THE CLONING AND EXPRESSION OF THE cDNA FOR OVINE STEM CELL FACTOR (KIT-LIGAND) AND CHARACTERIZATION OF ITSIN VITROHAEMATOPOIETIC ACTIVITY

Article No. cyto.1998.0430, available online at http://www.idealibrary.com on

THE CLONING AND EXPRESSION OF THE cDNA FOR OVINE STEM CELL FACTOR (KIT-LIGAND) AND CHARACTERIZATION OF ITS IN VITRO HAEMATOPOIETIC ACTIVITY Colin J. McInnes, David Deane, Jackie Thomson, Andrea Broad, David M. Haig The cDNA encoding the soluble form of ovine stem cell factor (SCF) has been cloned and expressed. The soluble protein is predicted to be 165/166 amino acids in length, one more than the human and murine SCFs with which it shares 87% and 81% identity respectively. Ovine SCF has 98.5%, 95% and 91% identity with cattle, pig and dog SCF, respectively. The recombinant ovine (rov) SCF protein has been expressed in Chinese hamster ovary (CHO) cells, purified, and its biological activity on ovine bone marrow cells compared with that of interleukin 3 (rovIL-3), granulocyte–macrophage colony-stimulating factor (rovGM-CSF), interleukin 5 (rovIL-5), human macrophage colony-stimulating factor (M-CSF) and human erythropoietin (epo). On its own rovSCF supported the development of small numbers of neutrophil, macrophage, eosinophil, granulocyte-macrophage, mixed cell phenotype, haemopoietic blast cell and basophilic granular cell colonies in a soft agar clonogenic assay. In combination with each of the above cytokines rovSCF supported an increase in the number and size of the lineage-specific colony types that were stimulated by the other cytokines on their own. In an assay for precursors of multipotential colony-forming cells (multi-CFC), rovSCF in combination with rovIL-3 (but neither cytokine alone) supported the development of these early haematopoietic progenitor cells  1999 Academic Press

Stem cell factor (SCF), also known as kit-ligand, steel factor and mast cell growth factor is the ligand for the c-kit tyrosine kinase receptor (reviewed in Ref. 1). SCF is a pleiotropic cytokine which has a profound effect on a number of different cell types, including an important function in stimulating haematopoiesis. In the mouse SCF is encoded by the Steel locus (Sl), mutations in which lead to anaemia, impaired development of melanocytes and in extreme cases sterility due to an absence (or reduction in numbers) of germ cells.2–5 Steel mice are deficient in mast cells, indicating a role for SCF in the generation and/or survival of these cells.6 Human and rodent SCFs support the survival of haematopoietic stem and progenitor cells, and act synergistically with other cytokines in vitro to increase the numbers and size of a variety of different cell colonies in agar or methylcellulose cultures of From the Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, UK Correspondence to: C. J. McInnes, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK Received 1 December 1997; accepted for publication 15 May 1998  1999 Academic Press 1043–4666/99/040249+08 $30.00/0 KEY WORDS: haematopoiesis/ovine/stem cell/SCF CYTOKINE, Vol. 11, No. 4 (April), 1999. pp 249–256

haematopoietic progenitor cells.7–10 SCF along with IL-3 in particular is important in regulating the survival, proliferation and phenotypic heterogeneity of mast cells1,711 and is up-regulated during, and involved in, the generation of host anti-nematode parasite inflammatory cell responses in vivo.12 Alternative splicing of the SCF gene gives rise to two products.13–15 Both are membrane-bound proteins, the larger of which contains a proteolytic cleavage site for generating a soluble form of the factor. The cDNAs for SCF have been cloned from a number of species including: man,9 mice,14 rats,9 dogs,16 cats,17 chickens,18 pigs19 and cattle20 amongst others. However whilst the recombinant rodent proteins are active on human cells the recombinant human protein is only minimally active on mouse or rat cells.9 Neither human nor rodent SCFs appear to be active on ovine haematopoietic cells (unpublished observations). The purpose of this study was to clone and express the cDNA for ovine SCF and determine whether ovine recombinant SCF exhibits similarities or differences to SCF activity in other species in appropriate haematopoietic progenitor cell clonogenic assays. The sheep is a useful species for studying aspects of: xenogeneic haematopoietic stem cell adoptive transfer,21 249

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Figure 1.

