Combined measurement of growth and differentiation in suspension cultures of purified human CD34-positive cells enables a detailed analysis of myelopoiesis.
In this study we have made a detailed analysis of growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte colony-stimulating factor [G-CSF], and macrophage colony-stimulating factor [M-CSF])-induced proliferation and differentiation of highly purified CD34+ committed human myeloid progenitor cells in suspension cultures. The results were compared with colony formation in semisolid medium. Proliferation in suspension cultures was determined by means of incorporation of [3H]thymidine, differentiation by flow cytometric immunophenotyping using a panel of monoclonal antibodies against monomyeloid antigens, and by morphology. A good correlation was found between the number of granulocyte-macrophage colony-forming units (CFU-GM) in semisolid medium and [3H]thymidine incorporation in suspension (r = 0.82), both assessed at day 11. Moreover, the frequency of proliferating cells as determined in suspension cultures by limiting dilution analysis was similar to the frequencies of CFU-GM as measured in semisolid medium. Studies on GM-CSF- and G-CSF-induced cell-growth kinetics revealed distinct proliferation patterns. Immunophenotypically the subsequent induction of the mature granulocytic antigens CD15 and CD67 was observed to be accompanied by a gradual loss of the HLA-DR antigen, whereas little monocytic differentiation was observed. M-CSF, although inducing no colony formation of CD34+ cells and minimal proliferation in suspension, induced monocytic differentiation, demonstrated by the expression of HLA-DR, CD14, and CD36 in the absence of CD15 and CD67. The observed immunophenotypical profiles were confirmed by the results of cytological characterization. Thus, the combined measurement of growth factor-induced proliferation and differentiation of progenitor cells in suspension cultures can be a useful alternative for the CFU-GM assay. Moreover, because small numbers of cells are required, it allows for detailed studies on cell-growth kinetics and developmental stages within the granulocytic and monocytic lineages.
Kerst
J M
JM
Department of Immunohematology, Central Laboratory of the Red Cross Blood Transfusion Service, Amsterdam, The Netherlands.
Slaper-Cortenbach
I C
IC
von dem Borne
A E
AE
van der Schoot
C E
CE
van Oers
R H
RH
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D001853:Bone Marrow / Q000276:immunology / Q000378:metabolism; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006410:Hematopoiesis / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018952:Antigens, CD34; D014316:Tritium; D016179:Granulocyte Colony-Stimulating Factor; D016173:Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D013936:Thymidine