Limiting-dilution analysis of the effects of colony-stimulating factors, phytohemagglutinin, and hydrocortisone on hematopoietic progenitor cell growth

Abstract

The effects of colony-stimulating factors (CSFs), phytohemagglutinin (PHA), and hydrocortisone on the growth of human bone marrow hematopoietic progenitor cells (granulocyte-macrophage; GM) were analyzed in a limiting-dilution assay (LDA). Both low-density bone marrow cells separated by discontinuous Percoll gradients and a T cell- depleted and progenitor-enriched cell fraction obtained by the combination of counterflow elutriation centrifugation and Percoll gradients were examined in LDA. GCT (monocytoid cell line-conditioned medium containing GM-CSF), human placenta-conditioned medium, bladder carcinoma cell line 5637-conditioned medium (containing GM- and G-CSF), and recombinant CSF (G-CSF) directly induced proliferation of progenitors with single-hit kinetics. In some instances, however, PHA- stimulated lymphocyte-conditioned medium (containing G- and GM-CSF) showed deviation from single-hit kinetics, which demonstrated the presence of factor(s) suppressive to progenitor growth. In a T cell- depleted, progenitor-enriched fraction, PHA alone was found to suppress progenitor growth at a level as low as 100 ng/mL. The addition of hydrocortisone (10(-6) mol/L) increased the progenitor frequency but suppressed progenitor growth at 10(-4) mol/L. LDA appears to be a valuable method for exploring mechanisms of factors regulating hematopoietic cell growth.

Activity of the ligand for c‐mpl, thrombopoietin, in early haemopoiesis

“…Limiting dilution assay. Limiting dilution analysis was performed, using a modification of the technique previously described (Taswell, 1981;Takaue et al, 1987). 100 ml of serum-free culture medium containing 4-100 cells/well together with designated growth factors were plated in 96well microplates (Nunc, Roskilde, Denmark) and incubated in the same manner as the methylcellulose clonal culture.…”

Impaired production of burst promoting activity by blood mononuclear cells from chronic uremic patients

“…MNC were separated in a 60% Percoll gradient (d = 1.077) as reported previously [7]. After centrifugation at 400g for 20 min, cells at the interface were harvested, washed twice with phosphate buffered solution (PBS), and resuspended in Iscove's modified Dulbecco's medium (IMDM) at the optimal concentration for each experiment.…”

Cell‐mediated suppression of human hematopoiesis: Evaluation by limiting‐dilution analysis of hematopoietic progenitors

“…Moreover, deviation from linearity in this assay allows analysis of helper or suppressor accessory functions on progenitor growth. We have found LDA to be useful in analyzing the regulatory effects of phytohenimaglutinin, hydrocortisone, and colony-stimulating factors [3] and of recombinant colony-stimulating factors [4]. In the present study, we have adapted LDA to investigate the mechanism of ccllmediated suppression of progenitor growth.…”