CYTOKINE, Vol. 11, No. 4 (April, 1999: 249–256)

The nucleotide and conceptual amino acid sequence of the extracellular domain of ovine SCF.

Primer sequences are indicated in bold as are the amino acids derived from the human sequence. Numbering is with respect to the predicted start of the mature protein, indicated, with a #. The extra Glu130 residue (cf. the human and rodent sequences) is marked by an asterisk. The –COOH terminus of the soluble from of SCF is predicted to be between, or after, residues Ala165 and Ala166 and is marked with arrows (?).

reproduction,22,23 cloning by nuclear transfer24 and host inflammatory responses to parasites (Ref. 25 and MacAldowie et al., in press). Ovine SCF will prove valuable in studies of these different processes.

RESULTS Cloning and sequencing of the ovine SCF cDNA The SCF cDNA was amplified by PCR from RNA isolated from primary ovine keratinocyte cultures and cloned into the PCR cloning vector pCR2.1. Two clones picked at random were sequenced in their entirety and found to be identical. The sequence corresponds to the first 201 amino acids of the human and rat proteins. The ovine sequence is presented in Figure 1 together with its conceptual translation. It contains an additional three nucleotides, by comparison to the human sequence, encoding a Glu residue found at position 130 with respect to the predicted N-terminus of the mature protein. Overall the ovine SCF sequence

was found to have between 68% and 98% nucleotide identity and 54% to 98.5% amino acid identity with other SCF sequences previously reported. The highest identity was with the cattle SCF whereas the lowest was with chicken SCF. The first two amino acids and the last six amino acids given for the ovine SCF are derived from the primer sequences (and therefore the human sequence). A comparison of the ovine sequence with that of the cattle, pig, dog, human, mouse, rat, and chicken SCFs is shown in Figure 2. The ovine SCF cDNA sequence was deposited in the Genbank/EMBL nucleotide database under the Accession No. Z50743.

Expression of rovSCF and characterisation of its in vitro haematopoietic activity Northern analysis confirmed the expression of ovine SCF mRNA in the CHO cells transfected with the pEE14SCF plasmid, but not those transfected with pEE14 control plasmid (data now shown). Stem cell factor cytokine activity was purified by FPLC anion exchange and gel filtration chromatography (Fig. 3),

Biological activity of ovine SCF / 251

Figure 2. A comparison of the predicted amino acid sequences of the first six exons of the SCF gene from sheep, cattle, pigs, dogs, humans, mice, rates and chickens. The sequences are listed in descending order of identity with the ovine sequence. Amino acids common to all the sequences are indicated by asterisks. The predicted site of proteolytic cleavage in exon six is between, or after, Ala165 and Ala166 (numbering according to the mature protein) and is indicated by ##. The predicted site of signal peptide cleavage is indicated by a+.

and activity assayed in the soft agar clonogenic assay. Recombinant ovine SCF stimulated a range of myeloid cell colonies in the soft agar clonogenic assay (Table 1). BFU-E, however, were detected only when rovSCF was used in combination with epo. At concentrations that stimulated maximum numbers of colonies (5–10 U/ml), rovSCF did not stimulate as many colonies as equivalent concentrations of rovIL-3, rovGM-CSF or M-CSF. This indicated that only a proportion of ovine progenitor colony-forming cells (CFC) formed colonies in the presence of rovSCF alone. When used at sub-optimal concentration (1U/ml), rovSCF synergised with rovIL-3, rovGMCSF, and M-CSF to stimulate significantly more colonies than the sum of those stimulated by the cytokines individually (P<0.05 for all comparisons,

Table 1). In addition, the majority of colonies were increased in size in the cultures containing rovSCF in combination with each of these cytokines compared to those in cultures containing each of the cytokines alone. The combination of rovSCF and epo stimulated the largest BFU-E colonies observed in this study. The differential colony analysis revealed that rovSCF together with each of rovIL-3, rovGM-CSF, M-CSF, rovIL-5 and epo supported an increase in the number and size of lineage specific colony types that were supported by the latter cytokines on their own (Table 1) as well as supporting in addition an increase in the number of mixed and haematopoietic blast colonies. For example, rovSCF and IL-5 increased the frequency and size of eosinophil colonies compared to IL-5 alone. Mixed and haematopoietic blast colonies

