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来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， 用于烧伤和创伤的细胞治疗。具体的问题是，通常用于获得皮肤的细胞， 体外缺乏生成皮肤附属物的能力，如毛囊和汗腺，以及 辅助细胞，如黑素细胞和脂肪细胞。识别关键细胞信号传导机制 需要从祖细胞（如人类多能干细胞）分化出带有附件的皮肤 （hPSC）或刺激来自成体皮肤细胞的皮肤附属物诱导是进展的关键障碍。 表皮细胞可以在体外从hPSC有效地产生;然而，目前还不清楚如何调节表皮细胞的生长。 真皮祖细胞的诱导。因此，我们的长期目标是确定化学和物理 再现人类皮肤祖细胞发育以重建复杂皮肤所需的信号。我们 该项目建立在初步数据的基础上，展示了一种新型的多阶段3D培养系统，该系统使用hPSC， 生成包含正常皮肤的大部分初级和特化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， 表面外胚层和颅神经嵴细胞-两种对面部皮肤发育至关重要的细胞类型。目标1： 将使用单细胞RNA测序和药物治疗来测试FGF-WNT-TGF信号转导的假设， 动力学是颅神经嵴细胞群多样化的基础，包括真皮的，软骨的，神经的， 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不依赖于真皮细胞的假设。 谱系，并可推广到其他解剖位置。我们将利用中胚层的定向分化- 衍生的真皮细胞和微孔类器官重建用于这些实验。我们预计这些 研究将导致新的方法来创建特定部位的皮肤移植。最后，在目标3中，我们将检验以下假设： 可以在异种移植小鼠模型中组合单个皮肤类器官以形成多类器官移植物。这些 实验将提供原理性证据，证明皮肤类器官可用于皮肤重建 手术考虑到人类胚胎在很大程度上无法进行科学分析，我们的类器官系统 将为研究人类发育的最早阶段的研究人员提供一个前所未有的窗口， 皮肤器官发生此外，我们的努力可能导致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.