Growth and Differentiation of a Murine Interleukin-3-Producing Myelomonocytic Leukemia Cell Line in a Protein-Free Chemically Defined Medium


Yasuhiko Kajigaya, et al

Abstract.  We established the continuous growth of WEHI-3B D+ cells in protein-free chemically defined F-12 medium by stepwise decreases in the concentration of fetal calf serum.  This cell line, designated as WEHI-3B-Y1, has now been propagated in protein-free F-12 medium for 3 years.  The population-doubling time of the cells in culture is about 24 hours.  WEHI-3B-Y1 cells are immature undifferentiated cells which show positive staining for naphthol ASD chloroacetate esterase and ƒż-naphthyl butyrate esterase and spontaneously exibit a low level of differentiation to mature granulocytes and macrophages.  Medium conditioned by WEHI-3B-Y1 cells stimulated the proliferation of an interleukin-3 (IL-3)-dependent FDCP-2 cell line. This conditioned medium was shown to have erythroid burst-promoting activity when assayed using normal murine bone marrow.  The colony formation of WEHI-3B-Y1 cells in semi-solid agar culture was not stimulated by purified recombinant human granulocyte colony-stimulating factor (rhG-CSF).  However, in the presence of human transferrin, rhG-CSF enhanced the number of colonies of WEHI-3B-Y1 cells but did not induce their differentiation. These results suggest that WEHI-3B-Y1 cells cultured in protein-free medium produced murine IL-3. In addition, human G-CSF enhanced the clonal growth but did not induce the differentiation of WEHI-3B-Y1 cells cultured in serum-free medium.




 IL-3: interleukin-3

 rhG-CSF: recombinant human granulocyte colony-stimulating factor

 rhGM-CSF: recombinant human granulocyte-macrophage colony-stimulating factor

 FCS: fetal calf serum

 BSA: bovine serum albumin

 BPA: burst-promoting activity

 IMDM: Iscove's modified Dulbecco's medium

 CM: conditioned medium

 MLCM: mouse-lung-conditioned medium





   Murine leukemia cell lines are important models for studying the growth and differentiation of malignant hemopoietic cells (1).  WEHI-3B myelomonocytic leukemia cells constitutively produce interleukin-3 (IL-3) (2-4), that has erythroid burst-promoting activity and also supports the differentiation of multilineage hemopoietic progenitors (5,6).  It has been reported that WEHI-3B leukemia cells can be induced to differentiate into mature granulocytes and monocytes by incubation with either murine granulocyte colony-stimulating factor (G-CSF) or human G-CSF (D+ subline)(7-9).

   This report describes the growth and differentiation of a WEHI-3B D+ cell line in a protein-free chemically defined medium .  The new cell line was designated as WEHI-3B-Y1, and was morphologically similar to the original WEHI-3B D+ cell line.  We studied the effect of purified recombinant human G-CSF on the clonal growth and differentiation of these cells in serum-free medium, and also examined whether or not the cells could produce IL-3 when cultured without any protein supplementation. 



                  MATERIALS AND METHODS


   Cell lines.   A murine myelomonocytic leukemia cell line, WEHI-3B D+ (10), was kindly donated by Dr. S. Asano (Department of Medicine, Department of Pathological Pharmacology, Institute of Medical Sciences, University of Tokyo).  The cells were maintained in F-12 synthetic medium (Flow Laboratories, Irvine, Scotland) supplemented with NaHCO3, 0.75 g/liter, and 5% fetal calf serum (FCS) (HyClone Laboratories, Logan, Utah).  Medium conditioned by WEHI-3B D+ (WEHI-3B D+ CM) was collected after 3 days and centrifuged.  The IL-3-dependent cell line, FDCP-2 (11), was kindly donated by Dr. T. Suda (Department of Hematology, Jichi Medical School), and was maintained in F-12 with 10% FCS and 10% WEHI-3B D+ CM.  Both cell lines were cultured at 37Ž

in 25-sq cm tissue culture flasks (Corning Glass Works. Corning, NY) containing 8 ml of medium in a humidified atmosphere of 5% CO2/95% air.