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CYTOKINE, Vol. 11, No. 4 (April, 1999: 249–256)

were particularly frequent in cultures stimulated with rovSCF plus rovIL-3. The earliest haematopoietic progenitor cells that can be detected using the soft agar clonogenic assay are defined as multi-CFC,26 which give rise to large mixed cell phenotype colonies. In order to determine whether rovSCF supported the development of earlier haematopoietic cells, the ovine pre-multi-CFC assay was used. This assay detects the ability of cytokines to stimulate precursors of multi-CFC to develop into multi-CFC in liquid bone marrow cell culture, which are then A

detected using the soft agar clonogenic assay. This assay has been proved in detail elsewhere.27 Figure 4 shows that increased numbers of multi-CFC (detected as mixed colonies) developed over 6 days in cultures stimulated with rovSCF plus rovIL-3, and to a much lesser extent with rovSCF plus M-CSF or rovSCF plus rovGM-CSF. The combination of rovSCF plus rovIL-3 and M-CSF supported increased numbers of mixed colonies in all assays, but this was significant (P<0.05) in only one (not shown). However, there was a significant increase in the number of mixed colonies in all assays after liquid culture in the presence of the above cytokine combinations compared to the number detected prior to liquid culture (P<0.01, Fig. 4).

DISCUSSION

66.2 kDa

45 kDa

31 kDa

21.5 kDa

1

2

B 66.2 kDa

31 kDa

Ovine SCF cDNA was amplified from ovine keratinocytes. These cells reproducibly produced the greatest amount of SCF PCR product compared to other cell and tissue sources for reasons that are unknown, but is consistent with the finding that human keratinocytes have been shown to produce SCF.28 Although a full-length (protein coding sequence only) cDNA was amplified from ovine keratinocytes, only the cDNA sequence corresponding to the first six exons, representing the soluble form of SCF, is reported here. The ovine sequence has four Cys residues which are conserved in all SCF sequences previously reported and four putative Asn-linked glycosylation sites which are conserved in the mammalian species. In common with the cattle, pig, cat and dog SCF sequences, there is an extra Glu residue at position 130. Gentry et al.23 published the full coding sequence of an ovine SCF cDNA including 70 bases of the 5 non-coding region and the first 58 nucleotides of the 3 non-coding region. The sequence reported here

Figure 3. A: A 15% SDS-polyacrylamide gel of rovSCF expressed in CHO cells is shown.

31 kDa

21.5 kDa

1

2

A: Lane 1 contains CHO cell conditioned medium after anion exchange chromatography through Q-Sepharose (Pharmacia). Bound protein was eluted by application of a linear NaCl gradient from 0–0.5 M buffered with 20 mM Tris–HCl pH 8.0. The rovSCF eluted between 0.15 M and 0.16 M NaCl. Lane 2 contains material from lane 1 concentrated and applied to a 48-ml Sephacryl 200HR column (Pharmacia) and eluted using 0.15 M NaCl, 20 mM Tris– HCl pH 8.0. B: Western blot analysis of FPLC purified rovSCF. Lane 1 contains FPLC purified rovSCF as in (A) lane 2. Lane 2 contains affinity purified rovGM-CSF. Proteins were separated on a 15% SDS-polyacrylamide gel before transfer to a cellulose nitrate membrane. The membrane was blocked with 5% fat-free milk proteins in PBS and then washed in 20 mM Tris–HCl pH 8.0, 0.5 M NaCl and 0.5% Tween 80, before incubation with a goat polyclonal anti-rhuman SCF IgG fraction (2 ìg/ml; R&D Systems, Abingdon) for 1 hour followed by incubation with a HRP-conjugated anti-goat IgG reagent (Dako). Bound antibody was visualized by the ECL chemiluminescence method (Amersham International).

Biological activity of ovine SCF / 253

TABLE 1.

