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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要皮肤干细胞受到来自其生态位（包括不同成纤维细胞群）的信号的严重影响。虽然我们分离和分子分析不同细胞类型的能力在过去十年中得到了显着提高，识别驱动干细胞生态位相互作用的关键基因的一个主要障碍是漫长的过程生成所需的遗传模型（例如，细胞类型特异性 Cre 或 CreER，以及过度表达或敲除小鼠系）以在生理相关背景下以细胞类型特定的方式测试基因功能。虽然许多不同的细胞类型已被鉴定并进行分子分析，但驱动许多细胞的关键基因发育和再生过程仍然不完全清楚。对开发皮肤病疗法构成了重大障碍。为了解决这一差距并展示功能遗传学如何快速实现干细胞的新发现 - 利基相互作用，我们将首先构建腺相关病毒（AAV）工具包，以扩大该领域快速检测的能力这一目标扩展了我们目前使用 AAV 的成功。转导真皮细胞，目标是构建允许所有皮肤研究人员修改基因的工具我们最近开发了在真皮群体（例如真皮成纤维细胞和 DP）中快速表达的蛋白。并在皮肤上进行了 SHARE-seq，这是一种高通量单细胞测序方法，同时测量染色质可及性（单细胞 ATACseq）和基因表达（单细胞 RNAseq）相同的细胞。SHARE-seq 数据使我们能够通过计算推断关键的调控元件（增强子、启动子）。不同细胞类型的特征基因，这进一步使得构建细胞类型特异性 AAV 工具成为可能。我们知道所提出的策略是可行的，因为我们已经用它来构建可以操纵基因的工具在阻止菌毛肌 (APM) 中表达，这是一种目前缺乏特定 Cre/CreER 结构的细胞类型。 APM 是一种新兴的毛囊干细胞 (HFSC) 细胞类型，但其分子机制尚不清楚。 APM 是如何调节 HFSC 行为的仍然知之甚少，在 Aim2 中，我们将使用我们的 AAV 工具来实现。发现 APM 衍生的调节 HFSC 激活和维持的分泌因子。结果将为皮肤界提供急需的工具来加速不同主题的研究，以及由于 AAV 可能与了解和潜在治疗多种脱发病症相关。无毒且无免疫原性，并且由于许多关键的组织特异性调节元件保留了其特异性跨物种，将我们的生物学发现与技术进步相结合具有令人兴奋的潜力在不久的将来开发新的基因治疗策略来治疗这些皮肤病。