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(The FASEB Journal. 1999;13:263-272.)
© 1999 FASEB

RESEARCH COMMUNICATION

Self-renewal, maturation, and differentiation of the rat myelomonocytic hematopoietic stem cell

TREVOR LUCASa , WALTER KRUGLUGERa , PUCHIT SAMORAPOOMPICHITb , ROSWITHA GAMPERLc , HARTMUT BEUGd , OTHMAR FÖRSTERa and GEORGE BOLTZ-NITULESCUa , 1


a Institute of General and Experimental Pathology, A-1090 Vienna, Austria;

b Institute of Histology and Embryology, A-1090 Vienna, Austria;

c Institute of Medical Biology and Human Genetics, A-8010 Graz, Austria; and

d Research Institute of Molecular Pathology, A-1030 Vienna, Austria


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hematopoiesis is viewed as a differentiating system emanating from a pluripotent hematopoietic stem cell capable of both self-renewal and differentiation. By identifying and characterizing a novel and highly specific in vitro mitogenic response to the N-acetyl glucosamyl/sialic acid specific, stem cell-binding lectin wheat germ agglutinin (WGA), we demonstrate the existance of a rare (0.1%), plastic adherent precursor in rat bone marrow capable of proliferation (two to seven divisions) in response to WGA. Stimulated cells possess a lineage (lin)low/- immunophenotype and immature blastoid morphology (WGA blasts). A subsequent proliferative response to stem cell factor (SCF), the ligand for the proto-oncogene receptor tyrosine kinase c-kit, is characterized by an initial maturation in immunophenotype and subsequent self-renewal of cells (SCF blasts) without differentiation for at least 50 generations. Although granulocyte colony-stimulating factor (G-CSF), interleukin (IL) -6, IL-7, and IL-11 synergize with SCF to increase blast colony formation, cytokines such as granulocyte-macrophage CSF or IL-3 are without significant effect. At all time points in culture, however, cells rapidly differentiate to mature neutrophils with dexamethasone or to mainly monocytes/macrophages in the presence of 1{alpha},25-dihydroxyvitamin D3, characterized by cell morphology and cytochemistry. Removal of SCF during blast maturation, self-renewal, or induction of differentiation phases results in apoptotic cell death. Data indicate a pivotal role for SCF/c-kit interaction during antigenic maturation, self-renewal, and apoptotic protection of these lineage-restricted progenitors during non-CSF-mediated induction of differentiation. This approach provides a source of many normal, proliferating myelomonocytic precursor cells, and introduces possible clinical applications of ex vivo expanded myeloid stem cells.—Lucas, T., Krugluger, W., Samorapoompichit, P., Gamperl, R., Beug, H., Förster, O., Boltz-Nitulescu, G. Self-renewal, maturation, and differentiation of the myelomonocytic hematopoietic stem cell.


Key Words: lectins • stem cell factor • cell proliferation • cell differentiation • dexamethasone • calcitriol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MATURE BLOOD CELLS are generated from a pluripotent hematopoietic stem cell (PHSC)2 that both proliferates and differentiates to hierarchially produce antigenically heterogenous progenitor cell populations with little or no self-renewal capacity, lineage-restricted potentials, and antigenic profiles (1). The complex control of lineage-restricted progenitor expansion and mature cell development is principally under the control of growth factors and hormones that provide mitogenic stimuli, differentiation signals, and protection against apoptosis (2). A less well-characterized component in the organization of stroma compartments is the interaction of differentially expressed surface glycoconjugate structures with highly specific lectins that regulate cell adhesion and possibly transmit proliferative stimuli (3).

Production of monocytes and neutrophilic granulocytes from the myelomonocytic stem cell develops through an antigenically heterogeneous (4) common progenitor population known as the colony forming unit (CFU) granulocyte-macrophage (CFU-GM) (5, 6). Differentiation of CFU-GM in semisolid cultures results in the formation of either mixed or single lineage colonies (CFU-G, CFU-M) under the influence of a variety of growth factors (6). Examination of the processes governing the expansion and differentiation of a common progenitor to the differentiating myeloid CFU's has, however, been impeded by the extremely low frequency, inevitable heterogeneity of identifiable target populations, and spontaneous differentiation of normal progenitors in culture.

