An efficient method for single hematopoietic stem cell engraftment in mice based on cell-cycle dormancy of hematopoietic stem cells

Objective. To develop an efficient method for single hematopoietic stem cell (HSC) transplantation for high-level hematopoietic engraftment. Materials and Methods. 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. Results. 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 of <15 cells, often showed high-level multilineage engraftment, while clones consisting of >= 40 cells showed very low levels of engraftment. Daily observation of cell divisions of individual clones revealed that some HSCs are in the Go 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. Conclusions. 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. (C) 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.