Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/Fc epsilon RI- cell population

Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL- 3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4- week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.     

Thrombopoietin alone or in the presence of stem cell factor supports the growth of KIT(CD117)low/ MPL(CD110)+ human mast cells from hematopoietic progenitor cells

OBJECTIVES: Thrombopoietin (TPO) is known to promote platelet number, have growth-promoting potential for human megakaryocytes (HuMKs), and increase erythrocyte, monocyte, mast cell, and granulocyte numbers in the presence of additional growth factors. We explored the ability of TPO alone or in the presence of stem cell factor (SCF) to support human mast cells (HuMCs). METHODS: CD34+ pluripotent and CD34+/CD117+/CD13+ HuMC progenitor cells were cultured in rhTPO and examined for HuMCs. Similarly, we added rhTPO to CD34(+) cells cultured in stem cell factor (SCF), which promotes HuMC development. RESULTS: When CD34+ cells were cultured in 10 ng/mL rhTPO and 10 ng/mL rhSCF, TPO enhanced HuMC numbers compared to rhSCF alone. Higher concentrations of rhTPO (50 ng/mL) in the presence of 100 ng/mL rhSCF inhibited the rhSCF-dependent subpopulation of CD117high HuMCs, while promoting CD117low HuMCs. Human CD34+/CD117+/CD13+ cells cultured in rhTPO alone for 1 to 2 weeks differentiated into CD41+/CD110+ HuMKs (85-90%) and FcepsilonRI+/CD117low/CD13+ HuMCs (5-10%). RhTPO-induced HuMCs expressed the TPO (CD110) receptor, tryptase, and chymase and survived when recultured in rhSCF. CONCLUSION: The effect of TPO on HuMCs in the presence of rhSCF varies, depending on the relative concentration of each growth factor, while TPO alone or in combination with rhSCF supports a unique population of CD117low/CD110+ HuMCs.     

c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides.

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.     

Interleukin-13 is involved in functional maturation of human peripheral blood monocyte-derived dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are required for the initiation of the immune response. DCs have been shown to be generated from hematopoietic stem cells, but relatively little is known about the regulation underlying differentiation and activation of DCs. Here, we report that recombinant human (rh)IL-13 induces functional maturation of rhGM-CSF plus rhIL-4 generated monocyte-derived immature DCs. Incubation of these immature DCs with rhIL-13 or rhTNF-alpha for 2 days resulted in increased surface expression of CD1a, CD11c, CD86 and HLA-DR. The DCs treated with rhIL-13 or rhTNF-alpha, but not rhIL-4, for 2 days were more efficient than unstimulated DCs in the primary autologous/allogeneic T-cell response whereas the antigen (Ag)-specific T-cell response was suppressed. The treatment of DCs with rhIL-13 as well as rhTNF-alpha for 4 days down-modulated endocytic capacity for FITC-dextran (FITC-DX) and lucifer yellow (LY), and induced surface expression of CD83. Morphological, phenotypical, and functional analyses revealed that the monocytes cultured with rhGM-CSF plus rhIL-13 gave rise to a DC type more mature than rhGM-CSF plus rhIL-4-induced DCs. These findings revealed a new role for rhIL-13 in regulating both the maturation and activation of DCs.     

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45(+)/CD34(-) cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow-derived CD34(+)/CD38(-) stem/progenitor cells. The human CD45(+) cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45(+) cell were recovered from the marrow of bnx mice than from the marrow of human stem cell-engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45(+)/CD34(-) cells recovered from the bnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34(+) progeny following retransplantation. These studies demonstrate that the human CD34(+) population can act as a reservoir for generation of CD34(-) cells. In the current studies we demonstrate that human CD34(+)/CD38(-) cells can generate CD45(+)/CD34(-) progeny in a long-term xenograft model and that those CD45(+)/CD34(-) cells can regenerate CD34(+) progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.     

