Single cell characterization of the biomaterial immune and stromal response

使用单细胞RNA测序(ScRNAseq)分析单个细胞正在彻底改变我们对 发展和疾病。在这项提案中，我们将应用scRNAseq来创建一份细胞图谱，这些细胞对 诱导不同反应并作为修复组织微环境模型的生物材料 而不是纤维化。拟议的研究旨在利用单细胞分析来定义关键的亚群 淋巴、髓系和间质成纤维细胞对组织纤维化和修复的生物材料模型作出反应。 最低限度加工的生物支架诱导以白细胞介素4为特征的2型免疫反应 和组织修复，类似于肌肉修复过程。我们的初步数据描述了一种17型免疫和 诱导IL-17依赖的纤维包膜形成的人工合成植入物对衰老细胞的反应 举止。我们还展示了scRNASeq发现新的巨噬细胞群的能力 生物材料微环境。我们假设通过在活体内对FBR中的细胞亚群进行排序， 结合单细胞分析，我们将识别新的和稀有的种群，这将有助于阐明 并提供新的治疗靶点，以加强组织修复或减少纤维化。以下是 为实现这一目标提出了具体目标： 具体目标1.鉴定和鉴定分离的淋巴、髓系和成纤维细胞亚群 使用单细胞RNA测序分析合成和生物支架植入物。 具体目标2.在细胞类型内和跨细胞类型之间计算表型细胞群，以定义不同的 使用伪时间分析、RNA速度、差异表达和基因集的子集和相互作用模型 浓缩、用于预测独特表面标记/组合的聚类分析以及细胞相互作用分析。 具体目标3.从单细胞分析中确定独特的表面和细胞内标记，以识别 亚群使用标准的实验方法。新发现的免疫和成纤维细胞亚群 随着时间的推移，将在雄性和雌性小鼠身上进行评估，结果将得到不同材料的验证。 在拟议研究中创建的细胞图谱将使未来的机械研究和调查成为可能 潜在的广泛应用于伤口愈合、癌症和其他组织病理学。

Profiling single cells using single cell RNA sequencing (scRNAseq) is revolutionizing our understanding of development and disease. In this proposal, we will apply scRNAseq to create an atlas of cells that respond to biomaterials that induce divergent responses and serve as a model for tissue microenvironments of repair versus fibrosis. The proposed research aims to leverage single cell analysis to define key subpopulations in the lymphoid, myeloid and stromal fibroblasts response to biomaterial models of tissue fibrosis and repair. Minimally processed biological scaffolds induce a Type 2 immune response characterized by interleukin (IL)-4 and tissue repair, similar to muscle repair processes. Our preliminary data describes a Type 17 immune and senescent cell response to synthetic implants that induce fibrotic capsule formation in an IL-17-dependent manner. We also demonstrate the ability of scRNASeq to uncover new macrophage cell populations in biomaterial microenvironments. We hypothesize that by sorting cell subpopulations in the FBR in vivo, combined with single cell analysis, we will identify new and rare populations that will help elucidate mechanisms and provide new therapeutic targets to enhance tissue repair or reduce fibrosis. The following specific aims are proposed to accomplish this goal: Specific Aim 1. Identify and characterize lymphoid, myeloid, and fibroblast subpopulations isolated from synthetic and biological scaffold implants using single cell RNA sequencing analysis. Specific Aim 2. Computationally phenotype cell clusters both within and across cell types to define distinct subsets and interaction models using pseudotime analysis, RNA velocity, differential expression and gene set enrichment, cluster analysis to predict unique surface markers/combinations, and cell interactions analysis. Specific Aim 3. Define unique surface and intracellular markers from single cell analysis to identify subpopulations using standard experimental methods. Newly-identified immune and fibroblast subpopulations will be evaluated over time in male and female mice and results will be validated with diverse materials. The cell atlas created in the proposed research will enable future mechanistic studies and investigation into the potential broad applicability to wound healing, cancer and other tissue pathologies.