   Morphological examination.   Cultured cells were spun in a cytocentrifuge (Cytospin Centrifuge, Shandon Southern, Sewickley, PA)  and stained with Wright-Giemsa.  The cells were also evaluated for their staining with ƒż-naphthyl butyrate esterase and naphthol ASD chloroacetate esterase.


   Semisolid culture.   Cultured cells were mixed in F-12 medium containing 0.3% purified agar (Difco Laboratories, Detroit, Michigan), and 1-ml aliquots of medium were plated onto 35- x 10-mm plastic dishes.  Each dish contained 1~103 cells in 1-ml of medium with appropriate stimulators.  Incubation was performed in a humidified atmosphere of 5% CO2/95% air at 37Ž, and colonies containing >40 cells were counted after an incubation period of 14 days using an inverted microscope.  Individual colonies were removed with a fine pipette, placed onto microscope slides, and allowed to dry.  Cells were stained with 0.6% orcein in 60% acetic acid (Muto Pure Chemicals, Tokyo, Japan).  


   Recloning of colony cells.   Individual colonies were removed with a fine pipette and added to 5-ml of liquid agar medium.  Colony cells were resuspended by pipetting into agar medium, and the cell suspension was pipetted in 1-ml aliquots into new culture dishes.  Colony formation by these recloned cells was assessed after a further incubation period of 14 days.


   Materials.   We obtained purified recombinant human granulocyte colony-stimulating factor (rhG-CSF)(specific activity 1~108 units/mg protein) (9) and purified recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF)(specific activity 1~108 units/mg protein) from the Kirin-AMGen Company (Thousand Oaks, California). Partially purified human transferrin, fraction – bovine serum albumin (BSA), and partially purified bovine insulin were purchased from Sigma Chemical Company (St. Louis, Mo).  The transferrin was fully iron-saturated with FeCl3, and the BSA was deionized with ion exchange resin.


   Proliferation assay of IL-3-dependent FDCP-2 cells.   Assays were carried out in microtiter wells, using washed FDCP-2 cells suspended at 106 cells/ml in F-12 with 10% FCS.  Wells were set up in quadruplicate to contain 50 ƒĘl of FDCP-2 cells plus 50 ƒĘl of the test culture medium, and were incubated overnight at 37Ž in 5% CO2.  At 24-hour intervals, cultures were pulsed with [3H] thymidine (1.0 Ci in 10 ƒĘl), for 16 hours.  Cells were harvested onto filters using an automated cell harvester, and [3H] thymidine uptake was measured with a scintillation counter.


   Assay of erythroid burst-promoting activity (BPA).   Bone marrow cells were obtained from BALB/c female mice (8-12 weeks old) by flushing the bone marrow from the femur into Iscove's modified Dulbecco's medium (IMDM)(Flow Laboratories) through a 23-gauge needle; the bone marrow cells were then washed twice with the same medium.  The cells were cultured by the methylcellulose method of Iscove (12), and the BPA of the culture medium was assayed as follows.  Bone marrow cells (2~105) were cultured in 1-ml of IMDM containing 0.9% methylcellulose (Fisher Scientific Company, Norcross, GA), 1% deionized fraction – BSA , 2 units/ml of recombinant human erythropoietin (Kirin-AMGen Inc.) (12), 30% FCS, 5~10-5M 2-mercaptoethanol (Tokyo-Kasei Kogyo Company, Tokyo, Japan), and 10% culture medium.  Cultures were performed in triplicate with incubation being carried out in a humidified atmosphere of 5% CO2/95% air at 37Ž, and erythroid bursts were counted on day 11.  The BPA was expressed as the number of bursts observed.


   Mouse-lung-conditioned medium.   Mouse-lung-conditioned medium (MLCM) was prepared in vitro using lung tissue from C57BL mice (8-12 weeks old) preinjected with 5 ƒĘg of endotoxin (E. coli W, lipopolysaccharide; Sigma Chemical Company, St. Louis, Missouri)(14).  The MLCM was serially diluted using phosphate-buffered saline and filtered through a millipore filter before use. 