Recombinant human interleukin-11 synergizes with steel factor and interleukin-3 to promote directly the early stages of murine megakaryocyte development in vitro

The authors studied the role that interleukin (IL)-11 plays during the early stages of megakaryocyte (MK) development by investigating its in vitro effects on cell subpopulations enriched for bone marrow primitive progenitor cells and early and late committed progenitor cells. Progenitor subpopulations were isolated from bone marrow of normal or 5-fluorouracil (5FU)-treated mice and separated by sorting based on the surface antigens Sca-1, c-kit, and CD34. Functional analysis of the cell subpopulations, 5FU Lin(-)Sca-1(+)c-kit(+) or normal bone marrow (NBM) Lin(-)Sca-1(+)c-kit(+)CD34(-)cells, indicated that exposure of these cells to recombinant human (rh)IL-11 in combination with steel factor (SF) stimulates the formation of colonies in methylcellulose and their proliferation in single cell-containing liquid cultures. Kinetic studies of MK progenitor generation, in response to SF and rhIL-11, demonstrated that a significant number of the progenitors produced are committed to the MK lineage. RhIL-11 also synergized with both SF and IL-3 to stimulate MK colony growth from NBM Lin(-)Sca-1(+)c-kit(+) cells (early progenitors) and NBM Lin(-)Sca-1(-)c-kit(+) cells (committed late progenitors). In the presence of IL-3, NBM, Lin(-)Sca-1(-)c-kit(+) cells responded more strongly to rhIL-11 than SF. Consistent with these results is the observation that IL-11 receptor alpha chain mRNA is present in all the progenitor cells from which the MKs are derived. This cell culture and RNA analysis suggest that murine bone marrow primitive progenitor cells and early and late progenitor cells are direct targets of rhIL-11 and that rhIL-11 has the potential to promote megakaryocyte development at several very early stages. (Blood, 2000;95:503-509) (Blood. 2000;95:503-509)     

Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages.

The cDNA for human stem cell factor (hSCF) has been cloned and expressed in mammalian and bacterial hosts and recombinant protein purified. We have examined the stimulatory effect of recombinant human SCF (rhSCF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors (CSFs) and erythropoietin (rhEpo). RhSCF alone resulted in no significant colony formation, however, in the presence of rhGM-CSF, rhG-CSF or rhIL-3, rhSCF stimulated a synergistic increase in colony numbers. In addition, increased colony size was stimulated by all combinations. The morphology of cells in the colonies obtained with the CSFs plus rhSCF was identical to the morphology obtained with rhGM-CSF, rhG-CSF or rhIL-3 alone. RhEpo also synergised with rhSCF to stimulate the formation of large compact hemoglobinized colonies which stained positive for spectrin and transferrin receptor and had a morphological appearance consistent with normoblasts. RhSCF stimulation of low density non-adherent, antibody depleted, CD34+ cells suggests that rhSCF directly stimulates progenitor cells capable of myeloid and erythroid differentiation.     

Primitive acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo lack surface expression of c-kit (CD117)

A hierarchy of progenitor cells is thought to exist in human acute myeloid leukemia (AML), with only the most primitive cells capable of proliferating to maintain the malignant clone. To further characterize this AML cell hierarchy, we evaluated the coexpression of CD34 and c-kit (CD117) on cells that are capable of long-term proliferation in vitro and in vivo.AML cells were sorted for coexpression of CD34 and c-kit (CD117) using two c-kit monoclonal antibodies (mAbs), clones 95C3 and 104D2. Sorted subfractions were evaluated for the ability to produce colony-forming units (CFU) for up to 8 weeks in suspension culture (SC) and for the capacity to repopulate NOD/SCID mice.When expression of c-kit on blood cells from 19 AML patients at diagnosis was compared using both mAbs, expression defined by 104D2 (34% +/- 6% c-kit(+)) was somewhat higher than that defined using 95C3 (18% +/- 4%). AML cells were sorted for coexpression of CD34 and c-kit using both c-kit mAbs, and the subfractions were assayed in vitro and in vivo. Whereas the majority of AML blast cells lacked expression of CD34, most AML cells capable of proliferating to produce CFU after 4 to 8 weeks in SC were CD34(+)/c-kit(-). Cultures of sorted CD34(+)/c-kit(-) cells, supplemented with steel factor, were composed of a large proportion (18% to 87%) of CD34(+)/c-kit(+) cells after 1 week, suggesting that either c-kit expression was upregulated or CD34(+)/c-kit(+) cells were produced. Moreover, the CD34(+)/c-kit(-) subfraction was found to be capable of responding to steel factor alone to produce CFU after 4 weeks in SC. In most AML patients tested (11/15), the only sorted subfraction capable of engrafting NOD/SCID mice was CD34(+)/c-kit(-). The CD34(+)/c-kit(+) subfraction from only 2 of the 15 patients and CD34(-) cells from 3 patients also engrafted the NOD/SCIDs. Only the CD34(+)/c-kit(+) subfraction of normal bone marrow engrafted.These studies suggest that primitive AML cells capable of long-term proliferation in vitro and NOD/SCID repopulation differ from primitive normal progenitor cells in their lack of surface expression of c-kit.     

