(CANCER RESEARCH 46, 4357-4361, September 1986] Effects of Recombinant Human Tumor Necrosis Factor a, Recombinant Human 7- Interferon, and Prostaglandin E on Colony Formation of Human Hematopoietic Progenitor Cells Stimulated by Natural Human Pluripotent Colony- stimulating Factor, Pluripoietin a, and Recombinant Erythropoietin in Serum-free Cultures1 Li Lu,2 Karl Weite, Janice L. Gabrilove,3 Giao Hangoc, Edward Bruno, Ronald Hoffman, and Hal E. Broxmeyer Departments of Medicine (Hematology/Oncology) [L. L., G. H., E. B., R. H., H. E. B.J, Microbiology and Immunology [L. L., H. E. B.], the Walther Medical Research Institute, the Indiana Elks Cancer Research Center, Indiana University School of Medicine, Indianapolis, Indiana 46223, and Laboratories of Molecular Hematology {K. W.] and Developmental Hematopoiesis [J. L. G.], The Sloan Kettering Institute for Cancer Research, New York, New York 10021 ABSTRACT The influences of pure human pluripotent colony-stimulating factor, highly purified pluripoietin or, pure recombinant human tumor necrosis factor a, pure recombinant human -v-interferon, and natural prostaglandin Ki (PGEi) were evaluated on colony formation of multipotential and erythroid progenitor cells in the presence of recombinant erythropoietin and hemin and on colony formation of granulocyte-macrophage progeni tors in normal human marrow cultured in the presence or absence of serum. Serum was replaced by bovine serum albumin, iron-saturated transform!, cholesterol, and calcium chloride. Increasing concentrations of pluripotent colony-stimulating factor and pluripoietin a stimulated increasing numbers of colonies from nonadherent low-density T-lympho- cyte-depleted cells in the absence and presence of serum. Growth was usually greater in the presence of serum and on a unit basis pluripoietin a was more active than pluripotent colony-stimulating factor. Recombi- nant human tumor necrosis factor a and recombinant human 7-interferon suppressed colony formation colony forming unit-granulocyte-macro- phage, burst forming unit-erythroid, and colony forming unit-granulocyte- erythroid-macrophage-megakaryocyte; I'(,K, suppressed colony forma tion by colony-forming unit-granulocyte-macrophage, stimulated colony formation by burst forming unit-erythroid, and had no effects on colony formation by colony forming unit-granulocyte-erythroid-macrophage-me- gakaryocyte in both serum-containing and serum-free medium. The l'( ; I-, enhancing effects on erythroid colony formation required T-lymphocytes. Thus, results are similar using serum-containing and serum-free cultures of human bone marrow cells and serum-free defined culture medium can be used to study the mechanisms of action of purified natural and recombinant growth and suppressor molecules in vitro. INTRODUCTION The growth of multipotential, erythroid, and granulocyte- macrophage progenitor cells is influenced in vitro by specific stimulating and suppressing molecules (1-3). Some of the mol ecules implicated in the growth regulation of human hemato- poietic progenitor cells in vitro include PPO4 (4), PPO-a (5), Received 3/31/86; accepted 5/16/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1These studies were supported by USPHS Grants CA 36740 and CA 36464 to H. E. B. from the National Cancer Institute and by a grant from the Deutsche Forschungsgemeinschaft to G. H. 2To whom requests for reprints should be addressed, at Department of Medicine (Hematology/Oncology), the Walther Medical Research Institute, the Indiana Elks Cancer Research Center, Indiana University School of Medicine, 541 Clinical Drive, Indianapolis, IN 46223. ' Recipient of National Cancer Institute Clinical Investigator Award and a Junior Faculty Fellowship Award from the American Cancer Society. 4 The abbreviations used are: CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; BFU-E, burst-forming unit-erythroid; CFU-GM, colony-forming unit-granulocyte-macrophage; PPO, pluripotent col ony-stimulating factor; PPO-a, pluripoietin a; rEPO, recombinant erythropoietin; rHuTNF-a, recombinant human tumor necrosis factor a; r11u11N -:. recombinant human -Hnterferon; n, natural; PGE, prostaglandin E; FBS, fetal bovine serum; CM, conditioned medium; IMDM, Iscove's modified Dulbecco's medium. EPO (6), HuIFN-T (7-9), TNF (10, 11), and PGE (12-16). The complementary DNA and/or genes have been cloned and expressed for human EPO (17, 18), HuTNF (19-21), and HuIFN-7 (22). Studies evaluating the regulation of hematopoiesis in vitro have been performed mainly in the presence of serum, which contains complex mixtures of proteins and other defined and undefined molecules which vary between lots, and have included use of crude preparations of stimulating molecules. This makes it difficult to rule out potential influences of factors other than those one is attempting to evaluate. Culture conditions for the growth of hematopoietic cells under serum-free conditions have been reported (23-29) but these studies did not always use purified growth-stimulating molecules. With serum-free de fined culture conditions and purified regulatory factors it is now possible to more precisely evaluate the influence of specific regulatory molecules on the growth of hematopoietic progenitor cells in vitro. In this report we compare the effects of nPPO, nPPO-a, rEPO, rHuTNF-a, rHuIFN-f, and nPGE, on colony formation in vitro by hematopoietic progenitor cells present in low-density or nonadherent low-density T-lymphocyte-depleted normal hu man bone marrow grown either in the presence of FBS or in the presence of defined medium containing bovine serum al bumin, iron-saturated transferrin, cholesterol, and calcium chloride. MATERIALS AND METHODS Cells and Cell Separation Procedures Bone marrow cells were obtained by aspiration from the posterior iliac crest of healthy volunteers who had given informed consent. Unseparated nucleated huffy coat cells were obtained by centrifugation of whole marrow aspirates at 1SOOrpm for 10 min and aspiration of the WBC which overlay the RBC layer. Low-density cells (< 1.077 g/ ml) were obtained after separation on Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ) at 1500 rpm for 30 min, washed three times, and resuspended in IMDM (Gibco, Grand Island, NY) contain ing 10% FBS (Hyclone; Sterile Systems, Inc., Logan, UT). Cells were further separated into nonadherent and adherent cells after incubation on plastic tissue culture dishes (Falcon 3003; Falcon Plastics, Div., Becton Dickinson and Co., Rutherford, NJ) for 90 min at 37°Cunder 5% CO2. Nonadherent cells were collected by gently swirling the dishes and slowly removing the suspension cells. Nonadherent cells prepared in this manner routinely contained <2% a-naphthyl acetate esterase- positive cells. Nonadherent low-density cells were further separated into erythrocyte rosette-positive and negative populations as described elsewhere (15). The erythrocyte rosette positive fraction contained approximately 95% T-cells as determined using the OKTlla (anti- sheep RBC) pan T-cell antibody. The nonadherent low-density T- lymphocyte-depleted marrow cell fraction usually contained <5% 4357 Research. on January 26, 2016. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from