Activity of recombinant ovine Stem cell factor (rovSCF) in ovine bone marrow soft agar clonogenic assays

Sample (dilution/activity) CHO-con† (1:100) SCF (10 U/ml) SCF (5 U/ml) SCF (1 U/ml) SCF (0.5 U/ml) GM-CSF+CHO-con IL-3+CHO-con M-CSE+CHO-con IL-5+CHO-con epo+CHO-con‡ SCF§+GM-CSF SCF+IL-3 SCF+M-CSF SCF+IL-5 SCF+epo‡

Day 7 cols

Day 7 clus

Day 14 cols

Day 14 clus

12 44 48 28 18 94 80 55 22 14 136 130 124 48 46

24 76 84 42 26 120 86 72 74 36 164 174 126 124 82

6 48 52 30 20 98 92 52 48 16 144 176 138 88 58

14 58 61 46 18 102 64 66 64 28 142 136 102 104 62

mix*

GM

mac

neut

eos

blast

mast/bas

bfu-e

other

1 0

4 2

15 10

4 1

15 9

2 0

5 2

0 0

6 4

0 5 0 0

14 0 1 0

28 24 28 12

10 1 4 2

32 28 8 30

1 12 0 0

1 12 0 0

0 0 0 0

12 18 9 4

2 10 8 2 2

22 6 9 5 3

39 41 58 16 17

12 10 11 5 5

41 52 18 50 17

9 16 15 4 4

6 20 2 2 3

0 5 3 0 4

12 15 14 4 3

Mean (n=3 replicate plates) colonies (cols) and clusters (clus) per 105 bone marrow cells seeded on day 0. S.D. were within 10% of the values shown. This experiment is representative of three in total. Blank spaces=not determined. CHO-con=cell-free supernates (CFS) from mock-transfected CHO cells; Cytokine concentrations were: rovGM-CSF (10 U/ml), rovIL-3 (10 U/ml), rovIL-5 (10 U/ml and rhuM-CSF (100 U/ml). *Differential day 14 colony analysis was as described in Materials and Methods: Mix, Mixed colonies; GM, granulocyte-macrophage colonies; mac, macrophage colonies; neut, neutrophil colonies; eos, eosinophil colonies; blast, haematopoietic blast cell colonies; mast/bas, basophilic granular cell colonies; bfu-e, erythroid burst-forming units; other, colonies that could not be phenotyped. †CHO-control supernates and medium control cultures contained similar numbers of colonies and clusters. Only the CHO-con results are shown. ‡In cultures containing epo and SCF+epo there were 30628 and 32822 (S.D.) erythroid cluster-forming cells (CFC-E) respectively on day 5. §rovSCF used at 1 U/ml in combination with the other cytokines.

has two nucleotide changes compared to that reported by Gentry et al.,23 neither of which result in changes to the amino acid sequence. Recombinant ovine SCF demonstrated a spectrum of in vitro haematopoietic activities similar to those of SCFs in other mammalian species. In particular, the ability to synergize or co-operate with other haematopoietically active cytokines to stimulate the development of early haematopoietic pre-multiCFC and enhance the number and size of cell colonies in the soft agar clonogenic assay was observed. Synergy with IL-3, GM-CSF, M-CSF and epo in increasing colony numbers has been shown for SCF in other species.10,29,30 The combination of SCF and IL-3 (with or without additional cytokines) has also been shown to support haematopoietic stem cell survival and development.29,31 There is no reliable stem cell assay in sheep, and the pre-multi-CFC assay detects the earliest cells in ovine haematopoiesis that we are aware of. IL-3 is a mast cell growth factor in the sheep32 and in a separate study we have demonstrated that rovSCF alone will support mast cells in liquid cultures of ovine bone-marrow cells, and that a large increase in the numbers of these cells is obtained when SCF is used in combination with IL-3 (MacAldowie et al., in press). The rovSCF therefore has been shown to support the enhanced development of: neutrophils, macrophages, eosinophils, mast cells, and erythroid BFU-E when used in combination with