CD34 human hematopoietic progenitor cell line, MUTZ-3, differentiates into functional osteoclasts

OBJECTIVE: CD14(+) monocyte cell lines can differentiate into an osteoclast (OC)-like lineage. However, the identification of human cell lines with stem cell characteristics, capable of differentiating into OCs, would provide a tool for the study of the molecular mechanisms regulating their commitment, differentiation, and function. Since the human acute myeloid leukemia cell line MUTZ-3 contains both CD34(+) stem cell and CD14(+) cell populations, we investigated the capacity of the stem/progenitor CD34(+) population to differentiate into functional OCs. MATERIALS AND METHODS: Sorted MUTZ-3-CD34(+) and MUTZ-3-CD14(+) cells were cultured in presence of M-CSF, RANK-L, and TNF-alpha to generate OCs. Differentiation was evaluated by TRAP staining and RT-PCR, which assessed the expression of c-fms, RANK, MMP-9, CATK, TRAP, and CTR in -CD34(+)OC and -CD14(+)OC cells. Resorption pit formation was also evaluated. CD34, CD14, M-CSF-R, RANK, and CTR expression was assessed by FACS analysis. RESULTS: MUTZ-3-CD34(+) differentiated into OCs, displaying the full range of differentiation markers; MMP-9, CATK, TRAP, and RANK mRNA were detected from day 3 of culture, whereas CTR from day 12. Stimulated MUTZ-3-CD34(+) generated functional osteoclasts that formed extensive resorption lacunae on both mineralized surface and bone slices. Surprisingly, in both sorted populations we identified a population M-CSF-R(+)/RANK(+) that at the same time co-expressed CD14 and CD34. CONCLUSIONS: These findings demonstrate that MUTZ-3 cells constitute an invaluable model to study the expression pattern in different developmental stages of commitment and differentiation. Importantly, the data indicate that the CD14(+)CD34(+)M-CSF-R(+)RANK(+) population represents an intermediate stage of differentiation from CD34 precursors and monocytes to osteoclast.     

CD163/CD16 coexpression by circulating monocytes/macrophages in HIV: potential biomarkers for HIV infection and AIDS progression

Monocytes and macrophages play a prominent role in the establishment of HIV-1 infection, virus dissemination, and development of viral reservoirs. Like T cells, macrophages display immune polarization that can promote or impair adaptive immunity. We hypothesize that dysregulation of monocyte/macrophage activation and differentiation may promote immune dysfunction and contribute to AIDS pathogenesis. Using flow cytometry, we analyzed the frequency of monocyte subsets in human immunodeficiency virus type 1 (HIV-1) infection relative to seronegative controls, focusing on the CD163(+)/CD16(+) monocyte as a likely precursor of the "alternatively activated" macrophage. Individuals with detectable HIV-1 infection showed an increase in the frequency of CD163(+)/CD16(+) monocytes (CD14(+)) when compared to seronegative or HIV-1-infected persons with undetectable viral loads. A positive correlation between increased CD163(+)/CD16(+) monocyte frequency and viral load was revealed that was not seen between viral load and the number of CD4(+) T cells or frequency of CD16(+) monocytes (without CD163 subtyping). We also found a strong inverse correlations between CD16(+) monocytes (r = -0.71, r(2) = 0.5041, p = 0.0097) or CD163(+)/CD16(+) monocytes (r = -0.86, r(2) = 0.7396, p = 0.0003) and number of CD4(+) T cells below 450 cells/microl. An inverse relationship between CD163(+)/CD16(+) and CD163(+)/CD16() monocytes suggests the expanded CD163(+)/CD16(+) population is derived exclusively from within the "alternatively activated" (MPhi-2) subset. These data suggest a potential role for CD163(+)/CD16(+) monocytes in virus production and disease progression. CD163(+)/CD16(+) monocytes may be a useful biomarker for HIV-1 infection and AIDS progression and a possible target for therapeutic intervention.     

