Leukemia (1997) 11, 1753–1761 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 The role of soluble growth factors in inducing transient growth and clonal extinction of stroma cell dependent erythroblastic leukemia cells K Itoh1, J Friel1, C Laker1, W Zeller2, U Just1, S Bittner2, RJB Nibbs3, PR Harrison3, S-I Nishikawa4, KJ Mori5 and W Ostertag1 1Heinrich-Pette Institute for Experimental Virology and Immunology, Hamburg University; 2University Hospital Eppendorf, II Medical Clinics, Department of Oncology and Hematology, Hamburg, Germany; 3Cancer Research Campaign Beatson Laboratories, The Beatson Institute for Cancer Research, Glasgow, UK; 4Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University; and 5Department of Biology, Faculty of Science, Niigata University, Japan A coculture system of a murine erythroblastic leukemia cell line mia which meets many of these requirements.10,11 The leu- (ELM-D) with its supportive stromal cell line (MS-5) was estab- kemic cells require contact with primary cultured stromal lay- lished. Long-term growth of ELM-D cells is strictly stroma cell dependent. Interaction between stem cell factor (SCF) and its ers for growth in vitro and differentiate in response to Epo. A receptor, c-kit, was demonstrated to be important for stroma stroma-dependent cell line, termed ELM-D, was isolated from 12 cell-dependent growth by anti c-kit neutralizing monoclonal this erythroblastic leukemia. Utilizing this novel in vitro cul- antibody (mAb) inhibition experiments. Significantly, soluble ture system, we report the effect of soluble growth factors or growth factors such as granulocyte–macrophage colony- stromal elements on growth, differentiation and the induction stimulating factor (GM-CSF), interleukin-3 (IL-3) or SCF of MS- of cell death of these erythroid precursor cells. 5 stromal cells (MS-5 CM) could replace the requirement of stroma cells for a considerable period. However, ELM-D cells maintained in these growth factors underwent clonal extinction after 3–6 weeks unless contact with stroma was re-established. Materials and methods Furthermore, IL-3 or GM-CSF acted in a dominant manner in inducing cell death in the presence of stroma cells. Cells show- Growth factors, CM ing clonal extinction undergo programmed cell death and do not differentiate. These altered growth properties of ELM-D cells exposed to soluble growth factors or to stroma cells rGM-CSF and rEpo were gifts from Boehringer Mannheim appear to be analogous to those described for T or B cells (Mannheim, Germany). Conditioned media of Wehi-3D and 13 primed by antigen presenting cells and then grown in growth RAT1 GM 4 cells were used as sources of IL-3 or GM-CSF factors. unless indicated. Routinely, GM-CSF (5 U/ml), rGM-CSF Keywords: apoptosis; clonal extinction; erythropoiesis; stem cell (10 U/ml) and IL-3 (5 U/ml) were used after titration on FDC- factor; stroma cells P1 cells and rEpo (5 U/ml) after titration by CFU-e formation. MS-5 CM, used as the source of SCF, was concentrated 10- fold by ultrafiltration (Amicon, Witten, Germany). The amount Introduction of MS-5 CM applied was five-fold the amount required for half maximal growth stimulation of MC-9 cells. Three-fold Differentiation and maintenance of many, if not all, hemopoi- lower and higher amounts were also tested to ensure optimal etic cell types in vivo requires direct cell contact with stroma growth conditions. cells.1,2 The nature of these stroma cell interactions is as yet neither functionally nor molecularly defined, despite the recent progress in understanding the role of adhesion mol- Cells and culture conditions ecules in such systems.3–5 Mice mutated at the white spotted (W) or the Steel (Sl) locus have been invaluable for the Cell lines: MS-5 stromal and ELM-D cell lines were main- advancement of our knowledge of cell-to-cell interaction. tained in a-MEM medium supplemented with 20% horse These mice are defective for either the c-kit receptor6 or its serum of selected batches. ELM-D cells were maintained on ligand, SCF,7 and are severely anemic. This indicates that SCF irradiated MS-5 cell layers (16000 rad) by passaging every 2– acts selectively on primitive and erythroid progenitors. SCF 3 weeks and then cloned by serial transfers. One clone, ELM- induces in synergy with erythropoietin (Epo) extensive pro- D No. 6, was routinely used in further experiments. liferation of erythroid progenitor cells.8 Extensive studies have been carried out in order to under- stand the mechanisms of growth and differentiation of Evaluation of cloning efficiency: Cells were plated into erythroid cells utilizing erythroleukemia cell lines established 96-well plates by limiting dilution and cloning efficiencies with the Friend virus.9 One major problem is that these cells were evaluated after 2–3 weeks according to the