Monoclonal antibody BB9 raised against bone marrow stromal cells identifies a cell-surface glycoprotein expressed by primitive human hemopoietic progenitors

Objective. The identification of cell-surface antigens whose expression is limited to primitive hematopoietic progenitor cells (HPC) is of major value in the identification, isolation, and characterization of candidate stem c ells in human hemopoietic tissues. Based on the observation that bone marro w stromal cells and primitive HPC share several cell-surface antigens, we s ought to generate monoclonal antibodies to HPC by immunization with culture d human stromal cells. Methods. BALB/c mouse were immunized with human bone marrow (BM)-derived st romal cells. Splenocytes isolated from immunized mice were fused with the N S-1 murine myeloma cell line and resulting hybridomas selected in HAT mediu m, then screened for reactivity against stromal cells, peripheral blood (PB ), and BM cells. Results. A monoclonal antibody (MAb), BB9, was identified based on its bind ing to stromal cells, a minor subpopulation of mononuclear cells in adult h uman BM, and corresponding lack of reactivity with leukocytes in PB. BB9 bo und to a minor subpopulation of BM CD34(+) cells characterized by high-leve l CD34 antigen and Thy-1 expression, low-absent expression of CD38, low ret ention of Rhodamine 123, and quiescent cycle status as evidenced by lack of labeling with Ki67. CD34(+)BB9(+) cells, in contrast to CD34(+)BB9(-) cell s, demonstrated a capacity to sustain hematopoiesis in pre-CFU culture stim ulated by the combination of IL-3, IL-6, G-CSF, and SCF. BB9 also demonstra ted binding to CD34(+) cells from mobilized PB. Conclusion. Collectively, these data therefore demonstrate that MAb BB9 ide ntifies an antigen, which is selectively expressed by hierarchically primit ive human HPC and also by stromal cells. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.