A multi-modality approach to decode epithelial heterogeneity and function in metaplasia

1)背景、我们认识上的主要差距以及需要解决的重要挑战。响应于 损伤后，胃肠道中分化的成体分泌细胞发生化生，或转化为 一种细胞类型到另一种细胞类型。在胰腺中，这被称为"腺泡到导管化生"（ADM）， 在组织再生中的作用。我们实验室的目标是生成工具来理解 ADM，以鉴定出现的ADM细胞类型的联合体，并确定它们在ADM中的生理作用。 组织损伤2)主要研究者对近期进展的描述。在她的博士后工作结束时，PI显示 分化的分泌细胞类型，如簇状细胞，在胰腺中形成以响应ADM（DelGiorno 等，Frontiers in Physiology，2020）。簇状细胞是一种单独的化学感受细胞，在介导 炎症结合RNA测序，电子显微镜（EM）和疾病的小鼠模型，她 鉴定了簇状细胞的功能作用，这是在任何肿瘤发生模型中的首次描述（DelGiorno et al. 胃肠病学，2020）。使用单细胞RNA测序（scRNA-seq）和EM，她已经确定了 在ADM中形成了几个功能未知的额外分泌细胞群，目前正在研究中。 在DelGiorno实验室进行了研究。3)展望未来的研究计划。我们建议继续我们的 通过创建结合单细胞RNA-seq和EM分析（SEMA）的工作流程来研究ADM 胰腺损伤中的上皮异质性，有和没有经鉴定的细胞类型的遗传操作。 将使用体积电子显微镜（3DEM）作为基底，在其上绘制所提供的信息 通过scRNA-seq揭示细胞之间的空间关系并推断单个细胞类型的功能 通过研究它们的细胞器含量。我们的方法利用扫描电子显微镜（SEM）， 在相同的组织上进行各种实验，包括：[1]脱水的整体包埋样品的成像， [2]大面积连续切片的宽视场"切片映射"，以及[3]大规模连续切片方法 获取3DEM。使用相关光学和电子显微镜增强映射，以分配分子 在scRNA-seq中通过免疫荧光对具有SEM中定义特征的细胞鉴定的标志物（例如， 核的形状、线粒体的分布等）。我们的3DEM数据集适合高通量图像 处理（例如，分割、几何处理）。 这些工具共同提供了一种新颖的发现工作流程，将应用于我们的整个项目。 实验室旨在评估ADM的细胞异质性。我们未来五年的目标是使用这个 SEMA方法来构建细胞内容和空间关系的多维图谱， 缺乏单个ADM细胞亚型的小鼠模型将用于 评估表型对细胞组成、相对定位和疾病进展的影响。所有这些 这些研究将为ADM在胰腺组织愈合和再生中的作用提供宝贵的见解。

1) Background, key gaps in our understanding, and important challenges to be addressed. In response to injury, differentiated adult secretory cells in the gastrointestinal tract undergo metaplasia, or the conversion of one cell type to another. In the pancreas, this is called `acinar to ductal metaplasia' (ADM) and it is thought to function in tissue regeneration. The goals of our laboratory are to generate tools to understand the process of ADM, to identify the consortium of ADM cell types that emerge, and to determine their physiological role in tissue injury. 2) Description of recent progress by the PI. By the end of her post-doctoral work, the PI showed that differentiated, secretory cell types, such as tuft cells, form in the pancreas in response to ADM (DelGiorno et al. Frontiers in Physiology, 2020). Tuft cells are solitary chemosensory cells with myriad roles in mediating inflammation. Combining RNA sequencing, electron microscopy (EM), and mouse models of disease, she identified a functional role for tuft cells, the first description in any model of tumorigenesis (DelGiorno et al. Gastroenterology, 2020). Using single cell RNA sequencing (scRNA-seq) and EM, she has identified the formation of several additional secretory cell populations in ADM of unknown function, which are now being studied in the DelGiorno laboratory. 3) Overview of future research program. We propose to continue our work on ADM by creating a workflow combining Single cell RNA-seq and EM Analysis (SEMA) to study epithelial heterogeneity in pancreatic injury with and without the genetic manipulation of identified cell types. Volumetric electron microscopy (3DEM) will be used as a substrate on which to map the information provided by scRNA-seq to reveal the spatial relationships between cells and infer the function of individual cell types through the study of their organellar content. Our approach leverages scanning electron microscopy (SEM) to conduct a variety of experiments on the same tissue including: [1] imaging of dehydrated whole-mount samples, [2] wide-field “chip mapping” of large areas of ultrathin sections, and [3] large-scale serial section approaches for 3DEM acquisition. Mapping is enhanced using correlative light and electron microscopy to assign molecular markers identified in scRNA-seq by immunofluorescence to cells with defining characteristics in SEM (e.g., nuclear shape, distribution of mitochondria, etc). Our 3DEM datasets are amenable to high-throughput image processing (e.g., segmentation, geometry processing) using machine learning and deep learning tool kits. Together, these tools provide a novel discovery workflow, which will be applied to projects throughout our laboratory aimed at evaluating cellular heterogeneity in ADM. Our goals for the next five years are to use this SEMA approach to build a multi-dimensional atlas of the cellular content and spatial relationships of the various emergent cell types in ADM. Mouse models lacking individual ADM cell subtypes will be used to assess phenotypic effects on cellular makeup, relative positioning, and disease progression. Together, these studies will provide invaluable insight into the role of ADM in tissue healing and regeneration in the pancreas.