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

摘要 成年哺乳动物骨髓(BM)中的造血干细胞(HSC)是一种罕见的多潜能细胞 移植后可再生并维持多系造血。考虑到稀有和有限的 骨髓中HSC的可获得性，将HSC从骨髓动员到外周血的策略显示出很好的效果 承诺提高造血重建的效率。另一方面，重组 HSC的能力在老年或炎症性侮辱后可能会受到损害。HSC本地化在唯一的 功能性的BM隔间被称为利基。它的关键组成部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CXCL12，其 受体CXCR4有助于将HSC保留在骨髓中。然而，内源活体的形态和动力学 HSC及其与BM利基成分的相互作用尚未得到很好的描述。我们有 建立了一种成年小鼠HSC的特异性荧光标记系统，并将其用于活体内 骨髓内HSC的成像。初步研究揭示了一种出人意料的动态形态和 HSC在稳态时的复杂运动。此外，我们能够可视化之间的交互 活的HSC和基质细胞。我们将应用这一系统来系统地研究活的HSC的行为 以及它们与活体动物的生态位之间的相互作用。该项目涉及实验室与 在基因操作和分析HSC(Reizis)、活体骨髓显微镜方面的专业知识 (Fooksman)和细胞行为的计算分析和建模(Krichevsky)。在目标1中，我们将 观察骨髓中活的HSC在稳态和炎症过程中的行为。在目标2中，我们将 描述HSC与其BM环境的相互作用，无论是在正常条件下还是之后 来自BM的动员。在目标3中，我们将分析HSC动力学的分子基础并将其关联起来 具有支持造血的HSC功能。拟议的研究将解决我们在 基本的HSC生物学知识，并对HSC的临床相关过程提供新的见解 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.