Detection of Functional Hematopoietic Stem Cell Niche Using Real-Time Imaging

Hematopoietic stem cell (HSC) niches play an essential role in regulating adult stem cell self-renewal and differentiation. HSC niches have only recently been identified based on genetic models that showed a parallel increase in the numbers of both HSCs and osteoblasts, or by static immunolabeling of putative HSCs and the adjacent locations. However, osteoblasts and vasculature were viewed as separate niches with distinctive roles, their interrelationships and interactions with HSCs in vivo remain largely undefined. To study the dynamic interaction between the HSC and its niche, we developed a method of ex vivo imaging stem cells (EVISC) using two-photon microscopy. This method combined the technology of real-time imaging and the functional feature that Flk2−LSK HSCs can be prospectively isolated with high purity and can home to their niche upon transplantation into irradiated recipient mice. Using EVISC in combination with immunoassaying, we found that transplanted HSCs tended to home to the endosteal region predominantly in the trabecular bone area (TBA) compared to compact bone area (CBA) under irradiated conditions, but were randomly distributed and unstable under non-irradiated conditions. Mechanistically, we found that the ratio of expression level of SDF-1 between TBA and CBA was significantly increased in response to irradiation. By monitoring individual HSC behavior using EVISC ranging from minutes to 16-hours, we found that a small portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their initial expansion as measured by flow assay and immunoassay. Our findings suggest that the endosteal region in the trabecular bone area formed a special zone, which normally maintains HSCs but promotes their expansion in response to irradiation. This zone includes both osteoblastic and vascular components and signals, which both underwent dynamic changes from homeostatic to stressed conditions.