Detection and quantitation of long-term culture-initiating cells in normal human peripheral blood.
The objective of this study was to utilize the Dexter long-term culture system to characterize and quantitate early hematopoietic cells in normal human blood. Peripheral blood mononuclear cells were depleted of T cells. B cells, and monocytes, yielding a population (null cells) highly enriched for hematopoietic precursors. One to 2 x 10(7) null cells containing an average of 5.4 x 10(4) granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) were cultured on irradiated allogeneic marrow stromal layers in T-25 flasks. Nonadherent progenitors were detected for an average of 6.8 +/- 0.7 (standard error of the mean) weeks and exceeded 1 x 10(4) per flask for 4 weeks. Null cells were treated with 5-fluorouracil (5-FU) to determine whether long-term growth was due to survival of CFU-GM or to maturation of more primitive precursors. When 5-FU treatment killed greater than 99% of CFU-GM, an increase in progenitors was observed after 12 days of cultivation with stroma. CFU-GM cloning efficiency of 5-FU-treated cells peaked after 24 days of incubation, whereas controls peaked after 7 days. The total number of nonadherent CFU-CM in 5-FU-treated and control cultures converged during the 5-8 weeks of incubation. The frequency of long-term culture-initiating cells (LTCIC) in peripheral blood was determined by limiting dilution analysis in micro-stromal cultures. In two donors, the frequencies of LTCIC were 6.3 and 19.6 per 1 x 10(5) null cells. The CFU-GM:LTCIC ratios were 12.6:1 and 12.8:1. We conclude that normal peripheral blood contains primitive hematopoietic precursors demonstrable as 5-FU-resistant precursors of CFU-GM or as LTCIC. The frequency of the latter is 13-fold lower than day-14 CFU-GM.
Dooley
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American Red Cross, Jerome Holland Laboratory for Biomedical Sciences, Rockville, Maryland.
Law
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D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D005472:Fluorouracil / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects; D013997:Time Factors
D005472:Fluorouracil