Biophysical regulation of genome architecture in meniscus cells

项目摘要 我的长期职业目标是成为一名独立调查员和STEM领域的领导者 细胞工程和再生医学，重点是骨科应用。特别是，我将 有助于改善致密结缔组织修复和再生的细胞治疗策略， 包括半月板和肌腱。半月板损伤是膝关节损伤中最常见的损伤， 每年在欧盟和美国接受治疗的糖尿病患者达100万人。然而， 关于半月板细胞表型和发育中的机械调节， 在疾病期间/受伤后。很明显，基因组的动态时空组织是一个核心的 基因表达和细胞分化的决定因素，因此了解3D如何发挥作用非常重要 基因组结构随着半月板发育或对外源性化学机械作用的反应而改变 开发更好的细胞治疗策略的线索。我提出的K 01工作的目标是阐明如何 膝关节半月板内细胞的基因组组织随着发育和退化而变化， 动物和人类细胞。此外，我将确定如何化学机械线索（如基板刚度和 在发育或退化过程中发生变化的机械负荷）调节基因组的组织， 半月板细胞通过这项工作，我将获得3D基因组结构和技术方面的专业知识， 使用超分辨率随机光学重建显微镜定量染色质纤维组织 （风暴）。我还将使用ChIP-PCR评估表观遗传状态的变化，并评估基因表达 使用基于单细胞的荧光原位杂交（FISH）在半月板细胞中作为组织的功能， 发育或损伤后以及对机械输入的反应。目标1将确定染色质组织和 使用超分辨率在半月板发育和退化期间纳米级的组蛋白修饰 随机光学重建显微镜（STORM）成像和全基因组分析，包括ChIP- PCR和RNA-FISH。目标2将确定生物物理提示是否可以建立和/或恢复半月板 特定的基因组组织，表观遗传景观和表达模式，在分化和 退行性半月板细胞这项研究将为我们提供信息，以推进诊断和治疗策略， 致密结缔组织修复和再生。重要的是，这个K 01奖将使我能够提高我的 科学知识和技术，并将进一步为独立赠款提供重要的初步数据 应用.

Project Summary My long-term career goal is to become an independent investigator and a leader in the fields of stem cell engineering and regenerative medicine, with a focus on orthopedic applications. In particular, I will contribute to improving cell therapeutic strategies for the repair and regeneration of dense connective tissues, including meniscus and tendon. Meniscus injury and damage is the most common of knee injury, with over 1.8 million meniscal injuries being treated in the EU and the US each year. However, meniscal injuries have limited repair and little is known regarding meniscus cell phenotype and mechano-regulation with development and during disease/after injury. It is clear that the dynamic spatio-temporal organization of the genome is a central determinant of gene expression and cell differentiation, and so it will be important to understand how 3D genome architecture changes with meniscus development or in response to exogenous chemo-mechanical cues to develop better cell therapeutic strategies. The goals of my proposed K01 work is to elucidate how genome organization of cells within the knee meniscus change with development and degeneration, in both animal and human cells. In addition, I will determine how chemo-mechanical cues (e.g. substrate stiffness and mechanical loading) that change during development or degeneration regulate genome organization in meniscus cells. Through the work, I will acquire expertise in 3D genome-architecture and techniques to quantify chromatin fiber organization using super-resolution stochastic optical reconstruction microscopy (STORM). I will also evaluate changes in epigenetic status using ChIP-PCR, and will evaluate gene expression using the single cell-based fluorescent in situ hybridization (FISH) in meniscus cells as a function of tissue development or after injury and in response to mechanical input. Aim 1 will identify chromatin organization and histone modifications at the nanoscale during meniscus development and degeneration using super-resolution stochastic optical reconstruction microscopy (STORM) imaging and genome wide analyses including ChIP- PCR and RNA-FISH. Aim 2 will determine whether biophysical cues can establish and/or restore meniscus specific genome organization, epigenetic landscape, and expression patterns in differentiating and degenerative meniscus cells. This study will inform us to advance diagnostic and therapeutic strategies for dense connective tissue repair and regeneration. Importantly, this K01 award will allow me to improve my scientific knowledge and techniques, and will further provide important preliminary data for independent grant applications.