the more lineage-restricted cytokines. The abundance of eosinophils and their precursor CFCs in the bone marrow of sheep compared to the low frequencies of these in mouse and man33 has revealed that SCF and IL–5 co-operate in the development of eosinophil CFC, and that SCF allows IL-5-mediated eosinophil development from earlier precursors than IL-5 alone. Recombinant ovine SCF on its own supported a spectrum of detectable myeloid colonies, albeit at low frequency compared to IL-3, GM-CSF or M-CSF. This is consistent with the action of highly purified SCF in serum-free and accessory cell-depleted cultures.29,30,34 However, in this study we could not rule out the possibility that either serum used to support the cultures or certain cells in the cultures or both were the source of co-factors that might synergize with SCF to stimulate colony development. We have not found conditions for the growth of ovine bone-marrow cells in the absence of serum so cannot determine whether rovSCF functions to support the survival of stem and progenitor cells or supports limited proliferation in the absence of other potential co-factors. Ovine SCF has amino acid sequence and selected in vitro haematopoietic similarities to SCF in other mammalian species. The cytokine will be useful in studying aspects of haematopoiesis, immunology, embryology and development in which the sheep is an important experimental species.

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cDNA was primed using random hexanuleotides. The primers used for cDNA amplification were 5 GCG CTG CCT TTC CTT ATG AA3 , corresponding to position
15 to +5 of the human cDNA (the A of the start codon being numbered +1), and 5 TAT TAC TGC TAC TGC TGT CA3 , complementary to position +603 to +584 of the human sequence. Amplification of the ovine SCF cDNA was achieved using 25 cycles each of 1 min at 94C, 1 min at 50C and 1.5 min at 72C followed by a final 8-min incubation at 72C. PCR products were cloned into the pCR2.1 (TA) cloning vector (Invitrogen Inc.). Two clones were taken and their inserts sequenced in their entirety.

Liquid pre-culture IL-3/M-CSF IL-3/GM-CSF IL-3/GM-CSF/M-CSF SCF SCF/M-CSF SCF/GM-CSF SCF/IL-3 SCF/GM-CSF/M-CSF SCF/IL-3/GM-CSF SCF/IL-3/M-CSF 0

50

100 150 200 5 Colonies/10 BM cells

250

Figure 4. The effect of SCF alone or in combination with other cytokines on the development of precursors of multipotential colonyforming cells (pre-CFC) in cultures of ovine bone marrow cells (BM cells). BM cells were incubated for 5 days in liquid cultures with the cytokines shown (Liquid pre-culture). Concentrations of cytokines used in the liquid culture phase were: SCF, 5 U/ml; IL-3, 10 U/ml; GM-CSF, 10 U/ml; M-CSF, 50 U/ml (suppliers units). MultiCFC and total CFC were then detected by adding the cells to soft agar cultures, stimulated with SCF plus IL-3 plus GM-CSF plus epo and scoring the plates 14 days later. The results were standardised as the number of colonies per 105 cells added at the start of the soft agar clonogenic assaySD for triplicate cultures. This experiment is representative of three in total. The number of multipotential colonies (mixed phenotype) and total colonies detected in the bone marrow preparation prior to the liquid culture stage were 4 and 96 respectively. Colonies did not develop in unstimulated (medium control) soft agar cultures from the liquid culture stage. IL-3, GM-CSF or M-CSF did not support pre-CFC development on their own. ( ), mixed (multi-CFC); ( ), total colonies.

MATERIALS AND METHODS Animals and cells Suffolk cross lambs, aged 4–12 months, bred at the Moredun Research Institute were used as bone marrow cell donors and skin/hair follicle donors for the generation of keratinocyte cells in culture. Animals were treated with anthelmintic and housed for 6 weeks prior to use in order to minimize stimulation of bone marrow cells. Ovine keratinocyte cultures were developed from basal keratinocytes attached to plucked muzzle hairs in keratinocyte growth medium.35 After 4–6 weeks, cultures contained >95% keratinocytes as determined by pan-cytokeratin antibody (Sigma) immunohistochemistry, and <2% cells stained with an anti-vimentin antibody (present in fibroblasts).