Poisson have lost their normal requirements for soluble growth factors distribution. or stromal interactions and their ability to differentiate in response to physiological growth factors such as Epo in vivo. Thus, an in vitro culture system retaining these features of nor- Determination of the frequency of stroma cell independent mal erythropoiesis has been keenly sought for a long time. growth: Cells were cloned in semi-solid medium (103–105 Recently, we reported a novel murine erythroblastic leuke- cells/ml) or seeded into 96-well plates (0.5–104 cells/well) without stroma. After 3–4 weeks of culture, the frequency of independent growth was evaluated. Most of the colonies, Correspondence: W Ostertag, Heinrich-Pette Institute for Experi- found after 2–3 weeks of culture, degenerated. mental Virology and Immunology, Hamburg University, Martinistrasse 52, 20251 Hamburg, Germany First and second authors contributed equally Received 18 October 1996; accepted 18 May 1997 Semi-solid culture of two layers: The two layer method of Ligand-induced dominant extinction of myeloid cells K Itoh et al 1754 agar (0.3%) was performed as described by Sibley and evaluated by limiting dilution. In contrast to the high cloning Tomkins.14 Cloning efficiencies were evaluated after 2 weeks efficiency of ELM-D cells in coculture with irradiated stroma by counting colonies. (30–50%), a significantly reduced cloning efficiency (2–3%) was observed when ELM-D cells were cultured in growth media without stroma or growth factors (Table 1). Cloning Assay for long-term growth: Cells were cloned by limiting efficiencies in the presence of MS-5 CM or growth factors such dilution and cloned cells were transferred at a 1/10 dilution as GM-CSF or IL-3 were comparable to those obtained with into new wells every week. The percentage of positive wells stroma (Table 1). with growing cells was corrected by initial cloning efficiencies To analyze the growth dependency in more detail, the and presented as ‘% clones surviving’. To analyze the long- mutation frequency of ELM-D cells to stroma-independent term growth in more detail, changes of the cloning efficiencies growth was determined. In both semi-solid cloning and limit- were also examined. After cloning by limiting dilution, cloned ing dilution experiments in liquid culture, the survival rate of cells were recloned every 2–3 weeks and changes in the ELM-D cells was extremely low after 3–4 weeks of culture, cloning efficiencies were recorded. estimated to be ,3 × 10−5 in liquid and ,3.5 × 10−6 in semi- solid culture. Growth inhibition assay using ACK-2 mAb: Cells were plated into Terasaki plates at a concentration of 50 cells/well with 1:3 serial dilution of ACK-2 mAb15 (maximum concen- Conditioned media (CM) cannot support the long-term tration: 30 mg/ml). The number of cells/well were recorded growth of ELM-D cells after 5 days of culture. Since the initial cloning efficiency in the presence of MS-5 Morphological examination and detection of differentiated CM was comparable to that on stroma (Table 1), we examined cells: Cells were concentrated on slides and stained by whether the coculture with stroma is required for the long- May–Gru¨ nwald Giemsa and benzidine. Differentiated cells term growth of ELM-D or, alternatively, if CM is sufficient. All were determined by morphological examination. of 115 clones transferred in the presence of CM lost their self- renewal capacity and showed clonal extinction within 6 weeks, whereas all the clones transferred on irradiated MS-5 Reverse transcriptase (RT)-PCR analysis survived during the same time period (Figure 1). This demon- strates that although ELM-D cells can clone initially at high Detection of mRNA for SCF: Expression of mRNA for SCF efficiency in CM, CM is not sufficient to support long-term was examined by RT-PCR as described previously.16 Specific growth of ELM-D cells, suggesting the importance of direct DNA fragments were amplified by PCR for 25 cycles, separ- contact with stroma. ated, transferred, hybridized and exposed to X-ray films. Primer sets used in this study were SCF1: position 126–145 and 1041–1060 and SCF 2: position 845–863 and 1041– 17 1060. Table 1 ELM-D cells can be cloned in the presence of MS-5 CM, GM-CSF or IL-3 or MS-5 CM for short time only a Semi-quantified assay of -globin mRNA: Semi-quantitat- Condition Cloning efficiency (%) ive RT-PCR for a-globin was additionally performed. Various Mean (No. of experiments) cycle numbers had been previously tested to ensure a linear range of amplification and cDNAs were amplified for 15 ELM-Da ELM-D No. 6b cycles. Oligos used for a-globin expression were position 460–480 and position 930–950. After densitometric scanning On MS-5 layer of the autoradiographs, ratios of a-globin to b-actin18 were unirradiated 47 ± 20c (4) 54 ± 21 (2) calculated.