Rapid functional genetics to study stem cell-niche interactions in the skin

快速功能遗传学研究皮肤干细胞生态位相互作用

• 批准号: 10341428
• 负责人: Ya-Chieh Hsu
• 金额: $ 52.05万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-24 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10341428
• 关键词: ATAC-seq; Ablation; Address; Agonist; Alopecia; Automobile Driving; Behavior; Biological; Candidate Disease Gene; Cells; Chromatin; Communities; Data; Data Set; Dependovirus; Deposition; Dermal; Dermis; Development; Enhancers; Fibroblasts; Future; Gene Expression; Genes; Genetic; Genetic Models; Goals; Hair; Hair follicle structure; Homeostasis; Immunofluorescence Immunologic; Knockout Mice; Knowledge; LIF gene; Maintenance; Measures; Mediating; Mediator of activation protein; Methods; Molecular; Molecular Profiling; Mus; Muscle; Natural regeneration; Nerve; Organ; Physiological; Plasmids; Play; Population; Process; Publications; Regulatory Element; Research; Research Personnel; Role; Serotyping; Signal Transduction; Skin; Smooth Muscle; Specificity; Testing; Therapeutic; Time; Tissues; Tropism; Viral; Work; cell behavior; cell type; gene function; gene therapy; genetic manipulation; genetic signature; genetic testing; glial cell-line derived neurotrophic factor; hair erector muscle; improved; in vivo; inhibitor; innovation; insight; muscular system; nerve supply; new technology; novel; novel strategies; novel therapeutics; overexpression; promoter; repaired; repository; single cell sequencing; single-cell RNA sequencing; skin disorder; stem cell biology; stem cell niche; stem cells; success; therapy development; tongue papilla; tool; transcriptome sequencing; wound healing

Project Summary Skin stem cells are heavily influenced by signals from their niches including different fibroblasts populations. While our ability to isolate and molecularly profile diverse cell types has improved drastically in the past decade, a major roadblock in identifying key genes driving stem cell-niche interactions is the lengthy process of generating the genetic models needed (e.g., cell-type specific Cre or CreER, and overexpression or knockout mouse lines) to test gene functions in a cell-type specific manner in a physiologically relevant context. As such, while many different cell types have been identified and molecularly profiled, the critical genes that drive many developmental and regeneration processes remain incompletely understood. This substantial knowledge gap presents a significant impediment to developing therapies for skin diseases. To address this gap and to showcase how rapid functional genetics can enable new discoveries in stem cell- niche interactions, we will first build adeno-associated viral (AAV) toolkits to expand the field’s capacity for rapid functional genetics in multiple dermal cell types in mice. This aim expands on our current success in using AAVs to transduce dermal cells, with the goal of building tools that allows all skin researchers to modify gene expression rapidly in dermal populations such as the dermal fibroblasts and DP. We have recently developed and conducted SHARE-seq on the skin, a high-throughput single cell sequencing method that simultaneously measures chromatin accessibility (single cell ATACseq) and gene expression (single cell RNAseq) within the same cell. SHARE-seq data allow us to computationally infer key regulatory elements (enhancers, promoters) of signature genes for distinct cell types, which further enables the construction of cell-type specific AAV tools. We know the proposed strategy is feasible, because we have used it to build tools that can manipulate gene expression in the arrector pili muscles (APMs), a cell type that currently lacks specific Cre/CreER constructs. APMs are an emerging niche cell type for hair follicle stem cells (HFSCs). However, the molecular mechanisms by which APMs regulates HFSC behavior remain poorly understood. In Aim2, we will use our AAV tools to discover APM-derived secreted factors that regulate HFSC activation and maintenance. Collectively, these results will provide the skin community with much-needed tools to accelerate research in diverse topics, and may be relevant for understanding and potentially treating a wide range of alopecia conditions. Since AAVs are non-toxic and non-immunogenic, and since many key tissue-specific regulatory elements retain their specificity across species, there is an exciting potential to combine our biological findings with our technical advancements to develop novel gene therapy strategies to treat these skin diseases in the near future.

项目摘要 皮肤干细胞受到其壁ni的信号的严重影响，包括不同的成纤维细胞种群。 尽管在过去的十年中，我们分离和分子剖面多样的细胞类型的能力已大大提高，但 识别驱动干细胞纽带相互作用的关键基因的主要障碍是漫长的过程 生成所需的遗传模型（例如，细胞型特异性CRE或CRER，以及过表达或敲除 小鼠线）以在物理相关的上下文中以细胞类型的方式测试基因功能。像这样， 虽然已经鉴定出许多不同的细胞类型并分子进行了分析，但驱动许多的关键基因 发育和再生过程仍然不完全理解。这个巨大的知识差距 给开发皮肤疾病疗法带来了重大障碍。 解决这一差距并展示功能遗传学如何能够在干细胞中引起新发现 利基互动，我们将首先建立与腺相关的病毒（AAV）工具包，以扩大该领域的快速能力 小鼠多种真皮细胞类型的功能遗传学。这个目标扩展了我们目前使用AAVS的成功 翻译真皮细胞的目的是建立工具，使所有皮肤研究人员都可以修改基因 皮肤群体（例如皮肤成纤维细胞和DP）的表达迅速。我们最近开发了 并在皮肤上进行了股票，这是一种高通量单细胞测序方法，简单地 测量染色质的可及性（单细胞ATACSEQ）和基因表达（单细胞RNASEQ） 相同的单元。股票数据允许我们在计算上推断关键监管元素（增强子，启动子） 签名基因的不同细胞类型，这进一步构建了细胞类型的特定AAV工具。 我们知道提出的策略是可行的，因为我们已经使用它来构建可以操纵基因的工具 到达pili肌肉（APMS）中的表达，这是一种目前缺乏特定Cre/Creer构建体的细胞类型。 APM是用于毛囊干细胞（HFSC）的新兴细胞类型。但是，分子机制 APM调节HFSC行为的理解仍然很少。在AIM2中，我们将使用我们的AAV工具来 发现调节HFSC激活和维护的APM衍生的分泌因素。总的来说，这些 结果将为皮肤社区提供急需的工具，以加速潜水员主题的研究，并 可能与理解和潜在治疗多种脱发状况有关。因为AAV是 无毒和非免疫原性，并且由于许多关键组织特异性调节元素保留其特异性 在整个物种中，将我们的生物学发现与我们的技术进步相结合的令人兴奋的潜力 开发新型的基因疗法策略来治疗这些皮肤疾病。