Identification of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progenitor capable of activating or suppressing CD8(+) T cells

Apoptotic death of CD8(+) T cells can be induced by a population of inhibitory myeloid cells that are double positive for the CD11b and Gr-1 markers. These cells are responsible for the immunosuppression observed in pathologies as dissimilar as tumor growth and overwhelming infections, or after immunization with viruses. The appearance of a CD11b(+)/Gr-1(+) population of inhibitory macrophages (iMacs) could be attributed to high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vivo. Deletion of iMacs in vitro or in vivo reversed the depression of CD8(+) T-cell function. We isolated iMacs from the spleens of immunocompromised mice and found that these cells were positive for CD31, ER-MP20 (Ly-6C), and ER-MP58, markers characteristic of granulocyte/monocyte precursors. Importantly, although iMacs retained their inhibitory properties when cultured in vitro in standard medium, suppressive functions could be modulated by cytokine exposure. Whereas culture with the cytokine interleukin 4 (IL-4) increased iMac inhibitory activity, these cells could be differentiated into a nonadherent population of fully mature and highly activated dendritic cells when cultured in the presence of IL-4 and GM-CSF. A common CD31(+)/CD11b(+)/Gr-1(+) progenitor can thus give rise to cells capable of either activating or inhibiting the function of CD8(+) T lymphocytes, depending on the cytokine milieu that prevails during antigen-presenting cell maturation. (Blood. 2000;96:3838-3846)     

Antigenic analysis of human haemopoietic progenitor cells expressing the growth factor receptor c-kit

The cell surface molecule encoded by the protooncogene c-kit has recently been identified as the receptor for a growth factor variously termed stem cell factor (SCF), mast cell growth factor or steel factor. Using the c-kit antibody 17F11 we analysed, in triple staining experiments, the surface molecule profile and scatter characteristics of c-kit+CD34+ human haemopoietic progenitor cells. In 10 normal bone marrow samples we found 19-51% of CD34+ bone marrow progenitor cells to coexpress c-kit. These c-kit+CD34+ bone marrow cells turned out to represent a phenotypically heterogeneous population. A considerable proportion coexpressed CD33 (52 +/- 23%), and/or CD71 (62 +/- 26) antigens, marker molecules previously shown to be expressed by committed in vitro colony forming cells but not by their precursors. In line with a relatively differentiated phenotype c-kit+CD34+ cells also gave rise to on average higher forward and right-angle light scattering signals. The proportions of CD38 and/or HLA-D expressing cells were similar in the c-kit+ and in the c-kit- subsets of CD34+ progenitor cells. Coexpression of CD19 was found to be less frequent in the c-kit+ (4 +/- 5%) as compared to the c-kit- (17 +/- 14%) fraction of CD34+ cells. CD7+ CD34+ bone marrow cells were hardly detectable and their numbers too low to allow further subdivision in c-kit+ and c-kit- subsets.     

Human hepatic stem-like cells isolated using c-kit or CD34 can differentiate into biliary epithelium

BACKGROUND & AIMS: Recent reports suggest that after bone marrow transplantation into rodents and humans, hematopoietic stem cells migrate into the liver and give rise to oval cells, hepatocytes, and biliary epithelial cells. We investigated this hypothesis further in the human liver using the hematopoietic markers c-kit and CD34. METHODS: Immunofluorescence confocal microscopy was performed using cytokeratin 19 (CK-19; biliary cell marker) with either c-kit or CD34. Immunomagnetic separation was then used to select c-kit- or CD34-positive cells. After attachment, cells were cultured for up to 7 days, and their growth and phenotypic characteristics were examined. RESULTS: In cirrhotic tissue, c-kit- or CD34-positive cells were located in the portal tracts surrounding bile ducts. Occasionally c-kit- (but not CD34-) positive cells that coexpressed CK-19 were observed integrated into bile ducts. In vitro, immunoisolated c-kit or CD34 cells gave rise to colonies of at least 2 morphologies expressing CK-19 or CD31 (endothelial cell marker). CD34- or c-kit-positive cells with similar properties were also isolated from normal liver. CONCLUSIONS: These findings indicate that cells present in human liver that express the markers c-kit or CD34 have the capacity to differentiate into biliary epithelial cell lineage and may therefore represent human biliary epithelial progenitor cells.     

