RAS oncogene suppression induces apoptosis followed by more differentiated and less myelosuppressive disease upon relapse of acute myeloid leukemia

To study the oncogenic role of the NRAS oncogene (NRAS(G12V)) in the context of acute myeloid leukemia (AML), we used a Vav promoter-tetracycline transactivator (Vav-tTA)-driven repressible TRE-NRAS(G12V) transgene system in Mll-AF9 knock-in mice developing AML. Conditional repression of NRAS(G12V) expression greatly reduced peripheral white blood cell (WBC) counts in leukemia recipient mice and induced apoptosis in the transplanted AML cells correlated with reduced Ras/Erk signaling. After marked decrease of AML blast cells, myeloproliferative disease (MPD)-like AML relapsed characterized by cells that did not express NRAS(G12V). In comparison with primary AML, the MPD-like AML showed significantly reduced aggressiveness, reduced myelosuppression, and a more differentiated phenotype. We conclude that, in AML induced by an Mll-AF9 transgene, NRAS(G12V) expression contributes to acute leukemia maintenance by suppressing apoptosis and reducing differentiation of leukemia cells. Moreover, NRAS(G12V) oncogene has a cell nonautonomous role in suppressing erythropoiesis that results in the MPD-like AML show significantly reduced ability to induce anemia. Our results imply that targeting NRAS or RAS oncogene-activated pathways is a good therapeutic strategy for AML and attenuating aggressiveness of relapsed AML.     

Oncogenic NRAS rapidly and efficiently induces CMML- and AML-like diseases in mice

Activating mutations in RAS, predominantly NRAS, are common in myeloid malignancies. Previous studies in animal models have shown that oncogenic NRAS is unable to induce myeloid malignancies effectively, and it was suggested that oncogenic NRAS might only act as a secondary mutation in leukemogenesis. In this study, we examined the leukemogenicity of NRAS using an improved mouse bone marrow transduction and transplantation model. We found that oncogenic NRAS rapidly and efficiently induced chronic myelomonocytic leukemia (CMML)- or acute myeloid leukemia (AML)- like disease in mice, indicating that mutated NRAS can function as an initiating oncogene in the induction of myeloid malignancies. In addition to CMML and AML, we found that NRAS induced mastocytosis in mice. This result indicates that activation of the RAS pathway also plays an important role in the pathogenesis of mastocytosis. The mouse model for NRAS leukemogenesis established here provides a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing relevant therapies.     

Inhibiting the palmitoylation/depalmitoylation cycle selectively reduces the growth of hematopoietic cells expressing oncogenic Nras

The palmitoylation/depalmitoylation cycle of posttranslational processing is a potential therapeutic target for selectively inhibiting the growth of hematologic cancers with somatic NRAS mutations. To investigate this question at the single-cell level, we constructed murine stem cell virus vectors and assayed the growth of myeloid progenitors. Whereas cells expressing oncogenic N-Ras(G12D) formed cytokine-independent colonies and were hypersensitive to GM-CSF, mutations within the N-Ras hypervariable region induced N-Ras mislocalization and attenuated aberrant progenitor growth. Exposing transduced hematopoietic cells and bone marrow from Nras and Kras mutant mice to the acyl protein thioesterase inhibitor palmostatin B had similar effects on protein localization and colony growth. Importantly, palmostatin B-mediated inhibition was selective for Nras mutant cells, and we mapped this activity to the hypervariable region. These data support the clinical development of depalmitoylation inhibitors as a novel class of rational therapeutics in hematologic malignancies with NRAS mutations.     