   Stepwise decreases in the FCS concentration were made at weekly or biweekly intervals until there was no serum left in the medium: the time required could be as short as 3 months. Although the most of the cells were round in shape and remained in suspension, during adaptation a few cells adhered to the bottom of the culture flasks. The cells established in protein-free medium were designated as WEHI-3B-Y1 cells, and were morphologically similar to the original WEHI-3B D+ cells.  Most of the cells were undifferentiated blast cells with round or oval nuclei containing from one to several nucleoli (Fig. 1-A).  The cytoplasm was deeply basophilic.  These cells were weakly positive for naphthol ASD chloroacetate esterase and positive for ƒż-naphthyl butyrate esterase.  As shown in Figs. 1-B and 1-C, WEHI-3B-Y1 cells spontaneously exhibited a low level of differentiation to mature granulocytes (0.3}0.1%) and macrophages (0.2}0.1%).  The original WEHI-3B D+ clone also spontaneously developed 1.5}0.3% mature granulocytes and macrophages in serum-supplemented culture.  Our cell line has now been propagated continuously in protein-free F-12 medium for about 3 years and the cells used in these experiments were from the 60th-passage.  The in vitro growth characteristics of the WEHI-3B-Y1 and WEHI-3B D+ cells are shown in Fig. 2.  The experimental cultures were initiated with an inoculum of 3~104 cells/ml and growth curves were obtained by counting the number of cells per dish.  From the growth curve shown in Fig. 2, the population-doubling time of WEHI-3B-Y1 cells was estimated as 24 hours.  Conditioned medium (WEHI-3B-Y1 CM or WEHI-3B D+ CM) was collected after 3 days and cenrifuged.     We investigated the effect of WEHI-3B-Y1 CM on erythroid-burst formation of murine bone marrow cells.  WEHI-3B-Y1 CM significantly increased the number of erythroid bursts compared to the control (Table 1).  The effect of WEHI-3B-Y1 CM in a proliferation assay is shown in Table 2.  WEHI-3B-Y1 CM stimulated [3H]thymidine uptake by IL-3-dependent FDCP-2 cells. 

   When both WEHI-3B D+ and WEHI-3B-Y1 cells were cultured for 7 days in agar medium containing FCS and medium, the cells from both lines proliferated and formed colonies, the majority of which were tightly-packed spherical aggregates.  In contrast, when both cell lines were cultured in medium containing MLCM or rhG-CSF, most of the colonies formed were of the spreading type (Table 3).  Morphological analysis showed that the spreading colonies formed by both cell lines were composed of either granulocytes at varying stages of maturation or else differentiated macrophages.  The ability of these cells to form colonies containing differentiated cells was verified by means of the original serum-supplemented soft agar culture system (1,7,8).

   The WEHI-3B-Y1 cells could not proliferate in protein-free semisolid agar, but these cells proliferared to form colonies in the presence of human transferrin or FCS (Table 4).  Most of these colonies were compact and composed mainly of immature blast cells.  We examined the effects of rhG-CSF on the growth of clonogenic WEHI-3B-Y1 cells using an in vitro serum-free colony assay.  When rhG-CSF alone was added, no WEHI-3B-Y1 colonies were formed.  However, the number of WEHI-3B-Y1 colonies formed after the addition of rhG-CSF plus human transferrin was increased significantly in comparison to treatment with human transferrin alone.  To examine the correlation between the rhG-CSF concentration and the enhancement of WEHI-3B-Y1 colony formation, we then added various doses of rhG-CSF to cultures simultaneously supplemented with human transferrin at 200 ƒĘg/ml (Fig. 3).  Colony formation was enhanced at concentrations of rhG-CSF above 500 units/ml and marked enhancement was observed between 2,000 and 4,000 units/ml of rhG-CSF.  However, after 28 days of culture the colonies were tightly packed and consisted entirely of immature blasts that showed no signs of morphological differentiation, even in the presence of rhG-CSF.  When 14-day colonies were analyzed for their content of colony-forming cells by resuspending and reculturing each individual colony, it was found that the relative frequency of clonogenic cells per recloned colony was not reduced by rhG-CSF (Table 5).