Interaction between stem cell factor (SCF), also known as mast cell growth factor (7–12) or kit ligand (13, 14), and the proto-oncogene receptor tyrosine kinase c-kit (15) during myelomonopoiesis stimulates the formation of committed blast colonies (16). In synergy with other growth factors, SCF expands colony forming cells (17), activates noncycling progenitors, and directly differentiates CFU-GM (18, 19), most strikingly with GM-CSF, G-CSF, or interleukin 3 (IL-3) (20, 21). The potential oncogenicity of c-kit activation during myeloid development has also become apparent in mast cells (22) and transformed multipotent cells (23); gain of function point mutations causing ligand-independent autophosphorylation and constitutive c-kit activation (24) have been identified in myeloproliferative disorders (25) that also cause factor-independent growth in myeloid precursor cell lines (26). Recently, the characteristic of self-renewal, a property previously considered exclusive to the PHSC (1) and myeloid leukemias in blast crisis (27), has also been demonstrated in committed erythroid progenitor populations (28), suggesting that development of a leukemogenic phenotype and normal lineage-restricted progenitor expansion may be closely linked processess.

The discovery that stem cells express sialic acid residues (29) originally led to methods using WGA in the flow cytometric purification of stem cells (30–32). Although lectin stimulation of leukocyte proliferation in vitro are standard methods, mitogenic stimulation of stem populations has not yet been described.

The aims of the present study were to investigate the effects of lectin–carbohydrate interactions on the proliferation of myeloid stem cells under the influence of WGA and to characterize the role of SCF/c-kit signaling and steroid hormones on myeloid stem cell proliferation and differentiation. We demonstrate here the existence of an apparantly unique progenitor in rat bone marrow that has transiently proliferated in response to WGA, allowing the isolation of a population of lineage-restricted blast cells that undergo antigenic maturation followed by extensive self-renewal in the presence of SCF, differentiate in the presence of calcitriol or glucocorticoids, but are apoptotic in the absence of growth factor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Bone marrow cells (BMC) were prepared from 3- to 4-wk-old Louvain rats (Himberg, Austria) that were killed with diethyl ether, as described (33). Cells were incubated in erythrocyte lysis buffer (11.5 mM Tris, 10 mM NH4Cl) for 8 min at 4°C; after washing and filtration to remove aggregates, they were resuspended at a density of 2.5 x 106/ml in a modified Iscove's modified Dulbecco's medium (Vitromex, Selters, Germany) supplemented with 1% bovine serum albumin (Sigma Chemical, St. Louis, Mo.), Fifty U/ml penicillin, 250 µg/ml streptomycin, 2 mM glutamine, pH 7.2, further referred to as culture medium (CM) and 5 µg/ml WGA (Sigma). Cell suspensions (25 ml) were cultured in 150 cm2 tissue culture flasks (Costar, Cambridge, Mass.) under humidified 5% CO2 at 37°C for 7–8 days. All nonadherent cells were then removed by vigorous pipetting with cold medium, and adherent cells were harvested by scraping. Cells were then incubated in 0.25 M of the competing sugar N-acetyl-D-glucosamine (Sigma) in CM for 30 min at room temperature to remove the lectin, washed, and resuspended either in CM supplemented with 5 µg/ml insulin (CMI) obtained from Boehringer Mannheim (Mannheim, Germany) or in CMI containing 15% heat-inactivated fetal calf serum (CMIS) purchased from Sebak (Linz, Austria). Cell viabilities were estimated in a hemocytometer by trypan blue exclusion.