Fucoidan ingestion increases the expression of CXCR4 on human CD34+ cells

OBJECTIVE: Transplantation of hematopoietic progenitor stem cells (HPC) is an important treatment modality for a variety of neoplastic diseases. HPC collection for transplantation with granulocyte colony-stimulating factor may be unsuccessful in patients who have received prior chemotherapy or for other reasons. Methods to improve mobilization of HPCs are required. Disruption of the interaction between the cell surface receptor CXCR4 and its ligand stromal derived factor-1 (SDF-1) is a mechanism for HPC release from the bone marrow into the peripheral blood (PB). METHODS: We carried out a clinical trial to evaluate the effects of ingestion of a fucoidan, galactofucan sulfate (a putative HPC mobilizing agent) on circulating CD34(+) cells, CXCR4 expression, and levels of SDF-1, interferon gamma (IFN-gamma) and interleukin 12. RESULTS: Following ingestion of fucoidan, CD34(+) cells increased significantly in the PB from 1.64 to 1.84 cells/microL after 4 days. The proportion of CD34(+) cells that expressed CXCR4 increased from 45 to 90% after 12 days, the plasma level of SDF-1 increased from 1978 to 2010 pg/mL, and IFN-gamma level increased from 9.04 to 9.89 pg/mL. CONCLUSION: Oral fucoidan significantly amplified the CXCR4(+) HPC population. The ability to mobilize HPC using sulfated polysaccharides and mobilize more HPC with high levels of CXCR4 could be clinically valuable.     

Mutant N-ras preferentially drives human CD34+ hematopoietic progenitor cells into myeloid differentiation and proliferation both in vitro and in the NOD/SCID mouse

OBJECTIVES: Ras oncogene mutations are the most frequently observed genetic abnormality (20-40% of patients) in acute myeloid leukemia (AML), and in the preleukemic conditions myelodysplastic syndrome (MDS) and myeloproliferative disorder (MPD). We have previously shown that mutant N-ras (N-rasm) can induce myeloproliferative disorders and apoptosis in a murine reconstitution system. In the present study we investigated the effect of N-rasm in human primary hematopoietic progenitor cells (HPC). METHODS: Cord blood CD34+ hematopoietic progenitor cells (HPC) were transduced with retroviral vectors containing green fluorescence protein (GFP) alone, or in combination with N-rasm. Cells were then cultured in vitro with a cytokine supplement or cocultured with murine stroma MS-5 cells. The in vivo behavior of transduced cells was examined in the NOD/SCID mouse model. RESULTS: N-rasm-transduced cells exhibited greater proliferative capacity; a higher frequency of granulocyte-macrophage colony-forming unit (CFU-GM); and an increase in myelomonocytic lineage cells with a concomitant decrease in lymphoid and erythroid cells. Analysis of transduced HPC in NOD/SCID mice revealed higher bone marrow engraftment by N-rasm HPC and increased numbers of myeloid lineage cells. CONCLUSIONS: The results demonstrate that N-rasm in HPC induces myeloproliferation both in vitro and in the NOD/SCID mouse model as a primary event that does not appear to be dependent on cooperating transforming events.     

GPIIb (CD41) integrin is expressed on mast cells and influences their adhesion properties

OBJECTIVES: GPIIb integrin expression has been found on platelets and megakaryocytes, and more recently on immature hematopoietic progenitors. We set out to investigate expression of GPIIb in other hematopoietic cell lineages and, having detected it on mast cells, aimed to determine what possible role it might perform. METHODS: We have made use of cultured human and murine bone marrow mast cells (BMMC) in order to characterize the expression of GPIIb. Further, BMMC cultures from wild type and GPIIb deficient (gpIIb-/-) mice were used for comparison of the adhesive properties mediated by this receptor. Finally, peritoneal mast cells were analyzed from both wild type and (gpIIb-/-) mice. RESULTS: We demonstrate expression of GPIIb on cultured BMMC. Using cells derived from mice homozygous for a null allele of gbIIb we show that the absence of GPIIb has no effect on mast cells with respect to a number of measures of cell growth and differentiation. However, loss of GPIIb on BMMC results in an increase in surface expression of aV integrin, the alternative partner of GPIIIa. CONCLUSION: The results in this study demonstrate that GPIIb is expressed in human and murine mast cells. A function for GPIIb on mast cells is suggested by the altered adhesion of gbIIb-/- BMMC to fibronectin- and vitronectin-coated surfaces. Moreover, comparison of mast cells from the peritoneal cavity of wild type and gbIIb-/- mice indicates that GPIIb could influence the in vivo differentiation or homing of tissue mast cells.     

Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit

Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in cells of adult mice, including hematopoietic precursors, caused severe mastocytosis with 100% penetrance at young age frequently associated with additional hematopoietic (mostly B lineage-derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain-of-function mutation.     

G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow

Accumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)-induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF-induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.     

