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.


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 > or =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.


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.
Abe
Takanori
T
Department of Pathology and Laboratory Medicine, Medical University of South Carolina and Research Service, Department of Veterans Affairs Medical Center, Charleston, 29401-5799, USA.
Masuya
Masahiro
M
Ogawa
Makio
M
D000818:Animals; D002453:Cell Cycle; D005434:Flow Cytometry; D006085:Graft Survival; D049452:Green Fluorescent Proteins / Q000737:chemistry; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins