Self-organizing models of human trunk organogenesis recapitulate spinal cord and spine co-morphogenesis [scRNA-seq]

Integrated in vitro models of human organogenesis are needed to elucidate the multi-systemic events underlying development and disease. We report the generation of human trunk-like structures that model the co-morphogenesis, patterning, and differentiation of the human spine and spinal cord. We identified differentiation conditions for human pluripotent stem cells favoring the formation of an embryo-like extending antero-posterior (AP) axis. Single cell and spatial transcriptomics show that somitic and spinal cord differentiation trajectories organize along this axis and can self-assemble into neural tubes surrounded by somites upon extracellular matrix addition. Morphogenesis is coupled with AP patterning mechanisms which results, at later stages of organogenesis, in in vivo-like arrays of neural subtypes along a neural tube surrounded by spine and muscle progenitors contacted by neuronal projections. This integrated system of trunk development indicates that in vivo-like multi-tissue morphogenesis and topographic organization of terminal cell types can be achieved in human organoids, opening windows for the development of more complex models of organogenesis. Overall design: Single-cell RNA-sequencing analyses were performed on 26 pooled trunk organoids at day 7 of differentiation. Libraries were prepared following 10X Chromium Next GEM Single cell 3' v.3.1 manufacturer protocol. For the spatial transcriptomic study (Tomo-sequecing), two human trunk organoids at day 7, were freshly frozen and then sectioned. Each section was sequenced by bulk RNA sequencing. Libraries were prepared using Cel-Seq2 protocol and Illumina TruSeq small RNA adapters (RPI series).

为阐明驱动发育与疾病发生的多系统事件，亟需整合式的人类器官发生体外模型。本研究报道了人类类躯干结构（human trunk-like structures）的构建方法，该模型可复现人类脊柱与脊髓的共形态发生、模式形成与分化过程。我们鉴定出适配人类多能干细胞（human pluripotent stem cells）的分化培养条件，可诱导形成类似胚胎的延伸性前后轴（antero-posterior (AP) axis）。单细胞与空间转录组学分析显示，体节（somite）与脊髓的分化轨迹沿该轴有序排布，在添加细胞外基质（extracellular matrix）后可自组装为被体节包裹的神经管。形态发生过程与前后轴模式形成机制相耦合，在器官发生的后期阶段，可形成类似体内环境的神经亚型阵列：神经管被脊柱与肌肉祖细胞（muscle progenitors）包裹，且有神经元投射（neuronal projections）与其形成接触。这套整合式躯干发育系统表明，人类类器官（human organoids）中可实现类似体内环境的多组织形态发生与终末细胞类型的拓扑组织，为构建更为复杂的器官发生模型提供了研究契机。

实验整体设计：本研究于分化第7天，对26个混合的类躯干器官（trunk organoids）开展单细胞RNA测序（single-cell RNA-sequencing）分析。文库构建严格遵循10X Chromium Next GEM 单细胞3'端v3.1试剂盒厂商提供的实验流程。空间转录组学研究（Tomo-sequecing）部分：取2个分化第7天的人类类躯干器官，经新鲜冷冻后进行切片，对每一切片进行批量RNA测序（bulk RNA sequencing）。文库构建采用Cel-Seq2实验方案，并使用Illumina TruSeq 小RNA接头（RPI系列）。