SCF blast culture
All liquid cultures were seeded at an initial density of 7 x 105 blasts/ml. Stimulation by recombinant rat (rr) SCF, kindly provided by Amgen Biologicals (Thousand Oaks, Calif.), was analyzed by measuring radioactivity incorporated into 100 µl of 24 h stimulated liquid cultures further pulsed for 16 h with 1 µCi [3H]thymidine (Amersham, Little Chalfont, U.K.) in CMI. Blast cell colony formation was monitored by seeding 10,000 WGA blasts in 1 ml of 2% methyl cellulose (Fluka, Buchs, Switzerland) prepared in CMI in 24-well tissue culture plates (Costar) and counting colonies containing more than 30 cells on day 14. The influence of additional growth factors on blast colony formation was examined by supplementing SCF (100 ng/ml) -supported cultures with either 100 U/ml recombinant murine (rm) IL-1{alpha}, 100 U/ml rm IL-7 (Genzyme Corporation, Cambridge, Mass.), 500 U/ml rm GM-CSF, 5 U/ml rm erythropoietin (EPO), 100 U/ml rm IL-3 (Boehringer Mannheim), 100 U/ml recombinant human (rh) IL-6 (Sigma), 10 U/ml rh IL-11 (R&D Systems Inc., Minneapolis, Minn.), or 500 U/ml rh G-CSF (Amgen). SCF blasts were cultured long-term in CMIS supplemented with 100 ng/ml rr SCF. Cultures were harvested by gentle scraping, maintained below a 2 x 106/ml confluent density, and fed every second day with fresh medium and growth factor.

Immunofluorescence and flow cytometry
Surface immunophenotyping was performed on WGA blasts and 5 day SCF (100 ng/ml) stimulated cells (SCF blasts) by incubating cells in Hank's balanced salt solution supplemented with 0.3% bovine serum albumin and 0.1% sodium azide for 30 min on ice (34) with saturating dilutions of monoclonal antibodies (mAb) against the following rat antigens: CD2 (clone OX34) and the monomorphic class I determinant RT1.A (OX18) were obtained from Serotec (Kidlington, U.K.). mAb's directed against CD4 (OX35), CD45 (OX1) and the nonpolymorphic MHC class II determinants RT1.B (OX6) and RT1.D (OX17) were purchased from (Harlan Sera-Lab, Sussex, U.K.). Anti-CD3 (G4.18), CD5 (OX19), CD8 (OX8), CD11b/Mac-1 (WT.5), CD45R (HIS24) and CD90/Thy-1 (OX7) mAb's were obtained from Pharmingen (San Diego, Calif.). Anti-RM-1 mAb was purchased from Bachem (Bubendorf, Switzerland) and mAb HIS49 directed against erythroid cells was a kind gift from Dr. F. G. M. Kroese (University of Groningen, The Netherlands). Intracellular staining was performed with a cell permeabilization kit (An der Grub Bioresearch, An der Grub, Austria). Cells were washed twice between stages and unconjugated antibodies were further incubated with fluorescein isothiocyanate (FITC) -conjugated goat anti-mouse immunoglobulins previously absorbed with rat immunoglobulins (Pharmingen). Subcellular particles and dead and membrane compromised cells were excluded by scatter characteristics and propidium iodide (20 µg/ml) uptake; at least 104 gated events were analyzed on a FACScan (Becton Dickinson, San Jose, Calif.) with an argon laser tuned at 488 nm and expression compared to isotype matched controls using Lysis II software.

Freshly isolated and erythrocyte lysed BMC were stained with WGA-FITC (EY Labs. Inc., San Mateo, Calif.) and sorted into WGA+ and WGA- populations using a FACSVantage TSO (Becton Dickinson), as described elsewere (31).

Morphology and cytochemistry
Cell morphology was examined by light microscopy from cytospins stained with Hemacolor embedded in Entellan (Merck, Darmstadt, Germany) and compared to standard texts (35). Neutral benzidine (36) and {alpha}-napthyl butyrate nonspecific esterase (NSE) type-1 (37) staining were performed as described.

