Isolation of quiescent murine hematopoietic stem cells by homing properties

A major challenge facing investigators working in the field of hematopoietic stem cell (HSC) biology has been to develop a strategy to purify rare primitive HSCs from bone marrow. Several methods have been available including the commonly used technique of isolating HSCs based on a specific cell-surface phenotype. As surface marker expression is dynamic and may fluctuate depending on the proliferative or activation state of the cell, our laboratory has established a unique functional in vivo assay (the 2-day homing assay) to isolate murine HSCs. This protocol selects for HSCs on the basis of their ability to home to bone marrow and yields a population that can reconstitute the murine hematopoietic system with the transplantation of a single cell. In contrast to other methods that use specific cell-surface antigens to acquire HSCs, our functional assay aids in obtaining a primitive HSC that exhibits both hematopoietic and epithelial engraftment capabilities. The 2-day homing protocol involves harvesting whole bone marrow and performing a physical separation method (elutriation) to acquire a fraction of small-sized cells (fraction 25). Fraction 25 cells are then depleted of later progenitors and differentiated hematopoietic cells, labeled with a fluorescent tracking dye and transplanted into lethally irradiated recipient mice. Two days after transplantation, the bone marrow is harvested from the primary recipient, and HSCs that have homed to the bone marrow are collected by fluorescence-activated cell sorting. In addition to the traditional 2-day homing protocol, we have included in this chapter our recently developed method of using density gradient centrifugation to replace the elutriation step that also selects for a primitive HSC.