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的调查员和STEM领域的领导者 细胞工程和再生医学，重点是整形外科应用。尤其是，我会 有助于改进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。半月板损伤和损伤是膝关节损伤中最常见的，超过1.8 每年在欧盟和美国治疗的半月板损伤有数百万人。然而，半月板的损伤有限 修复和半月板细胞的表型和机械调节与发育和 疾病期间/受伤后。显然，基因组的动态时空组织是一个中心 基因表达和细胞分化的决定因素，因此了解3D 基因组结构随半月板发育或对外源化学机械的响应而改变 提示开发更好的细胞治疗策略。我提议的K01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，我将确定化学机械信号(例如底物硬度和 机械负荷)，在发育或退化过程中的变化调节基因组组织 半月板细胞。通过这项工作，我将获得3D基因组体系结构和技术方面的专业知识 用超分辨随机光学重建显微镜定量研究染色质纤维结构 (暴风雨)。我还将使用芯片聚合酶链式反应来评估表观遗传状态的变化，并将评估基因表达 半月板细胞单细胞荧光原位杂交(FISH)对组织的影响 发展或受伤后，对机械输入的反应。目标1将确定染色质组织和 利用超分辨技术在半月板发育和退变过程中组蛋白的纳米级修饰 随机光学重建显微镜(STORM)成像和全基因组分析，包括芯片- 聚合酶链式反应和RNA-FISH。目标2将确定生物物理线索是否可以建立和/或恢复半月板 特定的基因组组织，表观遗传格局和表达模式，在分化和 退变的半月板细胞。这项研究将为我们提供信息，以提高诊断和治疗策略 致密的结缔组织修复和再生。重要的是，这个K01奖将让我提高我的 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 申请。

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.