Induction of differentiation
The effects of dexamethasone (DEX) obtained from Sigma and 1{alpha},25-dihydroxyvitamin D3/calcitriol (Hoffman-La-Roche Ltd., Basel, Switzerland), prepared as stock solutions in ethanol, were estimated by harvesting SCF blasts in logarithmic growth and reculturing in fresh CMIS supplemented with 100 ng/ml rr SCF and the indicated steroid. Proliferation was assayed after 24 h with [3H]thymidine, as above. Flow cytometric and cytochemical analyses were routinely performed between days 3 and 4 for DEX-induced differentiation and on days 6 to 8 for calcitriol-stimulated cultures; fluorescence intensities compared with SCF-stimulated controls containing appropriate ethanol concentrations.

Apoptosis
DNA was prepared by standard methods from 3 x 106 cells by resuspending cell pellets in 500 µl of lysis buffer (5 mM Tris-HCl, pH 7.4, 1% sodium dodecyl sulfate, 5 mM EDTA, pH 7.4, and 100 µg/ml proteinase K) and incubated at 56°C for 1 h. Samples were extracted once with chloroform, precipitated with ethanol, resuspended in TE buffer containing 5 µg/ml DNase-free RNase, incubated at room temperature for 30 min, and separated on 1% agarose gels containing 0.5 µg/ml ethidium bromide at 1 V/cm in TBE buffer (38, 39).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of blast cell proliferation by WGA
Serum-free culture of BMC for 1 wk results in the limited proliferation and survival of morphologically mature and immature cell types (Fig. 1 ).Addition of WGA induces a marked and concentration-dependent decrease in the cellularity of cultures harvested on day 7 (Fig. 1) . However, as can be seen from Fig. 2a ,WGA is also mitogenic for a minor subset of BMC resulting in the outgrowth of strongly plastic adherent colonies (containing >4 cells). At optimal concentrations of WGA (4–6 µg/ml), a maximal yield of cells are harvested from cultures that contain only plastic adherent colonies. Under these conditions, between 3–7 x 105 cells per 108 seeded BMC can be harvested after 1 wk of culture with a colony frequency of approximately 10-3. Longer culture results in a decrease in the number of cells harvested even in the presence of fresh CMI and lectin (data not shown). Isolated cells (WGA blasts) display an undifferentiated blastoid morphology with a basophilic cytoplasm and mostly large eccentric nuclei (Fig. 2b ).



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  		Figure 1. Influence of WGA and SCF on culture cellularity and morphology. BMC (108) were cultured under serum-free conditions for 7 days in the presence of SCF or different WGA concentrations (2.5, 5, 7.5, or 10 µg/ml). Cells were harvested, counted, and the percentage of immature cells estimated by morphological analysis. Data incorporate the standard deviation of two experiments. SCF alone causes a moderate expansion in culture cellularity. WGA induces a concentration-dependent increase in cytotoxicity but also causes the outgrowth of plastic adherent proliferating colonies of cells. At a WGA concentration of 5 µg/ml, cultures contain only proliferating adherent cells, which when harvested and stained display undifferentiated morphology (% immature cells).



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  		Figure 2. Cell culture and morphology. BMC stimulated with 5 µg/ml WGA for 7 days in serum-free cultures (CMI) results in the proliferation of plastic adherent colony forming cells (a, x40) with immature blastoid morphology (b, x100). Isolated cells (WGA blasts) further proliferate under the influence of 100 ng/ml SCF to cobblestone-like confluency (c, x40) in liquid culture (SCF blasts), retaining immature blastoid characteristics (d, x40). SCF blast cultures terminally differentiate into granulocytes after 5 day culture with 5 x 10-7 M dexamethasone (e, x100) or into mature macrophages on day 7 after stimulation with 5 x 10-8 M calcitriol (f, x100).

