Quantitative analysis of human notochord development

脊索祖细胞（NotoPs）对胚胎发育至关重要，是形成和维持椎间盘的细胞的前体。与其他结缔组织不同，椎间盘在童年时期就开始衰老。这种衰老过程是慢性腰痛的主要原因，影响了全球50%以上人口的生活质量，也是活动受限的主要原因。毫无疑问，无限制地使用NotoPs将为基础生物医学研究和再生医学提供许多机会。然而，不能从健康个体中获得NotoPs，目前也不可能从胚胎干细胞（ESCs）的分化中可靠地获得NotoPs。这是因为目前的策略是基于对早期脊索发育的不完整、仅定性的理解而制定的。为了应对这一挑战，规避罕见胚胎细胞群体研究固有的技术和伦理限制，我们开发了一种称为hAXIOMs的创新实验系统。这种新型的易于处理的体外系统使用微模式，这种技术使我们能够引导人类ESCs的发展成为脊索的标准化模式和围绕NotoPs出现的所有谱系。在这里，我们建议使用hAXIOMs和我们实验室开发的定量方法来确定组织组织和力学如何与信号动力学相互作用来定义NotoPs。我们将利用这一新知识为hESCs的高效衍生NotoPs提供一个健壮的协议。该项目将a)使NotoPs用于再生医学和基础生物医学研究，b)使我们了解和控制在NotoPs附近出现的其他治疗相关细胞类型，c)建立hAXIOMs作为一个范例实验系统，使我们能够在未来研究健康的轴向发育和脊柱侧凸（严重椎间盘退变的原因之一）的胚胎起源。

Notochord progenitors (NotoPs) are vital for embryonic development and are the precursors of the cells that form and maintain intervertebral discs. Unlike any other connective tissues, intervertebral discs start ageing early in life during childhood. This ageing process is the prevalent cause of chronic low back pain impacting the quality of life in more than 50% of the global population and a major cause of mobility limitations. There is no doubt that an unlimited access to NotoPs would open numerous opportunities for basic biomedical research and regenerative medicine. However, NotoPs cannot be obtained from healthy individuals and it is currently not possible to reliably obtain NotoPs from the differentiation of embryonic stem cells (ESCs). This is because current strategies have been devised on the basis of an incomplete, qualitative-only understanding of early notochord development.To tackle this challenge and circumvent the technical and ethical limitations inherent to research on rare embryonic cell populations, we have developed an innovative experimental system termed hAXIOMs. This novel tractable in vitro system uses micropatterning, a technology which enables us to guide the development of human ESCs into standardised patterns of notochord and all the lineages that surround the emergence of NotoPs.Here, we propose to use hAXIOMs together with quantitative methods developed in our lab to establish how tissue organisation and mechanics interplay with signalling dynamics to define NotoPs. We will leverage this new knowledge to produce a robust protocol for the efficient derivation of NotoPs from hESCs.This project will a) enable the use of NotoPs for regenerative medicine and basic biomedical research, b) inform our understanding and control over other therapeutically relevant cell types that emerge in the immediate vicinity of NotoPs and c) establish hAXIOMs as a paradigm experimental system allowing us in the future to investigate healthy axial development and the embryonic origins of scoliosis (one of the causes of severe intervertebral disc degeneration).