Generating morphogen gradients to engineer human integumentary organoids

项目摘要 从诱导多能干细胞的胚胎体（EB）产生人类类器官的最新成功 多能干细胞（iPSCs）的研究为理解人类器官形态发生和遗传疾病提供了新的工具。但 目前的类器官产生方法，其中细胞对称地暴露于分化， 在培养物中的生长因子/形态发生素仅允许产生组织的部分组分，从而不 支持多组分组织的空间控制生成。当前类器官面临的挑战 研究是为了实现细胞，组织成分和解剖结构的生理相关组织。 功能.在这个项目中，我们假设在EB中产生定义的不对称化学梯度将导致 涉及iPSC受控分化为包含解剖学特征的多组分类器官。这 这种方法将克服目前对称培养条件的局限性。皮肤代表了一个伟大的 模型器官来测试这一假设，因为（i）皮肤形态发生强烈依赖于细胞的相互作用 来自多个谱系;和（ii）毛囊和色素沉着的产生可用作功能读出 评估这种生物工程方法的稳健性，该方法以后可以适用于其他类器官系统。 在具体目标1中，我们将使用以下方法在单个EB水平上生成分化因子的精确交叉梯度： 微流体技术我们假设这将诱导表皮细胞和神经嵴细胞的同时产生- 衍生的黑素细胞和毛乳头细胞。我们的成功标准将是早期事件的重演 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。在特定 目标2，我们将扩展这种方法来模拟遗传疾病。我们将重点关注哈钦森-吉尔福德早衰症 综合征（HGPS），这是一种罕见的单基因早衰症，具有明显的皮肤异常，包括 硬皮病、色素沉着和脱发。我们将首先使用患者iPSC诱导皮肤形态发生， 形成皮肤病表型。随后，我们将使用我们的微生理皮肤模型来早期识别 发育异常的早衰症皮肤，这是很大程度上未知的人类，并进一步评估 三种具有不同分子靶点的药物对逆转HGPS皮肤表型的疗效和毒性， 人类相关语境。这种创新的方法是迈向工程全面发展的关键一步 皮肤类器官，并将对我们对人类的理解产生直接和压倒性的影响。 皮肤形态发生和发育性皮肤病。

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.