Recapitulating the Human Segmentation Clock with Pluripotent Stem Cells - RNAseq analysis of in vitro induced murine presomitic mesoderm (PSM) and identification of components of the in vitro mouse segmentation clock [Oscillation_Mm]

Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis and organ formation. Despite recent advances in the in vitro induction of major mesodermal lineages and mesoderm-derived cell types, experimental model systems that can recapitulate more complex biological features of human mesoderm development and patterning are largely missing. Here, we utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and DKK1, and identified novel oscillatory genes in human and mouse PSC-derived PSM. We furthermore determined the period of the human segmentation clock to be around five hours and showed the presence of dynamic traveling wave-like gene expression within in vitro induced human PSM. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with abnormal axial skeletal development such as spondylocostal dysostosis (HES7, LFNG and DLL3) have been reported. Subsequent analysis of patient-like iPSC knock-out lines as well as patient-derived iPSCs together with their genetically corrected isogenic controls revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating the overall utility of our model system, to recapitulate not only key features of human somitogenesis but also to provide novel insights into diseases associated with the formation and patterning of the human axial skeleton.

多能干细胞（Pluripotent stem cells, PSCs）已日益被广泛应用于胚胎发生与器官形成各环节的体外建模。尽管近年来在体外诱导主要中胚层谱系及中胚层来源细胞类型方面取得了诸多进展，但能够重现人类中胚层发育与模式形成复杂生物学特征的实验模型系统仍较为稀缺。本研究借助诱导多能干细胞（induced pluripotent stem cells, iPSCs），通过分步体外诱导体节前中胚层（presomitic mesoderm, PSM）及其衍生物，以模拟人类体节发生的不同特征。我们最初将研究焦点置于人类分节时钟（segmentation clock）的建模——这一核心生物学概念被认为是调控体节节律性、可控性产生的基础，而体节最终将形成脊椎动物轴向骨骼的节段模式。我们成功观测到包括HES7与DKK1在内的核心分节时钟基因的振荡表达，并在人类及小鼠多能干细胞来源的体节前中胚层中鉴定出新型振荡基因。此外，我们确定人类分节时钟的周期约为5小时，并证实体外诱导的人类体节前中胚层内存在动态的行进波样基因表达模式。随后，我们利用基于CRISPR/Cas9的基因组编辑技术，靶向编辑相关致病基因：此类基因突变可见于脊柱肋骨发育不良（spondylocostal dysostosis）等轴向骨骼发育异常患者，涉及HES7、LFNG及DLL3等基因。对模拟患者表型的iPSC敲除细胞系、患者来源iPSC及其经遗传校正的同基因对照进行后续分析后，我们发现了基因特异性的振荡、同步化或分化特性异常，验证了本模型系统的整体实用性：其不仅能够重现人类体节发生的关键特征，还可为人类轴向骨骼形成与模式异常相关疾病提供全新的研究见解。