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

1)背景、我们理解中的主要差距以及需要应对的重大挑战。作为对.的回应 损伤，胃肠道中分化的成体分泌细胞经历化生，或转化为 从一种细胞类型到另一种细胞类型。在胰腺，这被称为“腺泡到导管的化生”(ADM)，被认为是 在组织再生中的作用。我们实验室的目标是开发工具来理解 ADM，以鉴定出现的ADM细胞类型，并确定它们在 组织损伤。2)PI对最新进展的描述。在她博士后工作结束时，PI显示 在胰腺中形成的分化的、分泌细胞类型，如簇状细胞，以回应ADM(DelGiorno 等人的研究。生理学前沿，2020)。簇状细胞是一种孤立的化学感受性细胞，具有多种调节作用 发炎。结合RNA测序、电子显微镜(EM)和小鼠疾病模型，她 确定了丛生细胞的功能作用，这在肿瘤发生的任何模型中都是第一次描述(DelGiorno等人。 胃肠病学，2020)。使用单细胞RNA测序(scRNA-seq)和EM，她已经确定了 在功能未知的ADM中形成几个额外的分泌细胞群，目前正在 在德尔乔尔诺实验室学习。3)对未来研究计划的概述。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，是否存在已鉴定细胞类型的基因操作。 将使用体积电子显微镜(3DEM)作为衬底，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，推断单个细胞类型的功能 通过对它们细胞器内容物的研究。我们的方法利用扫描电子显微镜(SEM)来 在同一组织上进行各种实验，包括：[1]脱水整装样品的成像， [2]大面积超薄切片的广场“芯片测绘”，[3]大规模连续切片方法 用于3DEM收购。使用相关的光学和电子显微镜来指定分子，以增强作图 通过免疫荧光对扫描电子显微镜中具有明确特征的细胞鉴定的scRNA-seq中的标记(例如， 细胞核形状、线粒体分布等)。我们的3DEM数据集适用于高通量图像 使用机器学习和深度学习工具包进行处理(例如，分割、几何处理)。 这些工具共同提供了一个新颖的发现工作流，该工作流将应用于我们的 旨在评估ADM细胞异质性的实验室。我们未来五年的目标是利用这一点 SEMA方法，以建立一个多维地图集，其中包括 ADM中的各种突起细胞类型。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。加在一起，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.