Generation of human skin organoids from pluripotency

摘要 皮肤形态发生和稳态需要来自不同胚胎起源的不同细胞的组装。 这对在体外构建复杂且易于组装的皮肤提出了工程挑战，这可能是 用于烧伤和伤口的细胞疗法。具体问题是，通常用于衍生皮肤的细胞 体外缺乏产生皮肤附属物的能力，例如毛囊和汗腺，以及 辅助细胞，例如黑素细胞和脂肪细胞。关键细胞信号传导机制的识别 需要将附肢皮肤与祖细胞（例如人类多能干细胞）区分开来 （hPSC），或刺激成体皮肤细胞诱导皮肤附属器是进展的关键障碍。 hPSC 可以在体外高效生成表皮细胞；但目前尚不清楚如何监管 真皮祖细胞的诱导。因此，我们的长期目标是定义化学和物理 重现人类皮肤祖细胞发育以重建复杂皮肤所需的信号。我们的 该项目建立在初步数据的基础上，展示了一种新颖的多阶段 3D 培养系统，该系统使用 hPSC 生成包含正常皮肤大部分主要和特殊细胞成分的全层皮肤， 包括毛囊和感觉神经。在培养系统中，我们调节信号通路以共同诱导 表面外胚层细胞和颅神经嵴细胞——这两种细胞类型对于面部皮肤发育至关重要。在目标 1 中，我们 将使用单细胞 RNA 测序和药物治疗来测试 FGF-WNT-TGF 信号传导的假设 动力学是颅神经嵴细胞群多样化的基础，其中包括真皮、软骨、神经、 和神经胶质祖细胞。这些实验将使我们能够更好地控制皮肤中发育的细胞类型 类器官。在目标 2 中，我们将检验皮肤类器官形成独立于真皮细胞的假设 谱系并可推广到其他解剖位置。我们将使用中胚层的定向分化- 用于这些实验的衍生真皮细胞和微孔类器官重建。我们期望这些 研究将带来创建特定部位皮肤移植的新方法。最后，在目标 3 中，我们将检验以下假设： 在异种移植小鼠模型中，单个皮肤类器官可以组合形成多类器官移植物。这些 实验将提供皮肤类器官可用于皮肤重建的原理证明证据 手术。考虑到人类胚胎基本上无法进行科学分析，我们的类器官系统 将为研究人员提供一个前所未有的窗口来了解人类发展的最早阶段 皮肤器官发生。此外，我们的努力可能会带来用于皮肤伤口愈合的新型细胞疗法。

ABSTRACT Skin morphogenesis and homeostasis requires the assembly of diverse cells from different embryonic origins. This poses an engineering challenge for building complex and easily assembled skin in vitro, which could be used for cell therapy for burns and wounds. The specific problem is that cells commonly used to derive skin in vitro lack the potency to generate skin appendages, such as hair follicles and sweat glands, as well as accessory cells, such as melanocytes and adipocytes. Identification of the key cell signaling mechanisms needed to differentiate appendage-bearing skin from progenitor cells, such as human pluripotent stem cells (hPSCs), or to stimulate skin appendage induction from adult skin cells is a critical barrier to progress. Epidermal cells can be efficiently generated in vitro from hPSCs; however, it is unclear how to regulate the induction of dermal progenitor cells. Therefore, our long-term goal is to define the chemical and physical signals required to recapitulate development of human skin progenitor cells to reconstitute complex skin. Our project builds on preliminary data demonstrating a novel multi-stage 3D culture system that uses hPSCs to generate full-thickness skin containing most of the primary and specialized cellular components of normal skin, including hair follicles and sensory nerves. In the culture system, we modulate signaling pathways to co-induce surface ectoderm and cranial neural crest cells—two cell types critical for facial skin development. In Aim 1, we will use single-cell RNA-sequencing and drug treatments to test the hypothesis that FGF-WNT-TGF signaling dynamics underlie diversification of the cranial neural crest cell population containing dermal, chondral, neural, and glia progenitor cells. These experiments will allow us to better control the types of cells that develop in skin organoids. In Aim 2, we will test the hypothesis that skin organoid formation is independent of dermal cell lineage and is generalizable to other anatomical locations. We will use directed differentiation of mesoderm- derived dermal cells and micro-well organoid reconstitution for these experiments. We expect that these studies will lead to new ways to create site-specific skin grafts. Finally, in Aim 3, we will test the hypothesis that individual skin organoids can be combined to form multi-organoid grafts in a xenograft mouse model. These experiments will provide proof-of-principle evidence that skin organoids could be used for skin reconstructive surgery. Considering that the human embryo is largely inaccessible for scientific analysis, our organoid system will provide an unprecedented window into the earliest stages of human development for researchers studying skin organogenesis. In addition, our efforts could lead to novel cell therapies for skin wound healing.