Project 1 - Spleen

自古代首次发现脾以来，它一直被认为是一个神秘的器官。 时代。脾特异性地控制着异常红细胞(RBC)和颗粒的吞噬清除 物质，来自红细胞的铁储存，对循环抗原的初始免疫反应，以及胎儿造血。血样 过滤在解剖学上与红髓有关，同时检测到感染颗粒并引发 免疫反应是通过白髓中生发中心的形成而发生的。人体的显微解剖 然而，脾主要是从对啮齿动物的广泛研究中推断出来的。最近的研究由Dr。 Birte Steiniger(德国马尔堡大学)实际上已经指出，作为一个例子，井- 白髓和红髓之间有明显的B细胞室，称为边缘区。 人类的脾。因此，正常人脾的三维(3D)组织图的生成 无论是从科学角度还是从病理角度来看，都是及时的。我们的一个明显优势是覆盖面广 使用高水平质量控制采购和处理移植质量的人体器官的经验 措施(AIM I)。我们提出的目标是首先了解整体的一致性 以及使用磁共振成像对人的脾进行宏观解剖(AIM IIA)。然后我们设计了互动组织 处理(福尔马林固定石蜡包埋[FFPE]，最佳切割温度[OCT]包埋， 组织清除和扩张)和光学显微镜(随机光学重建显微镜[STORM]， 共聚焦、多光子、光片荧光显微镜[LSFM])管道，它将解决 脾的显微解剖从纳米到毫米的分辨率(AIM IIB-D)，与已知的叠加 脾生物标记物。分离/分散的脾细胞将与外周血单核细胞进行比较 从同一患者的血液(AIM III)中提取的(PBMC)和分离的免疫细胞亚群将受到RNA- SEQ(AIM IVB)。成像质量细胞术(IMC)将提供最终的联合注册 单个细胞的生物分子/生物标志物(AIM IVA)。同样，我们将使用多路传输的小分子 荧光原位杂交(FISH)和最终高通量多重错误稳健FISH (MerFish)将mRNA表达共同注册到同源细胞类型(AIM IVB)。数据来自RNA-Seq 单个细胞将进一步提供新的脾生物标志物，以反馈到AIMS IIB-D和IVA。有能力 根据所有提议的管道重建整体3D脾组织图基于通用文件格式 用于数据核心中描述的所有光学显微镜、IMC和FISH应用。有了所有这些 个人和合作的优势，我们准备完成正常人类脾的3D组织图 可以与所有HuBMAP、HIVE和TMC调查人员共享，最终，整个科学 社区。

The spleen has been considered an enigmatic and mysterious organ since its original discovery in the ancient era. The spleen specifically controls phagocytic removal of abnormal red blood cells (RBCs) and particulate matter, iron storage from RBCs, initial immune response to circulating antigens, and fetal hematopoiesis. Blood filtration is anatomically associated with the red pulp whilst detection of infectious particles and initiation of an immune response occurs via formation of germinal centers in the white pulp. The microanatomy of the human spleen, however, has been primarily extrapolated from extensive studies in rodents. Recent studies by Dr. Birte Steiniger (University of Marburg, Germany), have in fact indicated that as one example, the well- delineated B-cell compartment, known as the marginal zone, between white and red pulp is distinctly absent in the human spleen. The generation of a three dimensional (3D) tissue map of normal human spleen is therefore timely from both the scientific and pathologic perspectives. One of our obvious strengths is extensive experience with procurement and handling of transplant quality human organs using high level quality control measures (AIM I). Our proposed AIMs are designed to first obtain an understanding of the overall uniformity and macro-anatomy of the human spleen using MRI (AIM IIA). We have then designed interactive tissue handling (formalin-fixed paraffin embedded [FFPE], optimal cutting temperature [OCT] compound embedded, tissue clearing, and expansion) and optical microscopy (stochastic optical reconstruction microscopy [STORM], confocal, multiphoton, light sheet fluorescence microscopy [LSFM]) pipelines, which will resolve the microanatomy of the spleen from nanometer to millimeter in resolution (AIM IIB-D), superimposed with known spleen biomarkers. Isolated/dispersed splenocytes will be compared to peripheral blood mononuclear cells (PBMCs) from the same patient's blood (AIM III), and isolated immune cell subsets will be subjected to RNA- Seq (AIM IVB). Imaging mass cytometry (IMC) will provide the ultimate co-registration of biomolecules/biomarkers to individual cells (AIM IVA). Similarly, we will use multiplexed small molecule fluorescence in situ hybridization (FISH) and ultimately high throughput multiplexed error-robust FISH (MERFISH) to co-register mRNA expression to the cognate cell types (AIM IVB). Data from RNA-Seq of individual cells will further provide new splenic biomarkers to feed back to AIMs IIB-D and IVA. The ability to reconstruct an overall 3D spleen tissue map from all proposed pipelines is based on the common file format used for all optical microscopy, IMC and FISH applications as delineated in the Data Core. With all of these individual and cooperative strengths, we are poised to complete a 3D tissue map of the normal human spleen that can be shared with all HuBMAP, HIVE and TMC investigators and ultimately, the entire scientific community.