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.