   The WEHI-3 myelomonocytic leukemia was originated in BALB/c mice by the injection of mineral oil, and a B-subline has been maintained by serial transplantation in syngeneic mice (10).  The WEHI-3B subline forms colonies in semi-solid agar culture, and was established as a cloned continuous cell line in suspension culture (15).  These cells are responsive to the induction of differentiation (D+ subline) (7-9). Partially purified BPA from WEHI-3B-conditioned medium has been shown to support the differentiation of multilineage hemopoietic progenitors (5).  The protein responsible for this activity was purified to apparent homogeneity, and termed interleukin-3 (IL-3) (4).  IL-3 is a glycoprotein with a molecular weight of 28,000-30,000 daltons that has a variety of biological activities, including induction of the enzyme 20-alpha-hydroxy steroid dehydrogenase in cultured nu/nu splenic lymphocytes, stimulation of the proliferation of an FDCP-2 cell line, stimulation of mast cell growth, and stimulation of P cells (3).

   Our studies have shown that WEHI-3B D+ leukemia cells can be propagated in protein-free chemically defined F-12 medium without serum components or growth factors.  The WEHI-3B-Y1 cells established in such protein-free medium were morphologically similar to the original WEHI-3B D+ cells, had a 24-hour population-doubling time, and spontaneously exhibited a low level of differentiation to mature granulocytes and macrophages.  This spontaneous differentiation may corroborate the autoinduction of differentiation in WEHI-3B leukemia cells cultured under serum-supplemented conditions (16,17). 

   Our study revealed the presence of erythroid BPA in WEHI-3B-Y1 CM, and showed that WEHI-3B-Y1 CM could stimulate the proliferation of IL-3-dependent FDCP-2 cells.  These results indicate that WEHI-3B-Y1 cells retain the characteristic function of producing murine IL-3 and also that they can produce IL-3 in the protein-free culture.

   Since the growth of cultured cell lines generally depends upon serum components, serum-free and protein-free cultures are required to examine the effects of various factors on cellular growth and differentiation.  It has been reported that a human erythroleukemia cell line (K-562-T1) (18), a human myelocytic leukemia cell line (HL-60) (19), and the T3M-1-T2 cell line (20) that produces a colony-stimulating factor can all propagate continuously in serum-free and protein-free media without the need for insulin, transferrin, albumin, or other growth factors and hormones.  These cell lines are useful tools for obtaining a better understanding of cell growth and differentiation (18,19,20).

   Murine leukemia cell lines, including WEHI-3B D+, are important models for studying malignant hemopoietic cell growth and differentiation (1).  It was reported previously that WEHI-3B D+ cells can be induced to differentiate into mature granulocytes and monocytes by incubation with murine G-CSF or human G-CSF (8,9).  However, B*hmer and Burgess (21) observed that at low cell densities (<105/ml) neither murine G-CSF nor human G-CSF altered the rate of WEHI-3B D+ growth, nor did these factors induce the differentiation of WEHI-3B D+ cells.

   In the present studies, we examined the effects of purified rhG-CSF on the growth of clonogenic WEHI-3B-Y1 cells with a serum-free colony assay.  When rhG-CSF alone was added, no colonies were formed but colony formation was supported by human transferrin or FCS.  The number of WEHI-3B-Y1 colonies stimulated by rhG-CSF plus human transferrin was increased significantly in comparison with cultures supplemented with human transferrin alone.  The growth of WEHI-3B-Y1 colonies was enhanced as the concentration of rhG-CSF increased. However, even after 28 days of culture in the presence of rhG-CSF the colonies were tightly packed and consisted only of immature blasts, with no signs of morphological differentiation.  Furthermore, successful recloning of colony cells was not impaired by culture with rhG-CSF.  These results suggest that rhG-CSF enhances the clonal growth of WEHI-3B-Y1 leukemia cells, but does not induce their differentiation under serum-free conditions.  Therefore, the assessment of human G-CSF stimulated growth using WEHI-3B-Y1 cells in serum-free medium should be more sensitive than assays using WEHI-3B (D+) cells in serum-supplemented medium.




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