Interaction of Human Bone Marrow Fibroblasts With Megakaryocytes: Role of the c-kit Ligand zyxw By Hava Avraham, David T. Scadden, Sherry Chi, Virginia C. Broudy, Krisztina M. Zsebo, and Jerome E. Groopman Human kit ligand (KL), also known as stem cell factor (SCF), steel factor, or mast cell growth factor, is a recently identified hematopoietic growth factor whose receptor is the product of the c-kit proto-oncogene. Alternative splicing of the pre- mRNA of KL/SCF results in secreted and membrane-bound forms of the protein. We and others have recently shown that the c-kit gene product is expressed on human megakaryo- cytes and that soluble KL/SCF in combination with granulo- cyte-macrophage colony-stimulating factor, interleukin-3 (IL- 3). or IL-6 increased megakaryocyte progenitor colony formation (CFU-MEG) and stimulated mature megakaryo- cytes. Here we show that adhesion of human megakaryo- cytes to bone marrow stromal fibroblasts, which express the membrane-bound form of KL/SCF (mKL/SCF), is mediated HE zyxwvutsrqpo HUMAN kit ligand (KL), also known as stem cell T factor (SCF), mast cell growth factor, or steel factor has recently been identified as the product of the S1 The cell surface receptor for KL/SCF is the product of the c-kit proto-oncogene. Mice with mutations in the SI locus have abnormalities in hematopoiesis, germ cells, and mel- anocytes. Two forms of KL/SCF have been described that arise through differential RNA processing: a membrane- bound species and a soluble secreted form.3 Soluble KL/ SCF in vitro increased the growth response of progenitor cells to later-acting cytokines such as interleukin-3 (IL-3), erythropoietin, and granulocyte colony-stimulating factor (G-CSF).1-3 It has recently been observed that colony- forming unit-megakaryocyte progenitors (CFU-Meg) and mature human megakaryocytes proliferated in response to soluble recombinant KL/SCF.4,5 Cos cells expressing surface murine KL/SCF adhered with murine mast cells: suggesting that the membrane- bound molecule may have a different function from the soluble species. A study of murine mast cell adhesion to mesenchymal cells derived from Sl/Sl mice found that the extracellular domain of mKL/SCF was required to mediate this adhesion.’ The interaction of human megakaryocytes with adhesion molecules present on bone marrow stromal cells has not been defined. To explore the potential role of mKL/SCF as an adhesion structure for megakaryocytes and other c-kit expressing hematopoietic cells, we studied adhesive interac- tions of human megakaryocytic cells with bone marrow stromal fibroblasts. Our studies showed that adhesion of human megakaryo- cytes could be mediated in part via their c-kit receptor binding to membrane-associated KL/SCF expressed by bone marrow fibroblasts. Further, direct interaction be- tween stromal fibroblasts and megakaryocytes induced DNA synthesis as measured by thymidine incorporation in the megakaryocytic cells. The dual function of mKL/SCF as both an adhesion structure and a regulator of proliferation is novel among hematopoietic growth factors, and provides a model whereby hematopoietic cells, such as megakaryo- cytes, mast cells, and early hematopoietic progenitors may in part by the interaction between mKL/SCF and the c-kit protein. This interaction also results in marrow fibroblast- stimulated proliferation but not an increase in ploidy of megakaryocytes; when the two cell types were separated by a transoluble membrane, proliferation did not occur. Adhe- sion and proliferation of human megakaryocytesto an immor- talized murine stromal cell line zyxw SI/SI lacking the KL/SCF gene was impaired, whereas transfection of SI/SI cells with human mKL/SCF significantly increased both adhesion and proliferation. Marrow stromal fibroblast mKL/SCF may serve both as an adhesion structure and as a growth-potentiating factor for megakaryocytes in the bone marrow. zyx 0 1992 zyxwv by TheAmerican Society of Hematology. be anchored to stromal elements within the bone marrow microenvironment and positioned to respond to cytokines. MATERIALS AND METHODS zyx Cells. Human bone marrow was obtained by aspiration from the iliac crest of normal donors who gave informed consent in a protocol approved by the New England Deaconess Hospital Institutional Review Board. The marrow was aspirated into preser- vative-free heparin (Sigma Chemical zyx CO, St Louis, MO) and separated by centrifugation through Ficoll-Hypaque (Pharmacia, Piscataway, NJ) at 1,200g at room temperature for 30 minutes. After two washes with sterile zyxw lx phosphate-buffered saline (PBS), the cells were resuspended in Iscove’s modified Dulbecco’s me- dium (IMDM) with 20% fetal calf serum (FCS), penicillin/ streptomycin (PIS), and L-glutamine; seeded onto T-75 tissue culture flasks (Corning, Corning, NY); and incubated at 37°C in 5% COz. After 48 hours, the nonadherent cells were gently removed, and the adherent cells were refed with fresh media, The cells were refed with fresh medium every 3 days and trypsinized and split after 1 week or when confluent. Cells underwent three cycles of trypsinization and splitting before characterization or use in experimental protocols. Cultures of marrow stromal fibroblasts prepared by this method were uniformly positive for vimentin, negative for cytokeratin, negative for von Willebrand’s factor, and negative for nonspecific esterase by immunochemical or histochem- From the Division of HematologyIOncoIogy, Department of Medi- cine, New England Deaconess Hospital, Harvard Medical School, Boston, zyxwvu 11.24; the Division of Hematology, University of Washington, Seattle, WA; and Amgen, Inc, Thousand Oaks, CA. Submitted February 20,1992; accepted June 8, 1992. Supported in part by grants zyxwv from the National Institutes of Health HL33774, HL42112, HL43510, HL46668, AI-29847, CA5.5520, and the Department of Defense 17-90-CO106. Presented in part at the meeting of the American Society of Hematology, December 1991, Denver, CO. Address reprint requests to Jerome E. Groopman, MD, New England Deaconess Hospital, Division of HematologylOncologv, I IO Francis St, Suite 4A, Boston, 11.24 02215. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with I8 U.S.C. section I734 solely to indicate this fact. 0 1992 by The American Society of Hematology 0006-4971I92 l8007-0006$3.00/0 Blood, Vol80, No 7 (October I), 1992: pp 1679-1684 1679 Downloaded from http://ashpublications.org/blood/article-pdf/80/7/1679/608434/1679.pdf by guest on 26 February 2023