Preserving chromatin nano-structure to enhance chondrocyte therapeutic potential for cartilage repair

摘要 关于软骨细胞如何失去其表型和基质产生的知识存在空白 体外扩增过程中的能力。这种知识上的差距源于缺乏直接 询问单个细胞中软骨细胞的基因组结构和转录图谱，以获取固有的 细胞分化的异质性。为了填补这一空白，我们将使用最先进的超分辨率成像技术， 单细胞RNA测序、高通量RNA-FISH和代谢标记 (FUNCAT)技术，在逐个细胞的水平上，将染色质纳米结构、转录输出、 体外扩增单个软骨细胞的表观遗传修饰和基质生成能力 不同的表观遗传学和化学物理线索。我们将进一步开发机器学习模型来预测 用超分辨率图像研究软骨细胞染色质纳米结构的表型。我们的中央 假设有不同的染色质纳米结构排列和转录特征 与具有高基质生产能力的软骨细胞相关联，且染色质纳米结构可以 通过表观遗传和化学物理线索的组合以预测性的方式进行操纵，以改善 软骨细胞的治疗潜力。这一假设的基础是我们的初步超分辨率数据 体外扩增软骨细胞和基质干细胞中染色质纳米结构的研究 具有不同的硬度，并受各种化学物质的影响。拟议的工作意义重大，因为它将产生 染色质纳米结构和表观遗传景观如何调节基质产生的新认识 软骨细胞的能力，以及如何通过操纵染色质和 表观遗传状态。我们的目标是： 矩阵 染色质纳米结构和转录是否可以预测软骨细胞 生产能力 目标1：确定 目的2：确定化学物理和表观遗传线索如何影响染色质纳米... 软骨细胞的结构和基质产生 目的3：确定预测线索是否能提高软骨细胞的治疗效果 综上所述，我们期望为鉴定新的体外扩增条件做出贡献 幼稚的软骨细胞表型，增强其治疗潜力。拟议的研究具有创新性，因为 它通过应用多方面的、基于单细胞的成像和 确定软骨细胞染色质与表观遗传状态关系的测序技术， 转录活动和基质生产。如果成功，这项工作可能会通过提供 改善软骨修复的细胞群。

Summary There is a gap in the knowledge about how chondrocytes lose their phenotype and matrix production capacity during in vitro expansion. This gap in knowledge stems from the paucity of studies that directly interrogate chondrocyte genome architecture and transcriptional profiles in single cells to capture the inherent heterogeneity of cell differentiation. To fill this unmet gap, we will use state-of-the-art super-resolution imaging, single cell RNA Sequencing (RNA-Seq), high-throughput RNA-FISH (MERFISH) and metabolic labeling (FUNCAT) technologies to relate, on a cell-by-cell level, the chromatin nano-structure, transcriptional output, epigenetic modifications and matrix production capacity of single chondrocytes expanded in culture under different epigenetic and chemo-physical cues. We will further develop machine learning models to predict chondrocyte phenotype using super-resolution images of chondrocyte chromatin nano-structure. Our central hypothesis is that there are distinct chromatin nano-structural arrangements and transcriptional signatures associated with chondrocytes that have high matrix production capacity, and that chromatin nano-structure can be manipulated in a predictive manner via the combination of epigenetic and chemo-physical cues to improve chondrocyte therapeutic potential. The basis for this hypothesis is our preliminary super-resolution data of chromatin nano-structure in in vitro expanded chondrocytes and mesenchymal stem cells grown on substrates of varying stiffness and subjected to various chemical cues. The proposed work is significant as it will generate new knowledge about how chromatin nano-structure and epigenetic landscape regulates matrix production capacity of chondrocytes and how this capacity can be enhanced through manipulation of chromatin and epigenetic states. Our Aims are: matrix whether chromatin nano-structure and transcription are predictive of chondrocyte production capacity Aim 1: Determine Aim 2: Determine how chemo-physical and epigenetic cues impact transitions in chromatin nano- structure and matrix production in chondrocytes Aim 3: Determine whether predicted cues improve chondrocyte therapeutic efficacy In summary, we expect to contribute to the identification of new in vitro expansion conditions that maintain naïve chondrocyte phenotype and enhance their therapeutic potential. The proposed research is innovative as it represents a drastic departure from the status quo by applying multi-faceted, single-cell based imaging and sequencing technologies to determine the relationship between chondrocyte chromatin and epigenetic state, transcriptional activities, and matrix production. If successful, this work may change clinical practice by providing improved cell populations for cartilage repair.