SCF responsiveness
After lectin removal by sugar competition with N-acetyl-glucosamine, isolated WGA blasts further proliferate in medium containing rr SCF in semisolid and liquid cultures under both serum-supplemented and CMI. Cells initially seeded at a minimum density of 7 x 105/ml are strongly plastic adherent and grow to cobblestone-like confluency (Fig. 2c ). At cell densities between 1.5 and 2 x 106/ml, adherance is largely lost and growth continues in suspension. Stimulation of WGA blasts by SCF, assessed both by [3H]thymidine incorporation in liquid cultures and as the number of blast colonies developing in semisolid cultures in methyl cellulose, shows maximal stimulation between 75 and 125 ng/ml (Fig. 3 )with a seeding efficiency in methyl cellulose of approximately 12%. SCF blasts can be easily bulk cultured in CMIS supplemented with 100 ng/ml rr SCF. Insulin increases proliferation rates in these cultures by two- to threefold (data not shown). Under these conditions, SCF blasts have been cultured with a population doubling time of approximately 3–4 days (Fig. 4 )for up to 50 generations.



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  		Figure 3. Effects of SCF concentration on WGA blast proliferation. WGA blasts were incubated in CMI supplemented with the indicated SCF concentrations. Data show radioactivity incorporated into 24 h-stimulated liquid cultures, further pulsed for 16 h with 1 µCi [3H]thymidine ({blacktriangleup}), from one representative experiment. Blast cell colony formation was monitored by seeding 10,000 WGA blasts in 2% methyl cellulose prepared in CMI and counting colonies containing more than 30 cells on day 14. Data incorporate the standard deviation of 3 experiments ({blacksquare}).



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  		Figure 4. Long-term proliferation of SCF blasts. WGA blasts were seeded on day 0 in the presence of 100 ng/ml rrSCF. Cells were harvested at the indicated times, counted ({blacksquare}), and subcultured. Data incorporate the standard deviation of three experiments. At three time points, expression levels of Thy-1 ({blacklozenge}) and CD45 ({blacktriangleup}) antigens were monitored by flow cytometry. Data show the mean channel fluorescence (MCF) intensities from one representative experiment.

In additional experiments, the influence of SCF on the cellularity of BMC culture was investigated. After 7 day cultures of BMC in CMI alone or in the presence of SCF, a similar number of cells was obtained (Fig. 1) . To examine whether a rare progenitor is being stimulated, cells from both cultures were harvested and incubated with SCF under semisolid conditions. The number of colonies that subsequently developed from cells initially cultured in CMI, in the presence or absence of SCF, was 32 ±7 and 8 ±3 per 10,000 cells seeded, respectively (mean ±SD of two experiments).

Immunophenotypic maturation of WGA blasts
The results presented in Table 1 show that WGA blasts stain negatively for erythroid (acid benzidine and mAb HIS49), T cell (CD2, CD4, CD5, CD8, and intracellular CD3), pre-B, B cell (CD45R), and natural killer cell (CD8, CD45RC) markers, and NSE activity. But cells express CD90/Thy-1, MHC class I (RT1.A), CD45, CD11b, the myeloid marker detected by mAb RM-1, and very low levels of MHC class II (RT1.B and RT1.D) molecules.


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  		Table 1. Immunophenotypes of WGA blasts, SCF blasts, and SCF blasts differentiated with DEX or calcitriola

After SCF stimulation of WGA blasts, cells retain an undifferentiated blastoid morphology (Fig. 2d ) and a normal karyotype (data not shown). Monitoring of the immunophenotype in liquid cultures indicates that stimulation of WGA blasts with SCF is accompanied by increases in the surface density of CD90, CD45, RM-1, CD11b, RT1.A RT1.B, and RT1.D expression (Fig. 5 and Table 1 ).Cells retain negativity for erythroid and lymhopoietic markers. The immunophenotype of SCF blasts is not significantly influenced during prolonged expansion in SCF (Table 1) , shown graphically for CD45 and Thy-1 in Fig. 4 .



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  		Figure 5. Changes in cell immunophenotype. Representative fluorescence histograms showing increases in Thy-1/CD90, CD45/LCA, RM-1 antigen, Mac-1/CD11b, RT1.A/MHC class I, and MHC class II (shown for RT1.B) expression from a WGA blast (open) to an SCF blast (shaded) phenotype after 5 days in culture in CMI containing 100 ng/ml SCF. The same instrument settings were used, and 99% of all isotype controls stained within the first decade of the logarithmic scale (not shown).