Granulocytic and monocytic differentiation of CD34hi cells is associated with distinct changes in the expression of the PU.1- regulated molecules, CD64 and macrophage colony-stimulating factor receptor

The present study investigated the possibility that macrophage colony- stimulating factor (M-CSF) responsiveness of hematopoietic progenitor cells is regulated at the level of receptor expression and that M-CSF receptor (M-CSFR) may be used as an early marker of monocyte lineage commitment. Immunofluorescence measurements with an anti-M-CSFR antibody showed that 44% +/- 5% of CD34hi cells expressed the receptor. The M-CSFR was present on progenitor cells that were positive for the granulo-monocytic marker CD64, but not on primitive, erythroid, or lymphoid progenitors. The CD34hiCD64+ population could be divided into subsets of M-CSFRhi and M-CSFRlo cells. In addition, a subset of CD34hiCD64-M-CSFRhi cells was found. CD34+ cells that were positive for M-CSFR, CD64, or both gave rise exclusively to granulo-monocytic cells, and 65% of the granulomonocytic colony-forming cells in the CD34+ population were recovered from these cells. Approximately 70% of the colony-forming cells (CFCs) derived from CD34hiM-CSFRhi cells were macrophage colony-forming units (CFU-M), whereas 91% of the CFCs in the CD34hiCD64+M-CSFRlo population were granulocyte colony-forming units (CFU-G). The M-CSFRhi cells with the highest frequency of colony- forming and bipotent cells and largest average colony size were found in the CD64- subset, indicating that M-CSFR appears earlier than CD64 during monocyte development. After 60 hours in culture, a subset of the CD34hiM-CSFRhi cells had downmodulated M-CSFR (29% to 38%). This population gave rise almost exclusively to granulocytes, whereas the cells that remained M-CSFRhi gave rise exclusively to monocytes. In all experiments, the M-CSFRhi population responded to M-CSF, whereas minimal responses were observed among M-CSFRlo cells. These results suggest that M-CSF target specificity among human hematopoietic progenitor cells is determined by lineage-specific regulation of the M- CSFR and show that M-CSFR is a useful marker to discriminate between monocytic and granulocytic progenitor cells.     

Differentiation and maintenance of mast cells from CD34(+) human cord blood cells

OBJECTIVE: Establishment of a stable umbilical cord blood CD34(+) (UCB CD34(+)) cell culture system and identification of the cells in the cobblestone area differentiated from UCB CD34(+) long-term culture cells. MATERIALS AND METHODS: Human UCB CD34(+) cells were cultured on MS-5 mouse stroma cells in the presence of stem cell factor (SCF), flt-3 ligand (FL), and thrombopoietin (TPO) for 4 to 16 weeks. Cells in the culture medium and in the cobblestone area were collected and characterized by flow cytometry and microscopy. RESULTS: CD34(+) cells were stably expanded by culturing on MS-5 stroma cells in the presence of SCF, FL, and TPO for more than 4 months. Cells highly expressing CD117 (c-kit) appeared in the cobblestone area after 2 weeks and stably expanded. Isolation of cells highly expressing CD117 by fluorescence-activated cell sorter (FACS) revealed the cells were tryptase-positive and Fc epsilon receptor 1-negative mast cells. They showed typical mast cell morphology and released histamine upon stimulation by substance P or compound 48/80 in vitro. CONCLUSION: Human UCB CD34(+) cells were stably expanded on MS-5 stroma cells in the presence of SCF, FL, and TPO. Under this condition, multipotent CD34(+) cells and mast cells differentiated from UCB CD34(+) cells were expanded in the cobblestone area. The expanded mast cells showed histamine release after substance P or compound 48/80 stimulation. These human mast cells will be useful as a source of human cells for evaluating the allergic effects of drugs.     

Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture.

In the murine system, a number of cytokines (including interleukin-3 [IL-3], IL-4, and stem cell factor [SCF]) promote the growth of mast cells (MCs). However, so far little is known about factors controlling differentiation of human MCs. Recent data suggest that human MCs express receptors (R) for SCF. The aim of the present study was to investigate whether recombinant human (rh) SCF induces differentiation of human MCs from their precursor cells. For this purpose, bone marrow (BM; normal donors, n = 6) and peripheral blood (PB; normal donors, n = 11) mononuclear cells (MNC) were cultured in the presence of rhSCF, rhIL-3, rhIL-4, rhIL-9, recombinant human macrophage colony-stimulating factor (rhM-CSF), or control medium in long-term (8 weeks) suspension cultures. After 4 weeks, up to 5% of the MNC (BM and PB) cultured in the presence of rhSCF, but not in the presence of other cytokines, were found to exhibit the characteristics of MCs. These MCs expressed the YB5.B8-reactive domain of the SCF R as well as IgE R, as determined by combined toluidine blue/immunofluorescence staining. Myeloid antigens, likewise expressed on human basophils (ie, CD11b, CDw65, and Bsp-1), could not be detected on these cells. Furthermore, rhSCF, but not rhIL-3, rhIL-4, rhIL-9, or rhM-CSF, induced dose- and time-dependent increases in the formation of cellular tryptase (an MC-specific enzyme) (rhSCF [100 ng/mL], 1,308 +/- 679 ng/mL v control medium, 18 +/- 6 ng/mL tryptase on day 35 of PB cell cultures), as well as an increase in cellular histamine. After 6 to 8 weeks, when other mature hematopoietic cells decreased, MCs still could be detected in culture, with up to 40% of all cells being MCs. To test whether rhSCF also activates tissue MCs, we performed histamine release experiments (dispersed tissue; lung, n = 3; uterus, n = 3). SCF was found to enhance (by up to 3.4-fold) the capacity of the MCs to release histamine upon cross-linkage of IgE R with anti-IgE. Together, these observations suggest that rhSCF induces in vitro differentiation of human MCs from their BM and PB precursor cells in long-term culture and upregulates MC releasability.     

Study of early hematopoietic precursors in human cord blood.

Human cord blood is a source of transplantable stem cells. These stem cells express the antigen CD34, are resistant to treatment with 4-hydroperoxycyclophosphamide (CD34+/4-HCres), and do not give rise to colonies when plated in clonogenic assays. We studied the number of CD34+ cells present in cord blood and developed a two-step assay for early precursors (pre-colony-forming units, pre-CFU) capable of giving rise to committed progenitors. In this assay CD34+/4-HCres cord blood cells were cultured in suspension with different growth factors. After 7 days in suspension the remaining cells were plated in clonogenic assays, for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and mixed lineage colony-forming units (CFU-MIX), in the presence of pure factors or a combination of recombinant human (rh) interleukin 3 (IL-3) and medium conditioned by the PU34 primate cell line. Pre-CFU for all precursors were identified. These pre-CFU developed into committed progenitors in response to rhIL-3. The combinations of rhIL-3 plus rh interleukin 1 (IL-1) or rhIL-3 plus rh interleukin 6 (IL-6) did not enhance recovery of progenitors. The developing CFU-GM were responsive to rh granulocyte-macrophage colony-stimulating factor (GM-CSF) and rh granulocyte colony-stimulating factor (G-CSF) but much less so to rhIL-3. BFU-E and CFU-MIX developed in suspension but could only be detected when cells were replated in the presence of a combination of rhIL-3 and PU34 but not rhIL-3 alone. This assay may be useful in evaluating the number of early hematopoietic precursors present in cord blood samples and in defining growth factor combinations that could enhance hematopoietic recovery after cord blood stem cell transplants.     

Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34 cells after ex vivo expansion

OBJECTIVE: The relationship between phenotype and function in ex vivo-cultured human hematopoietic stem cells (HSC) remains poorly understood. We investigated the effects of a short-term serum-free culture on the relationship between stem cell phenotype, cell division history, and function in human CD34(+) cells. METHODS: G-CSF-mobilized peripheral CD34(+) cells were cultured for 4 days with stem cell factor, flt-3 ligand, and thrombopoietin. The phenotype (CD34, CD38, HLA-DR, c-kit), cell division history, colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and NOD/SCID repopulating activities were evaluated at Day 0 and 4. RESULTS: We observed a loss of CD38, HLA-DR, and c-kit surface expression resulting in a drastic increase in CD34(+)CD38(-), CD34(+)HLA-DR(-), and CD34(+)c-kit(-/low) cells at Day 4. In contrast, the frequency of Thy-1(+) cells was maintained. We observed a 1.3-fold expansion of CFC, a 4.8-fold increase in LTC-IC, and an overall maintenance of the NOD/SCID repopulating cell activity. CD34(+)CD38(-) and CD34(+)HLA-DR(-) cells detected at Day 4 displayed the most active pattern of division (4 to 5 divisions) whereas 60% of CD34(+)Thy-1(+) cells divided 0 to 2 times during the same period. At Day 4, the NOD/SCID repopulating activity was associated with Thy-1(+) cells with no more than 2 divisions. CONCLUSIONS: Our results show that the relationship between stem cell phenotype and function is dramatically altered in cultured CD34(+) cells. Thy-1 expression and cell division history appear to be superior to CD38, HLA-DR, and c-kit, or to homing molecules (CXCR4, VLA-4) as predictors of the repopulating activity of cultured peripheral CD34(+) cells.     

Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.

The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli. The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs. The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer. Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog. Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures. When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks. The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6. Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF. Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors. It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues. In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.     

Recombinant human interleukin-3 and granulocyte-macrophage colony- stimulating factor show common biological effects and binding characteristics on human monocytes

Two human hemopoietic growth factors involved in monocytopoiesis, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied for their ability to stimulate blood monocytes and to bind to the monocyte membrane. Both cytokines maintained monocyte/macrophage numbers during long-term culture and increased cell size as compared with controls. Effects on cell numbers were present at low cytokine concentrations (6 to 20 pmol/L), whereas enhanced 3H-thymidine incorporation was observed only at higher concentrations (greater than or equal to 60 pmol/L). Autoradiographic studies showed only 1% to 3% of stimulated monocytes with nuclear grains. These results suggest that the primary mechanism for IL-3 and GM-CSF-induced maintenance of monocyte/macrophage numbers in humans is through an effect on cell survival. Surface receptors for both IL-3 and GM-CSF were studied by using 125I-labeled recombinant human (rh) cytokines and performing Scatchard analyses. Both cytokines showed curvilinear Scatchard plots, and computer analyses favored a two-site binding model. High-affinity binding data for 125I rhIL-3 (Kd 7.7 to 38.2 pmol/L; receptor number/cell 95 to 580) and for 125I rhGM-CSF (Kd 4.7 to 38.9 pmol/L; receptor number/cell 8 to 67) show similar binding affinities for the two cytokines but a lower receptor number/cell for 125I rhGM-CSF. Low-affinity binding characteristics for 125I rhIL-3 (Kd 513 to 939 pmol/L; receptor number/cell 179 to 5,274) and for 125I rhGM- CSF (Kd 576 to 1,120 pmol/L; receptor number/cell 130 to 657) show a similar pattern for the two cytokines. Specificity of 125I rhIL-3 and 125I rhGM-CSF binding to monocytes was established by the ability of the homologous cytokine to inhibit binding and the inability of a range of other cytokines to compete at 100-fold excess molar concentration. It is important, however, that binding of 125I rhIL-3 was partially inhibited by rhGM-CSF and that rhIL-3 partially inhibited binding of 125I rhGM-CSF to the monocyte membrane under conditions shown to prevent receptor internalization. The degree of inhibition varied between 25% and 80% in different experiments, and quantitative inhibition experiments showed that 1,000-fold excess concentrations of competitor failed to inhibit binding of the heterologous ligand completely. These results demonstrate that human IL-3 and GM-CSF have similar effects on growth and survival of human monocytes in vitro and suggest that these and other common biological effects may be mediated either through a common receptor or through distinct receptors associated on the monocyte membrane.