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]. 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. Overall design: In order to identify the molecular components of the mouse segmentation clock, mouse epiblast stem cell (EpiSCs)-derived presomitic mesoderm (PSM) samples were collected in timely manner utilizing a luciferase reporter line of the key oscillatory segmentation clock gene Hes7 (Hes7-reporter). Two independent sets of 16 samples each of entrained oscillating mouse in vitro induced PSM samples were analyzed using RNAseq.

多能干细胞（pluripotent stem cells, PSCs）已日益被用于模拟胚胎发生与器官形成的不同方面。尽管当前在体外诱导主要中胚层谱系及中胚层来源细胞类型领域已取得显著进展，但能够重现人类中胚层发育与模式形成更复杂生物学特征的实验模型系统仍极为匮乏。本研究利用诱导多能干细胞（induced pluripotent stem cells, iPSCs），通过分步体外诱导体节前中胚层（presomitic mesoderm, PSM）及其衍生细胞，以模拟人类体节发生的不同特征。我们最初聚焦于模拟人类分节时钟——这一核心生物学概念被认为是调控体节节律性、有序发生的基础，而体节最终将形成脊椎动物轴向骨骼的节段性模式。我们成功观测到包括HES7和DKK1在内的核心分节时钟基因的振荡表达，并在人类与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，对与异常轴向骨骼发育患者（如脊柱肋骨发育不良症（spondylocostal dysostosis））中已报道存在突变的基因（包括HES7、LFNG及DLL3）进行靶向操作。对构建的患者样诱导多能干细胞敲除细胞系以及患者来源诱导多能干细胞，结合其经遗传矫正的同基因对照进行后续分析，结果显示不同基因在振荡、同步化或分化特性上存在基因特异性改变，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形成相关疾病提供全新见解。整体实验设计：为鉴定小鼠分节时钟的分子组分，我们利用关键振荡分节时钟基因Hes7的荧光素酶报告细胞系（Hes7-reporter），按时收集小鼠上胚层干细胞（epiblast stem cell, EpiSCs）来源的体节前中胚层样本。我们采用两组独立的体外诱导振荡小鼠体节前中胚层样本，每组各16份经同步化的振荡样本，通过RNA测序（RNAseq）进行分析。