Effect of cytokines on SCF blasts
To examine the effects on blast cell colony formation of individual growth factors known to synergize with SCF to regulate hematopoiesis, WGA blasts were seeded in serum-free methyl cellulose cultures supplemented with 100 ng/ml SCF and one additional cytokine; blast colonies containing more than 40 cells were counted on day 14. Addition of G-CSF induced a substantial synergistic increase in the number and size of colonies. Moderate increases in the seeding efficiency of WGA blasts in semisolid cultures were also induced by IL-6, IL-7, and IL-11 (Fig. 6 ).Cell morphology in all cultures was scored as blastoid.



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  		Figure 6. Influence of cytokines on SCF-supported blast colony formation in serum-free methyl cellulose cultures (CMI). The number of blast colonies containing more than 30 cells on day 14 of culture was counted and expressed as a percentage of SCF-stimulated controls. Data incorporate the standard deviations of 5 experiments.

Induction of differentiation
Differentiation induction in SCF blasts is generally characterized by concentration-dependent decreases in proliferation (Fig. 7 ),development of characteristic mature cell morphology, reduction in Thy-1, MHC class I and CD45 antigenic densities, and the differential regulation of RM-1 and MHC class II antigen expression and lineage-specific NSE type-1 activity (Table 1) . Treatment of SCF blasts with 5 x 10-7M DEX in liquid cultures containing SCF results in preferential, rapid differentiation divisions along the granulocyte lineage. Cells grow initially as small plastic adherent colonies; after 4 to 5 days, cultures are comprised almost exclusively of terminally differentiated neutrophils (Fig. 2e ). Maturing granulocyte lineage cells are specifically characterized by decreases in the antigenic densities of RM-1 molecules and both RT1.B and RT1.D determinants (Table 1) .



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  		Figure 7. Influence of steroid hormones on cell proliferation. SCF blasts were cultured in the indicated concentration of dexamethasone ({blacksquare}) or calcitriol ({blacktriangleup}) for 24 and 48 h, respectively, before pulsing with [3H]thymidine for an additional 16 h. Data show representative experiments incorporating the standard deviation of triplicate values.

Stimulation of SCF blasts with 5 x 10-8 M calcitriol results in the development of an almost pure population of monocytes/macrophages on day 7 of culture, distinguished by characteristic morphology (Fig. 2f ) maintenance or increases in RT1.B, RT1.D, and RM-1 antigen expression and induction of NSE activity (Table 1) . In contrast to the exclusively lineage restricted DEX-mediated differentiation, some granulocytic development is also evident during early stages of calcitriol-induced cell differentiation.

SCF withdrawal and apoptosis
Withdrawal of growth factor from SCF blasts leads to a complete loss in proliferative capacity after 12 h (data not shown). The crucial role of SCF as a survival factor during differentiation induction is also evidenced by abrogated mature cell development and cell death. During both blast expansion and induction of differentiation phases, electrophoresis of DNA clearly demonstrates endonuclease-induced oligonucleosomal fragmentation characteristic of apoptosis (Fig. 8 ).



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  		Figure 8. Effect of SCF removal on DNA fragmentation. SCF blasts were cultured in the presence (+) or absence (-) of SCF (100 ng/ml), dexamethasone (DEX; 5 x 10-7 M), or calcitriol (VD3; 5 x 10-8 M) for 24 h. DNA electrophoresis was performed as described in Materials and Methods. In the absence of SCF, cells undergo apoptosis as shown by oligonucleosomal fragmentation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have identified and characterized a novel mitogenic response to the plant lectin WGA in serum-free rat BMC cultures. Proliferating cells isolated are blastoid in appearance, with the immunophenotypical and morphological characteristics of early progenitors. By identifying this novel, transient, and highly specific proliferative BMC response, we not only demonstrate selective lectin-mediated mitogenic stimulation of stem cells, which suggests a possible functional role for the differential expression of glycoconjugate structures during hematopoietic development (32), but also provide a source to isolate large numbers of normal, proliferating myelomonocytic precursor cells.

