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

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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.