Generation of human skin organoids from pluripotency

摘要 皮肤的形态发生和动态平衡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和容易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治疗。具体的问题是，通常用于衍生皮肤的细胞 体外培养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。关键细胞信号机制的识别 需要区分带有附件的皮肤和祖细胞，如人类多能干细胞 (HPSCs)，或刺激皮肤附属物从成人皮肤细胞诱导是进展的关键障碍。 从hPSCs体外可以有效地产生表皮细胞；然而，目前还不清楚如何调节 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 重述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，展示了一种使用hPSCs的新型多阶段3D培养系统 生成包含正常皮肤的大部分主要和专门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 面部皮肤发育的两种细胞--表面外胚层细胞和脑神经脊细胞。在目标1中，我们 将使用单细胞RNA测序和药物治疗来验证以下假设 包含真皮，软骨，神经， 和神经胶质前体细胞。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 有机化合物。在目标2中，我们将检验皮肤类器官的形成独立于真皮细胞的假设。 并可推广到其他解剖部位。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有机体重建。我们预计这些 研究将导致创造特定部位皮肤移植的新方法。最后，在目标3中，我们将检验假设 在异种移植小鼠模型中，单个皮肤有机化合物可以组合形成多个有机类化合物移植物。这些 实验将提供皮肤有机物质可用于皮肤重建的原则证据 做手术。考虑到人类胚胎在很大程度上无法进行科学分析，我们的有机系统 将为研究人员提供一个了解人类最早发展阶段的前所未有的窗口 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.