Intravital Imaging of Young and Aged Stem Cells in the Marrow of Long Bones: Visualizing Mammalian Stem Cell Behavior in Real-Time in Vivo

Hematopoiesis, the process in which blood cells are generated from hematopoietic stem and progenitor cells (HSPCs) is primarily confined to the bone cavities. The interactions of hematopoietic cells with stroma cells forming niches inside the bone cavities are central to hematopoiesis, as these regulate cell proliferation, self-renewal and differentiation. Hematopoietic cell/stroma interactions have thus been, in analogy to the immunological synapse, named stem/progenitor cell synapses. So far, visualization of the behavior of somatic stem and progenitor cells in an undisturbed in vivo environment has not been reported for the mammalian system and consequently, the cellular dynamics of stem, progenitor and differentiated cells in vivo are only poorly defined. We developed and performed intravital time-lapse 2-photon microscopy in the marrow of the long bones (tibia) of mice to study the behavior and dynamics of differentiated hematopoietic cells as well as HPCs and HSCs in close vicinity to the endosteum in vivo over time. We demonstrate that HPCs as well as HSCs reside in close vicinity to the endosteum, further supporting the notion of an endosteal stem cell niche, and that they are, in contrast to differentiated macrophages and dendritic cells, solitary and immobile. Both HPCs and HSCs occupy distinct positions relative to the endosteum and show cell protrusion movement consistent with an active stem/progenitor cell synapse. Lastly, we report that aged HSCs show increased protrusion movement and localize more distantly to the endosteum compared to young HSCs. In addition, aged HSCs present with reduced adhesion to stroma as well as reduced polarity upon adhesion in vitro, implying a connection between altered stem cell dynamics in vivo and stem cell aging. The intravital imaging technology developed might establish a basis for further delineating additional important questions in stem cell biology like cellular mechanisms of hematopoietic stem cell self-renewal and differentiation in the context of the stroma/niche in vivo.