Quantitative analysis of the effect of colony-stimulating factors on human marrow progenitor growth in liquid-suspension cultures: application of limiting dilution assay.
Proliferation of human marrow progenitors in liquid cultures can be quantitated by limiting dilution clonal analysis (LDA) of progenitors in microwells. In this study, we have used LDA to study the effect of purified native or recombinant granulocyte colony-stimulating factors (G-CSFs) and recombinant granulocyte-macrophage CSF (GM-CSF) on progenitor growth. These results were compared to those of simultaneous cultures in methylcellulose. In LDA, single-hit kinetics were obtained with up to 500 U/ml of the recombinant preparation. In LDA with recombinant GM-CSF, progenitor growth conformed to single-hit kinetics from 100 to 2000 U/ml with maximum progenitor frequency at 500 U/ml. In simultaneous methylcellulose cultures with recombinant GM-CSF, colony formation reached a plateau at 100 U/ml. In LDA, purified native G-CSF was shown to support progenitor growth with single-hit kinetics at 100 U/ml, but at greater concentrations (greater than 150 U/ml), it suppressed progenitor growth with almost complete inhibition at a concentration of 200 U/ml. However, this dose-response effect was not observed in either simultaneous methylcellulose culture with G-CSF or in LDA with a purified recombinant preparation of the corresponding G-CSF. In methylcellulose cultures, colony formation reached a maximum at 100 U/ml and maintained a plateau up to 1000 U/ml. Hence, liquid culture allowed detection of contaminating suppressive activity in the G-CSF preparation that was not detected by methylcellulose assay. LDA may be more sensitive than methylcellulose culture for screening factors regulating human hematopoietic cell growth.
Watanabe
T
T
Department of Pediatrics, University Hospital of Tokushima, Japan.
Takaue
Y
Y
Kawano
Y
Y
Koyama
T
T
Suzue
T
T
Abe
T
T
Satoh
J
J
Shimokawa
T
T
Iishi
Y
Y
Mizuguchi
T
T
D001853:Bone Marrow; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000302:isolation & purification / Q000494:pharmacology*; D016179:Granulocyte Colony-Stimulating Factor; D006410:Hematopoiesis / Q000187:drug effects*; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008747:Methylcellulose; D011994:Recombinant Proteins / Q000494:pharmacology; D013535:Suspensions
D003115:Colony-Stimulating Factors; D011994:Recombinant Proteins; D013535:Suspensions; D016179:Granulocyte Colony-Stimulating Factor; D008747:Methylcellulose