Characterization of primitive hematopoietic cells in normal human peripheral blood.
The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC medium. We now show that the number of clonogenic cells present in such reconstructed LTC after 5 weeks is linearly related to the input number of peripheral blood (PB) cells over a wide range of cell concentrations, thereby permitting the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults to be 2.9 +/- 0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming units-erythroid plus colony-forming units [CFU] granulocyte-macrophage plus CFU-granulocyte, erythroid, monocyte, megakaryocyte) and represents a frequency of LTC-IC relative to all nucleated cells that is approximately 100-fold lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC to be small (low forward light scatter and side scatter), CD34+, Rh-123dull, HLA-DR-, and 4-hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal marrow. Isolation of the light-density, T-cell-depleted, CD34+, and either HLA-DR(low) or Rh-123(dull) fraction of normal blood yielded a highly enriched population of cells that were 0.5% to 1% LTC-IC (approximately 1,500-fold enriched beyond the light-density, T-cell-depletion step), a purity comparable to the most enriched populations of human marrow LTC-IC reported to date. However, purification of PB LTC-IC on the basis of these properties did not allow them to be physically separated from a substantial proportion (> 30%) of the clonogenic cells in the same samples, in contrast to previous findings for LTC-IC and clonogenic cells in marrow. These studies show the presence in the blood of normal adults of a relatively small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties with marrow LTC-IC, a cell type thought to have in vivo reconstituting potential.(ABSTRACT TRUNCATED AT 400 WORDS)
Udomsakdi
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C
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Lansdorp
P M
PM
Hogge
D E
DE
Reid
D S
DS
Eaves
A C
AC
Eaves
C J
CJ
D000328:Adult; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D002452:Cell Count; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology; D015672:Erythroid Precursor Cells / Q000166:cytology; D005456:Fluorescent Dyes; D006098:Granulocytes / Q000166:cytology; D006684:HLA-DR Antigens / Q000032:analysis; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D008264:Macrophages / Q000166:cytology; D008533:Megakaryocytes / Q000166:cytology; D009000:Monocytes / Q000166:cytology; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism
D015703:Antigens, CD; D018952:Antigens, CD34; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens; D012235:Rhodamines; D020112:Rhodamine 123