Searching for hematopoietic stem cells. II. The heterogeneity of Thy-1.1(lo)Lin(-/lo)Sca-1+ mouse hematopoietic stem cells separated by counterflow centrifugal elutriation.
Mouse hematopoietic stem cells (HSCs) are highly enriched in the rare (approximately 0.05%) Thy-1.1(lo)Lin(-/lo)Sca-1+ fraction of hematopoietic tissues. It has been demonstrated that Thy-1.1(lo)Lin(-/lo)Sca-1+ cells are the only HSCs in C57BL/Ka-Thy-1.1 bone marrow. In this study, we separated C57/Ka-Thy-1.1 bone marrow cells by counterflow centrifugal elutriation (CCE) into four fractions and characterized Thy-1.1(lo)Lin(-/lo)Sca-1+ cells in each eluted fraction. The peak number of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells was highly enriched in one eluted fraction, which was also highly enriched for day-12 to -13 CFU-S. Activities for day-13 CFU-S, radioprotection, and long-term multilineage reconstitution correlated with, and could be generally predicted by determining, the frequency of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells in a given eluted fraction. However, the fraction that was highly enriched for blast cells and contained a low frequency of Thy-1.1(lo)Lin(-/lo)Sca-1+ cells, only provided short-term but not long-term radioprotection, with a predicted cell number (100 cells) that should have protected > or = 95% (PD95) of hosts. Still, when 300 Thy-1.1(lo)Lin(-/lo)Sca-1+ cells from this fraction (3 X PD95 for unfractionated HSCs) were injected, mice were radioprotected and donor cells provided long-term multilineage reconstitution. We propose that such blast cells may contain two Thy-1.1(lo)Lin(-/lo)Sca-1+ subsets, one providing short-term and the other long-term multilineage sustained hematopoiesis, the latter presumably due to HSC self-renewal.
Uchida
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Department of Pathology and Developmental Biology, Stanford University School of Medicine, CA 94305, USA.
Jerabek
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Weissman
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IL
D000818:Animals; D000950:Antigens, Ly / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis; D001854:Bone Marrow Cells; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods*; D002498:Centrifugation; D016238:Cytapheresis / Q000379:methods*; D005260:Female; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D016130:Immunophenotyping; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D011828:Radiation Chimera; D018800:Thy-1 Antigens / Q000032:analysis*
D000950:Antigens, Ly; D000954:Antigens, Surface; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D018800:Thy-1 Antigens