In contrast to the transient nature of the lectin-mediated mitogenic response, WGA blasts are capable of further proliferation for up to 50 generations in the presence of SCF, although similar to hematopoietic progenitors in culture (38) and factor-dependent cell lines (39), cells undergo rapid apoptosis on growth factor withdrawal. Induction of self-renewal in committed myeloid progenitors not only implies additional levels of control during hematopoietic development, but also introduces the possibility of ex vivo autologous myeloid stem cell expansion to counter the effects of myeloablative therapies or in retroviral-mediated macrophage/accessory cell somatic cell gene therapy.

To ascertain whether a WGA blast-type cell that is capable of long-term proliferation without differentiation under the influence of SCF could be isolated from BM without prior WGA stimulation, BMC were cultured for 7 days in CMI supplemented with SCF (Fig. 1) . Although SCF stimulation had little effect on BM culture cellularity, more SCF-responsive colonies subsequently developed in semisolid cultures from SCF-stimulated BM cultures. Attempts to replate these colonies and induce subsequent long-term SCF-mediated cell proliferation were unsuccessful. These data suggest a role for prior WGA stimulation in the generation of myelomonocytic precursor cells. Nevertheless, we cannot rule out the possibility that stem cells under certain culture conditions can be expanded in the absence of WGA stimulation.

When freshly isolated BMC were stained with FITC-conjugated WGA and sorted into WGA+ and WGA- populations, all cells that respond to the lectin in culture were contained within the WGA+ compartment representing 48% of BMC. This indicates that cells responding to WGA stimulation (WGA-CFC), which have a frequency of >10-3 in bone marrow, possess an unidentified WGA binding structure, the ligation of which may generate proliferative signals in myelomonocytic stem cells.

The long-term repopulating murine hematopoeitic stem cell has been purified to near homogeneity by multi-parameter flow cytometry (40). Committed myeloid stem cells and CFU-spleen have also been enriched by methods including negative selection for a variety of mature lineage (lin) -specific markers (41), c-kit positivity (31), high levels of MHC class I (42, 43), early induction of class II expression (44, 45), low levels of CD45 (46), and species-specific differential expression of the CD90 molecule (47, 48). To estimate the ontogenic relationship between WGA blasts and defined stem cells and progenitors, cells were phenotyped by immunofluorescence and cytochemistry. The WGA blast antigenic profile lacking class II antigens with low basal expression of CD45 and myeloid lineage markers combined with high class I and CD90 expression (Table 1) is therefore consistent with an early myeloid progenitor immunophenotype. Consequential increases in CD45, CD90, and RM-1 antigenic densities on SCF stimulation characteristic of maturation from a linlow myeloid progenitor phenotype are, however, contrasted by further enhancement of the constitutively high WGA blast expression of CD90 and MHC class I that are down-regulated during development from the PHSC (48, 43). Changes in antigenic densities within a population of nondifferentiating progenitors may also represent a role for SCF/c-kit interaction in the regulation of these molecules, contributing to the generation of an antigenic and therefore functional heterogeneity within the myelomonocytic stem cell compartment (4) that would influence the phenotype of corresponding mature populations. Analogous expression increases for example, have also been observed on mature rat macrophages generated in GM-CSF-driven, differentiating BMC cultures (33) novelly stimulated with additional SCF (data not shown).

