Intravital analysis of hematopoietic stem cells in their bone marrow niche - Resubmission - 1

摘要 成体哺乳动物骨髓（BM）中的造血干细胞（HSC）是罕见的多能细胞， 在移植后可以再生和维持多系造血。考虑到稀有和有限的 尽管骨髓中HSC的可用性，从骨髓“动员”HSC到外周血的策略显示出很大的优势， 有望提高造血重建效率。另一方面， HSC的能力可在老年或炎症损伤后受损。HSC位于一个独特的 功能性BM隔间称为壁龛。其关键成分是血管周围基质细胞网络 表达HSC生长因子干细胞因子（SCF或Kit配体）和趋化因子CXCL 12，其 受体CXCR4有助于将HSC保留在BM中。然而，内源性肝细胞的形态和动力学 HSC及其与BM生态位组分的相互作用尚未得到很好的表征。我们有 开发了一种用于成年小鼠HSC的特异性荧光标记的系统，并将其用于活体内 BM内HSC的成像。初步研究揭示了一种出乎意料的动态形态， 稳定状态下HSC的复合运动。此外，我们还可以看到 活的HSC和基质细胞。我们将应用这一系统对活HSC的行为进行系统的研究 以及它们与活体动物生态位的相互作用。该项目涉及实验室之间的合作， 在HSC（Reizis）的遗传操作和分析方面的专业知识，BM的活体显微镜检查 （Fooksman）和细胞行为的计算分析和建模（Krichevsky）。在目标1中，我们 可视化稳态和炎症期间BM中活HSC的行为。在目标2中，我们将 表征HSC与其BM环境的相互作用，无论是在正常条件下还是在 从BM开始移动。在目标3中，我们将分析HSC动力学的分子基础，并将其与 造血干细胞支持造血功能。拟议的研究将解决我们的主要差距， 基础HSC生物学知识，并提供对HSC临床相关过程的新见解 动员。

ABSTRACT Hematopoietic stem cells (HSC) in the adult mammalian bone marrow (BM) are rare multipotent cells that can regenerate and sustain multilineage hematopoiesis upon transplantation. Given the rarity and limited availability of HSC in the BM, strategies to "mobilize" HSC from the BM to the peripheral blood show great promise to enhance the efficiency of hematopoietic reconstitution. On the other hand, the reconstitution capacity of HSC can be impaired in old age or following inflammatory insults. HSC are localized in a unique functional BM compartment called the niche. Its key component is a network of perivascular stromal cells expressing the HSC growth factor stem cell factor (SCF or Kit ligand) and chemokine CXCL12, whose receptor CXCR4 helps retain HSC in the BM. However, the morphology and dynamics of endogenous live HSC and their interactions with the BM niche components have not been well characterized. We have developed a system for specific fluorescent labeling of adult murine HSC, and adapted it for intravital imaging of HSC within the BM. Preliminary studies revealed an unexpectedly dynamic morphology and complex motility of HSC in the steady state. Furthermore, we were able to visualize interactions between live HSC and the stromal cells. We will apply this system to systematically study the behavior of live HSC and their interaction with the niche in live animals. The project involves a collaboration between labs with the expertise in genetic manipulation and analysis of HSC (Reizis), intravital microscopy of the BM (Fooksman) and computational analysis and modeling of cell behavior (Krichevsky). In Aim 1, we will visualize the behavior of live HSC in the BM in the steady state and during inflammation. In Aim 2, we will characterize the interaction of HSC with their BM environment, both in normal conditions and after mobilization from the BM. In Aim 3, we will analyze the molecular basis of HSC dynamics and correlate it with HSC function in supporting hematopoiesis. The proposed studies would address major gaps in our knowledge of basic HSC biology, and provide novel insights into the clinically relevant process of HSC mobilization.