The origin of neoplastic mast cells in systemic mastocytosis with AML1/ETO-positive acute myeloid leukemia

OBJECTIVE: Systemic mastocytosis with associated clonal hematological non-mast cell lineage disease (SM-AHNMD) is a distinct entity that was defined by World Health Organization. Systemic mastocytosis with acute myeloid leukemia (AML) is frequently seen among SM-AHNMD. However, the pathogenesis or origin of neoplastic mast cells has not been fully elucidated in this category of diseases. METHODS: We examined KIT mutation, chimeric status, and AML1/ETO mRNA concerning mast cells and immature hematopoietic cells of the bone marrow in a patient with systemic mastocytosis with AML1/ETO-positive AML following allogeneic hematopoietic stem cell transplantation (HSCT). RESULTS: Mast cells of the patient displayed KIT D816Y mutation, and were derived from the recipient. In contrast, immature hematopoietic cells as defined by CD34+ CD117+ were derived from the donor, which did not possess detectable KIT D816Y mutation. The ratio of AML1/ETO to 18S rRNA of the mast cells was 7.53, whereas that of immature hematopoietic cells was 1.67. CONCLUSIONS: In a patient with SM-AHNMD who underwent allogeneic HSCT, the major source of the detectable AML1/ETO mRNA of the bone marrow after transplantation was neoplastic mast cells with KIT mutation, which were thought to be derived from CD34+ CD117+ immature leukemic cells of the recipient.     

Hematopoietic progenitors and stem cells in murine muscle

We investigated hematopoietic capabilities of murine skeletal muscle using methylcellulose culture and transplantation into lethally irradiated mice. Muscle mononuclear cells (MNC) contained colony-forming cells and long-term engrafting cells. Studies using chimeric mice indicated a bone marrow origin of the hematopoietic cells in the muscle. We then separated muscle MNC by FACS sorting into Ly-5-positive cells and Ly-5-negative cells and analyzed their hematopoietic capability in vitro and in vivo. The hematopoietic progenitors and stem cells were present only in the Ly-5-positive fraction.     

A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells

CD43 (the major sialoglycoprotein of leukocytes) is an adhesion molecule broadly expressed on hematopoietic cells. A monoclonal antibody recognizing this molecule induces apoptosis of lineage marker- negative bone marrow hematopoietic progenitor cells (HPCs) that express CD34 at a high density (CD34hiLIN-). However, not all cells within this population undergo apoptosis on stimulation via CD43. Dividing progenitor cells are most highly affected, whereas more primitive quiescent cells survive anti-CD43 monoclonal antibody treatment. These surviving cells (1) are enriched for cobblestone area-forming cells, (2) repopulate fragments for human fetal bone implanted into C.B-17 scid/scid mice, (3) have a potential to differentiate in vivo to myeloid and lymphoid cells, and (4) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate that cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis as compared with HPCs. Thus, CD43 may be specifically involved in the regulation of HPC proliferation.     

CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor

Controversy has existed about CD34 expression by hematopoietic stem cells. We recently reported that CD34 expression reflects the activation state of stem cells by using a murine transplantation model. It has been generally held that mobilized blood stem cells express CD34.However, it has also been reported that mobilized stem cells and progenitors are in G0/G1 phases of the cell cycle. To address the state of CD34 expression by the mobilized stem cells, we again used the mouse transplantation model. We prepared CD34(-) and CD34(+) populations of nucleated blood cells from granulocyte colony-stimulating factor-treated Ly-5.1 mice and assayed each population for long-term engrafting cells in lethally irradiated Ly-5.2 mice. The majority of the stem cells were in the CD34(+) population. The CD34 expression by mobilized stem cells was reversible because re-transplantation of Ly-5.1 CD34(-) marrow cells harvested from the Ly-5.2 recipients of CD34(+)-mobilized stem cells 8 months posttransplantation revealed long-term engraftment. These results may support the use of total CD34(+) cells in mobilized blood as a predictor for engraftment and CD34 selection for enrichment of human stem cells. (Blood. 2000;96:1989-1993)     

Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus

NRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-Nras(G12D) mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-Nras(G12D) mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-Nras(G12D) mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-Nras(G12D) mice is attenuated compared with Mx1-Cre, LSL-Kras(G12D) mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous Kras(G12D) expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1(+) cells, whereas Mx1-Cre, LSL-Nras(G12D) mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.     

AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice

Fanconi anemia (FA) is a heterogeneous inherited disorder characterized by a progressive bone marrow (BM) failure and susceptibility to myeloid leukemia. Genetic correction using gene-transfer technology is one potential therapy. A major hurdle in applying this technology in FA patients is the inability of granulocyte colony-stimulating factor (G-CSF) to mobilize sufficient numbers of hematopoietic stem (HSC)/progenitor cells (HPC) from the BM to the peripheral blood. Whether the low number of CD34(+) cells is a result of BM hypoplasia or an inability of G-CSF to adequately mobilize FA HSC/HPC remains incompletely understood. Here we use competitive repopulation of lethally irradiated primary and secondary recipients to show that in two murine models of FA, AMD3100 synergizes with G-CSF resulting in a mobilization of HSC, whereas G-CSF alone fails to mobilize stem cells even in the absence of hypoplasia.     

The number of nucleated cells reflects the hematopoietic content of umbilical cord blood for transplantation

A single umbilical cord blood (UCB) collection may contain sufficient hematopoietic stem cells to achieve engraftment and repopulation of the hematopoietic system of children and adults after myeloablative therapy. The hematopoietic potential of a UCB unit is often defined by the number of CD34+ cells or the number of colony-forming units as measured in semisolid hematopoietic progenitor cell (HPC) cultures. However, these assays are relatively difficult to standardize between UCB banks. The number of nucleated cells infused per kilogram body weight of the recipient is also reported to be a significant factor in the speed of recovery of neutrophils and platelets after transplantation. To analyze which parameters could be used to evaluate the hematopoietic potential of a UCB graft, we evaluated almost 300 UCB units that were collected for banking for unrelated transplantation. A strong correlation was found between the frequencies of CD34+ cells and the HPC as measured in semi-solid medium cultures. From the various leukocyte subpopulations, the concentration and total numbers of nucleated cells correlated best with both the HPC content and the number of CD34+ cells. Differentiation of these nucleated cells into subsets of leukocytes offered no advantage for better prediction of HPC or CD34+ cells. These results indicate that the nucleated cell count probably reflects the hematopoietic potential of a UCB graft, and may for that reason correlate with the speed of engraftment after transplantation.     

Clonal analysis of NRAS activating mutations in KIT-D816V systemic mastocytosis

Cooperating genetic events are likely to contribute to the phenotypic diversity of KIT-D816V systemic mastocytosis. In this study, 44 patients with KIT-D816V systemic mastocytosis were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations. Activating NRAS mutations were identified in 2 of 8 patients with advanced disease. NRAS mutations were not found in patients with indolent systemic mastocytosis. To better understand the clonal evolution of mastocytosis, we evaluated the cell compartments impacted by the NRAS and KIT mutations. Clonal mast cells harbored both mutations. KIT-D816V was not detected in bone marrow CD34(+) progenitors, whereas the NRAS mutation was present. These findings suggest that NRAS mutations may have the potential to precede KIT-D816V in clonal development. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two activating mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity.     

Characterization of CD34+, CD13+, CD33- cells, a rare subset of immature human hematopoietic cells

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cells that express CD34 are heterogeneous in their lineage affiliation and degree of maturation. Expression of CD13 and CD33 antigens indicates myeloid lineage association, but the precise sequence of expression of these two markers during differentiation is unclear. We noted the presence of CD34+ cells expressing CD13 but lacking CD33, a subset of cells not yet well characterized. In this report we describe the prevalence and the immunophenotype of this cell subset. DESIGN AND METHODS: We studied the immunophenotype of immature myeloid cells in human bone marrow samples from 11 healthy transplantation donors and in 4 cord blood samples. We used four-color flow cytometry and a large panel of monoclonal antibodies directed against lineage and differentiation-associated antigens. Three additional bone marrow samples were analyzed after immunomagnetic sorting of CD34+ cells. We focused our analysis on the subset of cells defined by the expression of CD34 and CD13 and the lack of CD33. RESULTS: We found CD34+, CD13+, CD33- cells in all 11 bone marrow and 4 cord blood samples studied. These cells represented 0.5 0.5% (mean SD) and 0.8 1.2% of mononucleated cells, respectively. CD34+, CD13+, CD33- cells appeared to be more immature than those expressing CD33 because of their light scatter characteristics (smaller size and lower granularity), the expression of markers associated with early hematopoietic cells (CD90, CD133 and CD117), and the absence of lineage-associated markers. INTERPRETATION AND CONCLUSIONS: These findings suggest that the expression of CD13 precedes that of CD33 during myeloid differentiation, and that CD34+, CD13+, CD33- cells are at an early stage of human myeloid cell differentiation.     

