An Efficient Method for Single Hematopoietic Stem Cell (HSC) Engraftment in Mice Based on Cell Cycle Dormancy of HSCs

To develop an efficient method for single hematopoietic stem cell (HSC) transplantation for high-level hematopoietic engraftment.We combined single-cell sorting with short-term culture of putative HSCs. Mouse bone marrow cells that had been highly enriched for HSCs were individually deposited into a 96-well culture plate and incubated in the presence of mouse c-kit ligand and either mouse interleukin-11 or human recombinant granulocyte colony-stimulating factor. One week later, the resulting clones of cells were individually transplanted into lethally irradiated recipients. We also carried out time-course analysis of proliferation of the individual clones. Finally, we used micromanipulation of the paired progenies of the single cells and studied self-renewal and differentiation potentials of HSCs again in combination with transplantation.There was a correlation between clone size at day 7 of culture and engraftment at 2 months post-transplantation. Small clones, such as those consisting of15 cells, often showed high-level multilineage engraftment, while clones consisting ofor =40 cells showed very low levels of engraftment. Daily observation of cell divisions of individual clones revealed that some HSCs are in the G(0) state for as long as 1 week, despite the presence of permissive cytokines. Studies using micromanipulation of paired progenies documented the ability of an HSC to generate two HSCs, as well as asymmetric cell divisions.Single-cell sorting combined with short-term culture of individual putative HSCs provides an efficient method for single HSC transplantation. Analyses of the kinetics of individual HSCs provided direct evidence for HSC cell-cycle dormancy, self-renewal, and expansion.