Effect of Erythropoietin on Cultured Human Leukocytes. ∗

Abstract

Phytohemagglutinin-stimulated cultures of mature human leukocytes produce a mitotically active cell which appears to be derived from lymphocytes (1,2). Since the lymphocyte has long been suspected of playing a role as stem cell(3,4), a primitive cell originating from lymphocyte precursors might be multi-potential. This possibility and evidence that the hemoglobin type can be altered in irradiated mice by transplantation of leukemoid leukocytes (5) prompted us to study the effect of erythropoietin on cultured human leukocytes.

Method. Human peripheral blood leukocytes were cultured in the presence of phyto-hemagglutinin according to the technique of Moorhead et al(6). Cultures were set up in duplicate in each experiment so that one culture could serve as a control for the eryth-ropoietin-treated culture. Erythropoietin was added in concentrations of .01, .05, 0.1, 0.5 and 2.5 units/ml culture medium at 0, 24 and 48 hours in separate experiments. At 66 hours colchicine was added to each culture and at 72 hours cells were harvested, exposed to hypotonic saline solution and fixed in methanol-acetic acid fixative. The cells were then resuspended and total cell counts determined. An aliquot of each culture was spread on a glass slide, air dried, stained with Orcein and studied for the per cent of cells in metaphase.

Results. Average total cell counts and per cent of cells in metaphase are shown in Table I. Erythropoietin appeared to have no significant effect on this cell system.

Comment. Erythropoietin has not shown a consistent stimulatory effect in all in vitro experiments. However, in those experiments in which it has been effective, results suggest that its major site of action is at the stem cell level where it induces erythroid differentiation (7, 8).