Human erythropoiesis in vitro and the source of burst-promoting activity in a serum-free system.

Bovine serum albumin (BSA) can markedly increase the number of size of erythropoietic bursts produced by mononuclear cells from human bone marrow and peripheral blood, and reduce the threshold amount of erythropoietin (Epo) required for initial burst formation. The purpose of this study was to determine a possible burst-promoting activity (BPA) of BSA. The experiments were performed in a miniaturized agar system, in which the addition of sheep Epo to cultures with or without BSA was delayed for five days. The results obtained have shown that, with or without BSA, Epo deprivation of up to five days (an epoprival state) did not markedly decrease the number of bursts produced by unfractionated peripheral mononuclear cells compared to the number produced in the presence of Epo from the beginning of culture. Similar results were found whether the fetal calf serum (FCS) concentration was 15% or 2%. The preservation of potential BFU-e formation during the epoprival state has therefore been attributed to the ability of T-lymphocytes and/or monocytes to supply BPA. In order to reduce the endogenous amount of BPA, a nonadherent, E-rosette-negative cell fraction was cultured in the presence of Epo, with or without BSA, in serum-free medium containing transferrin (TF). Under these conditions, an equal number of bursts was obtained in FCS and in serum-free medium containing Epo, BSA and TF, whereas no BFU-e growth was found in the presence of Epo and TF, but without BSA. If Epo was withheld for up to five days, the capacity to form erythroid colonies was still retained by the monocyte- and T-lymphocyte-depleted cell fraction in the continuous presence of BSA. However, BPA could not be detected in the BSA. This observation was further supported by experiments in serum-free medium using human recombinant Epo, in which no BFU-e colony formation could be detected in the presence of BSA. From our investigations carried out at limited cell density and in serum-free medium, it could be concluded that the crude Epo preparation was the source of BPA.