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

总结 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 在体外扩增期间的容量。这种知识上的差距源于缺乏研究， 在单个细胞中询问软骨细胞基因组结构和转录谱， 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， 单细胞RNA测序（RNA-Seq）、高通量RNA-FISH（MERFISH）和代谢标记 （FUNCAT）技术，涉及，在一个细胞一个细胞的水平，染色质纳米结构，转录输出， 表观遗传修饰和基质生产能力的单一软骨细胞扩大培养下 不同的表观遗传和化学物理线索。我们将进一步开发机器学习模型来预测 使用软骨细胞染色质纳米结构的超分辨率图像的软骨细胞表型。我们的中央 一种假设是，存在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，并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的方式进行操纵， 软骨细胞治疗潜力。这一假设的基础是我们初步的超分辨率数据， 基质上生长的体外扩增软骨细胞和间充质干细胞中的染色质纳米结构 硬度不同，并受到各种化学物质的影响。拟议的工作是重要的，因为它将产生 关于染色质纳米结构和表观遗传景观如何调节基质产生的新知识 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 产能 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结构的转变。 软骨细胞中结构和基质产生 目的3：确定预测的线索是否改善软骨细胞治疗效果 总之，我们希望有助于确定新的体外扩增条件， 幼稚软骨细胞表型并增强其治疗潜力。该研究具有创新性， 它通过应用多方面的、基于单细胞的成像， 测序技术来确定软骨细胞染色质和表观遗传状态之间的关系， 转录活性和基质产生。如果成功，这项工作可能会改变临床实践， 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.