The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines.

beta-Glucans stimulate leukocyte anti-infective activity, enhance murine hematopoietic recovery following bone marrow injury and mobilize murine progenitor cells from bone marrow. This study evaluated the in vitro hematopoietic potential of the beta-glucan, PGG-glucan, on human bone marrow mononuclear cells (BMMC) and CD34+ BMMC compared with protein cytokines. In the presence of submaximal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.5 ng/ml), PGG-glucan significantly increased BMMC myeloid colony formation comparable to the increase observed with either interleukin-3 (rhIL-3) or stem cell factor (rhSCF). Moreover, the addition of PGG-glucan to cultures containing GM-CSF + IL-3 or GM-CSF + SCF significantly augmented granulocyte-macrophage colony production above baseline, demonstrating that PGG-glucan acts independently of those early-acting cytokines and can enhance their activity in an additive manner. Anti-PGG-glucan monoclonal antibody specifically abrogated the growth-enhancing effect of added PGG-glucan in a saturable manner and other control carbohydrate polymers failed to affect colony formation. Further, PGG-glucan was not associated with induction of IL-6, GM-CSF production and removal of accessory cells by CD34+ cell isolation did not alter the PGG-glucan effect. These data demonstrate that PGG-glucan acts on committed myeloid progenitors to enhance human hematopoietic activity by a mechanism of direct action independent of IL-3 or SCF and independent of secondary cytokine stimulation.     

Polymorphic and monomorphic HLA-DR determinants on human hematopoietic progenitor cells

The expression of monomorphic Ia-like antigens and polymorphic (allotypic) HLA-DR determinants on CFU-GM, BFU-E, CFU-E, and CFU-GEMM was studied in bone marrow and peripheral blood cells from normal healthy individuals. Using various polyclonal and monoclonal anti-Ia- like antibodies, the presence of HLA-DR backbone antigens was shown on all hematopoietic progenitor cells (HPC) studied, both in complement- dependent cytotoxicity assays and in fluorescence-activated cell sorting (FACS). The expression of allotypic determinants was demonstrated on all HPCs, using the HLA-DR typing sera anti-HLA-DR1, 2, 3, 4, 5, and 7. The Class II antigen MT-2 was also shown on all HPCs, using both monoclonal and alloantisera, whereas the MB-1 (DC-1) determinant could not be demonstrated on HPCs. This might open the possibility of removing MB-1-positive malignant cells from the graft in autologous bone marrow transplantation.     

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 cells in human hemopoietic tissues. Based on the observation that bone marrow stromal cells and primitive HPC share several cell-surface antigens, we sought to generate monoclonal antibodies to HPC by immunization with cultured human stromal cells. METHODS: BALB/c mouse were immunized with human bone marrow (BM)-derived stromal cells. Splenocytes isolated from immunized mice were fused with the NS-1 murine myeloma cell line and resulting hybridomas selected in HAT medium, then screened for reactivity against stromal cells, peripheral blood (PB), and BM cells. RESULTS: A monoclonal antibody (MAb), BB9, was identified based on its binding to stromal cells, a minor subpopulation of mononuclear cells in adult human BM, and corresponding lack of reactivity with leukocytes in PB. BB9 bound to a minor subpopulation of BM CD34(+) cells characterized by high-level CD34 antigen and Thy-1 expression, low-absent expression of CD38, low retention of Rhodamine 123, and quiescent cycle status as evidenced by lack of labeling with Ki67. CD34(+)BB9(+) cells, in contrast to CD34(+)BB9(-) cells, demonstrated a capacity to sustain hematopoiesis in pre-CFU culture stimulated by the combination of IL-3, IL-6, G-CSF, and SCF. BB9 also demonstrated binding to CD34(+) cells from mobilized PB. CONCLUSION: Collectively, these data therefore demonstrate that MAb BB9 identifies an antigen, which is selectively expressed by hierarchically primitive human HPC and also by stromal cells.