Generation of a temporal, spatial, and molecular map of in situ hematopoiesis

(请保存在Word中，不要保存为PDF) 在此输入文本，它是您的应用程序的新摘要信息。此部分不得超过30行文本。 可以说，从发育和干细胞生物学的角度来看，造血系统一直是研究得最好的组织。从历史上看，造血研究一直利用流式细胞术和细胞移植方法作为检测功能行为的金标准分析方法。虽然这些研究为造血提供了一种优雅的教科书观点，但我们在这里认为，我们对早期血液形成如何工作的了解在很大程度上是不完整的。这主要是由于使用传统分析和读数的局限性，这些分析和读数扰乱了正常的骨髓组织结构，忽视了细胞的解剖定位。因此，在该领域的许多研究中缺少的一个关键生物学成分是空间组织。基本的问题，如何时何地发生造血命运承诺，干细胞和成熟子细胞之间的确切细胞中间体是什么，以及细胞微环境(生态位)在造血过程中的身份和作用在很大程度上仍未解决。在这里，我们提出了一种跨学科的方法，基于最先进的活体显微镜操作，结合新的造血命运记者，使全面的研究造血完全在原位。使用新的双光子引导直接从骨髓中收集干细胞克隆，并对干细胞后代进行纵向实时成像分析，我们的目标是在单细胞分辨率下生成空间、时间和分子的造血地图。将采用互补的基于邻近的光标记方法来对稳态、患病和老龄化环境中的生态位的细胞组成进行编目。通过从组织破坏性方法转向依赖于对骨髓中单个造血细胞的观察、操作和分析的系统，我们希望对血液产生的细胞组织获得独特的见解。

(PLEASE KEEP IN WORD, DO NOT PDF) Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The hematopoietic system has been, arguably, the best well studied tissue from a developmental and stem cell biology perspective. Historically, the study of hematopoiesis has taken advantage of flow cytometry and cell transplantation approaches as the gold standard assays to examine functional behaviors. While these studies have provided an elegant textbook view of hematopoiesis, we argue here that our knowledge of how early blood formation works is largely incomplete. This is predominantly due to the limitations of utilizing traditional assays and readouts that disrupt the normal marrow tissue archicture and neglect the anatomical localization of cells. Thus, one key biological component missing in many studies in the field is that of spatial organization. Basic questions such as when and where hematopoietic fate commitment occurs, what the exact cellular intermediates are between a stem cell and mature daughter cells, and what the identity and role of the cellular microenvironement (niche) is in the hematopoietic process remain largely unsettled. Here, we propose an interdisciplinary approach based on state-of-the-art live microscopy manipulations in combination with novel hematopoietic fate reporters to enable a comprehensive study of hematopoiesis entirely in situ. Using novel two photon-guided collection of stem cell clones directly from the bone marrow, and longitudinal live-imaging analysis of stem cell progenies, we aim to generate a spatial, temporal and molecular map of hematopoiesis at single cell resolution. Complementary proximity-based photolabelling approaches will be taken to catalogue the cellular composition of the niche in the steady-sate, diseased, and aged settings. By moving away from tissue disruptive approaches to a system that relies on observation, manipulation, and analysis of single hematopoietic cells within the bone marrow, we hope to obtain unique insights into the cellular organization of blood production.