous system，PNS）相关的结构。我们鉴定出可分化为与上游脊髓区域整合的外周神经元的神经嵴细胞；该过程发生于EMLO初始极化事件之后，且与原始肠管伸长及多细胞空间重排协同推进。我们对EMLO开展了为期40天的系统性评估，采用多谱系及功能性生物标志物免疫荧光检测，涵盖第16天的单细胞RNA测序（single-cell RNA-Seq，scRNA-Seq），并利用该模型研究μ阿片受体（mu opioid receptor）调控对神经元活动的影响。本综合性研究证实，这是首个可应用于生物医学研究的人类躯干早期器官发生的中枢-外周神经系统联合模型。实验设计：对源自人类诱导多能干细胞（human induced pluripotent stem cell，hiPSC）系H3.3.1的第16天EMLO样本进行10x单细胞RNA测序（10x scRNA-Seq）。