Regulation of hematopoiesis is achieved in part by multiple reactivity of cells to different cytokines (49). WGA blasts, however, show no proliferative response to the early-acting and lineage-specific growth factors IL-1 to IL-7, IL-11, EPO, GM-CSF, G-CSF, M-CSF either alone or in combination, or a crude spleen-conditioned medium (50) stimulated by pokeweed mitogen (data not shown). Morphological differentiation also is not seen in combinations of the above growth factors with SCF. The pleiotropic proliferative and differentiative effects of SCF on hematopoiesis in vitro are mediated primarily in synergy with certain other growth factors (reviewed in ref 51 ). In contrast to minor stimulatory effects of IL-6, IL-7, and IL-11, addition of G-CSF to SCF-supported semisolid cultures induced a substantial synergistic increase in the number and size of blast colonies (Fig. 6) . This implies that either WGA blasts are a nonhomogenous population containing both SCF and SCF/G-CSF responsive cells or that G-CSF is supporting SCF in the recruitment of WGA blast progenitors into proliferative colony-forming status similar to previously described progenitors (52).

In addition to peptide growth factors, the regulation of hematopoiesis is also differentially controlled by steroids at unique developmental stages. Rat stem cells are resistant to cortisone (48), which promotes the self-renewal of myeloid stem cells in long-term bone marrow cultures (53), whereas the synthetic glucocorticoid DEX induces the differentiation of myeloid progenitors (23) and preferential CSF-induced maturation of granulocyte over macrophage progenitors (54). Calcitriol (1,25-dihydroxyvitamin D3) is the most active metabolite of vitamin D and is produced from 25-hydroxyvitamin D3 by renal, placental, or macrophage expressed mitochondrial 1{alpha}-hydroxylases. Calcitriol preferentially induces macrophage colony formation (55), differentiates normal monocytes (56, 57) and myeloid leukemic cell lines to macrophages (57, 58), and has been used in the differentiation therapy of myelodysplasia (59). Full terminal myelomonocytic differentiation induced by DEX or calcitriol is only seen in WGA/SCF blasts in the presence of SCF as the single exogenously added growth factor. Requirement for SCF during differentiation induction indicates a pivotal role for SCF/c-kit interaction as a survival stimulus in the early stages of progenitor differentiation. Steroid-mediated differentiation induction in WGA/SCF blasts supports evidence for early myelomonocytic responsiveness to calcitriol and glucocorticoids, as well as a role for these compounds in the steady-state differentiation of myelomonocytic progenitors, and may lead to a better understanding of leukemogenic phenotypes in which gain of function point mutations causing ligand-independent autophosphorylation and constitutive c-kit activation (24-26) have been identified.

This study indicates a hitherto unknown role for lectins and SCF in the self-renewal, maturation, and steroid-driven differentiation of myelomonocytic progenitors. We describe a method to obtain homogenous cultures of self-renewing myelomonocytic progenitors that would have direct relevance to the construction of ex vivo immune cell expansion models.


   ACKNOWLEDGMENTS
 
We thank Amgen Biologicals (Thousand Oaks, Calif.) for recombinant rat stem cell factor, Dr. F. Kroese (University of Groningen, Holland) for mAb HIS49, Hoffman-La-Roche Ltd. (Basel, Switzerland) for calcitriol, and Magda Vermes for technical assistance. The authors are grateful to Dr. Peter Steinlein (Research Institute for Molecular Pathology, Vienna) for expert help in FACs sorting. This work was supported by Austrian Science Research Funds (project Nr 11728-MED) and grants from the Austrian National Bank (project Nrs 5546 and 4433).


   FOOTNOTES
 
1 Correspondence: Institute of General and Experimental Pathology, AKH-3Q, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: georg.boltz{at}akh-wien.ac.at

2 Abbreviations: BMC, bone marrow cells; CFU, colony forming unit; CM, culture medium; CMI, CM containing insulin; CMIS, CMI containing fetal calf serum; CSF, colony-stimulating factor; DEX, dexamethasone; EPO, erythropoietin; FITC, fluorescein isothiocyanate; G-CSF, granulocyte CSF; GM, granulocyte/macrophage CSF; IL, interleukin; lin, lineage; M, macrophage; mAb, monoclonal antibody; NSE, nonspecific esterase type-1; PHSC, pluripotent hematopoietic stem cell; SCF, stem cell factor; rh, recombinant human; rm, recombinant murine; rr, recombinant rat; WGA, wheat germ agglutinin.

Received for publication March 26, 1998. Revision received September 23, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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