Generating morphogen gradients to engineer human integumentary organoids

项目摘要 从诱导多能干细胞的胚胎体(EBS)生成类人器官的研究进展 细胞(IPSCs)为理解人类器官形态发生和遗传性疾病提供了新的工具。然而， 目前的类器官生成方法，其中细胞对称地暴露在分化中 培养中的因子/形态因子只允许产生组织的部分成分，因此不 支持空间控制的多组分组织的生成。有机化合物目前面临的挑战 研究是实现细胞、组织成分和解剖结构的生理学相关组织 功能。在这个项目中，我们假设在EBS中生成定义的非对称化学梯度将导致 本发明旨在控制IPSCs分化为包括解剖特征的多组分有机化合物。这 这种方法将克服目前对称培养条件的局限性。皮肤代表着一种伟大的 模型器官来验证这一假说，因为(I)皮肤的形态发生强烈地依赖于细胞的相互作用 来自多个谱系的；以及(Ii)毛囊的生成和色素沉着可用作功能读出 以评估这种生物工程方法的稳健性，这种方法稍后可以适用于其他有机系统。 在具体目标1中，我们将在单个EB水平上生成精确的差异因子交叉梯度 微流体学。我们推测，这将诱导表皮细胞和神经脊细胞的同时产生- 来源的黑素细胞和毛乳头细胞。我们的成功标准将是对早期事件的重演 皮肤形态发生的过程，如皮肤附属物的形成和表皮的色素沉着。具体而言 目标2，我们将把这种方法扩展到建立遗传病的模型。我们将重点关注Hutchinson-Gilford早衰症 综合征(HGPS)，这是一种罕见的单基因过早衰老疾病，具有明显的皮肤异常，包括 皮肤硬化、色素沉着和脱发。我们将首先使用患者的IPSCs诱导皮肤形态发生 形成皮肤病的表型。随后，我们将使用我们的微生理皮肤模型来早期识别 早衰症皮肤的发育异常，这在很大程度上是人类未知的，并进一步评估 三种不同分子靶点的药物逆转HGPS皮肤表型的疗效和毒性 与人相关的背景。这种创新的方法代表着向充分发展的工程迈出的关键一步 并将对我们对人类的理解产生直接和压倒性的影响 皮肤形态发生与发育性皮肤病。

Project Summary Recent success in generating human organoids from embryonic bodies (EBs) of induced pluripotent stem cells (iPSCs) offers a new tool to understand human organ morphogenesis and genetic diseases. However, the current organoid generating approaches, where cells are symmetrically exposed to differentiation factors/morphogens in culture, only allow for the generation of partial components of a tissue, thereby do not support the spatially-controlled generation of multicomponent tissues. The current challenge in organoid research is to achieve a physiologically-relevant organization of cells, tissue components, and anatomical features. In this project, we postulate that generating defined asymmetrical chemical gradients in EBs will lead to controlled differentiation of iPSCs into multicomponent organoids comprising anatomical features. This approach will overcome the current limitations of symmetrical culture conditions. The skin represents a great model organ to test this hypothesis, because (i) the skin morphogenesis strongly relies on interactions of cells from multiple lineages; and (ii) generation of hair follicles and pigmentation can be used as functional read-outs to assess the robustness of this bioengineering approach, which can later be adapted for other organoid systems. In Specific Aim 1, we will generate precise cross-gradients of differentiation factors at the single EB level using microfluidics. We postulate that this will induce simultaneous generation of epidermal cells and neural crest cell- derived melanocytes and dermal papilla cells. Our success criteria will be the recapitulation of the early events of skin morphogenesis, such as the formation of skin appendages and pigmentation of the epidermis. In Specific Aim 2, we will extend this approach to model a genetic disease. We will focus on Hutchinson-Gilford progeria syndrome (HGPS), which is a rare monogenic premature aging disease with distinct skin abnormalities including sclerotic skin, dyspigmentation, and alopecia. We will first induce skin morphogenesis using patient iPSCs to develop a skin disease phenotype. Subsequently, we will use our microphysiological skin model to identify early developmental abnormalities in progeria skin, which are largely unknown for humans, and further evaluate the efficacy and toxicity of three drugs with different molecular targets on the reversal of HGPS skin phenotype in a human relevant-context. This innovative approach represents a critical step towards engineering fully-developed integumentary organoids and will have an immediate and overwhelming impact on our understanding of human skin morphogenesis and developmental skin diseases.