Hematopoietic stem cells do not engraft with absolute efficiencies.
Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here, we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition, SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore, through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether, our results indicate that even an extremely homogeneous HSC population, based on phenotype and dye efflux, cannot reconstitute mice at absolute efficiencies.
Camargo
Fernando D
FD
Program in Cell and Molecular Biology and the Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77098, USA.
Chambers
Stuart M
SM
Drew
Erin
E
McNagny
Kelly M
KM
Goodell
Margaret A
MA
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D017690:Cell Transplantation; D002478:Cells, Cultured; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018800:Thy-1 Antigens / Q000235:genetics / Q000502:physiology*
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D018800:Thy-1 Antigens; D017493:Leukocyte Common Antigens; C017807:bisbenzimide ethoxide trihydrochloride