Characterization of stroma-adherent colony-forming cells: a clonogenic assay for early hemopoietic cells?
Hemopoietic cells that adhered to preformed selected marrow stromal cell layers were characterized on the basis of progenitor cell production capacity, colony-formation kinetics and forward light scatter (FLS) properties. It was shown that early hemopoietic cells attached to the stromal layers within 2 hours of incubation, and were responsible for the initial production of the more differentiated granulocyte-macrophage colony-forming cells (day 14 GM-CFC) in long-term cultures (LTC). In a clonogenic assay system, hemopoietic cells that adhere to stromal layers can be detected by the formation of small colonies of blast-like cells and are designated as stroma-adherent CFC. Cell fractionation on the basis of FLS and counting colonies on days 5, 14 and 21 revealed that there was a succession of colony formation, indicating that the stroma-adherent CFC consisted of subpopulations with different lag-phases before initiation of proliferation. Day 5, day 14 and day 21 stroma-adherent CFC were shown to have a high, intermediate and low FLS, respectively. The cells that produced GM-CFC by day 21 showed FLS properties similar to those of day 21 stroma-adherent CFC, suggesting a correlation between day 21 stroma-adherent CFC and CFC-producing cells in LTC. The CFC present on day 21 required the synergistic action of GM-CSF+IL-3 + stem cell factor (SCF) for optimal proliferation. The prolonged lag-phase, the low FLS and the multifactor-responsive progeny are properties similar to those reported for other early cells, and it is proposed that day 21 stroma-adherent CFC represent an early hemopoietic cell type whereas day 5 stroma-adherent CFC represent a more mature stage of differentiation.
Tucker
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Department of Clinical Haematology & Oncology, Royal Children's Hospital, Melbourne, Australia.
Bol
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Kannourakis
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D001853:Bone Marrow / Q000502:physiology; D001854:Bone Marrow Cells; D002448:Cell Adhesion / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D013997:Time Factors
D007377:Interleukin-3; D016178:Granulocyte-Macrophage Colony-Stimulating Factor