Isolation and sequencing of the ovine SCF165 cDNA Total RNA was isolated by acid-phenol extraction,36 from approximately 107 cultured ovine keratinocytes. Synthesis and amplification of the SCF-cDNA was essentially by the method outlined previously,37 but using oligonucleotides derived from a consensus of the human, murine and porcine SCF cDNA sequences. Single-stranded

Expression of recombinant ovine SCF (rovSCF) in CHO cells Stable expression of rovSCF was achieved in CHO cells using the Celltech pEE14 vector and recommended procedures.38,39 A stop codon was introduced at the end of the ovine SCF cDNA by performing a second PCR using a 3 primer containing a stop codon and a Hind III cloning site; 5 GGG AAG CTT TTA CCT ATT ACT GCT ACT GCT GTC3 . The M13 Reverse primer, which anneals on the 5 side of the SCF cDNA clone in pCR2.1 was used as the other primer. The PCR conditions were as outlined above. The resulting amplicon was digested with Hind III and cloned into the Hind III-digested pEE14 vector. Resultant clones were checked for appropriate orientation by restriction enzyme digest. The integrity of the clone chosen for expression was verified by sequencing. Following CaPO4-mediated DNA transfection of the pEE14SCF into CHO cells, transfected cells expressing elevated levels of the marker glutamine synthetase were selected in the presence of 25 ìM methionine sulfoxamine (MSX; Sigma, Poole, UK). The selected cells were subjected to increasing concentrations of MSX in order to obtain cells secreting high titre rovSCF. Expression of the SCF RNA was verified by Northern blot analysis after separation on a denaturing formaldehyde agarose gel. The rovSCF was purified to >90% homogeneity by FPLC using anion exchange and gel filtration columns (Pharmacia). For Western analysis, murine polyclonal antiserum and preimmunization serum from mice immunized with rovSCF using standard procedures were used on blots from CHOSCF and control supernates run on SDS-PAGE under reducing conditions. The ovIL-3 gene,32 the ovIL-540 and ovGM-CSF37 cDNAs were expressed in CHO cells. The rovGM-CSF and rovIL-3 were >90% pure as determined by SDS-PAGE.32 Purified recombinant human M-CSF was a gift from Cetus Laboratories (CA). Purified human urinary erythropoietin (epo) was obtained from the Terry Fox Laboratory (Vancouver, Canada). pEE14 plasmid-transfected CHO cell conditioned medium (CM) was prepared for use as a control in the bone marrow soft agar assays. This contained no detectable activity (when used alone or in combination with the other cytokines) compared to medium control cultures in any of the bioassay systems employed. The different CHOexpressed cytokines were assigned activities in Units/ml), where 1 Unit stimulates half maximum colony formation in the soft agar clonogenic assay (see below). M-CSF was

Biological activity of ovine SCF / 255

assayed by the manufacturer, and was active in the ovine soft agar clonogenic assay between 10 and 500 U/ml.

and Paul Wood (CSIRO, Australia) for the IL-5 cDNA.

Bone marrow haematopoietic cell cultures The soft-agar bone marrow cell clonogenic assays for ovine haematopoietic progenitor CFC has been described previously.32,33,41 Sternal bone marrow cells (5104/ml culture) in IMDM containing 20% V/V batch-tested FCS and bacto-agar (Difco) 3% were set up in 35-mm Petri dishes with or without cytokines and incubated in a highly humidified atmosphere of CO2 5% in air. Colonies (>40 cells) and clusters (3–40 cells) were analysed on days 7 and 14. Erythroid cluster-forming cells (CFC-E) were enumerated on day 5, and erythroid burst-forming units (BFU-E) on day 14 in unstained cultures. Differential analysis of other cell colonies was as described previously.27,33,41 Soft agar discs were fixed in paraformaldehyde 2% and stained for chloro-acetate esterase (neutrophils and mast cells), nonspecific esterase (macrophages and some undifferentiated haematopoietic blasts), luxol fast blue (eosinophils) and counterstained with toluidine blue or haematoxylin. Multipotential-CFC were identified by their mixed cell lineage colony progeny in 14 day cultures. These were of two types: (a) containing >3 separate cell lineages (usually containing erythroid series cells) that were >0.5 mm in diameter and either multi-centric or dispersed in appearance; and (b) large (>1 mm diameter), spherical and consisting predominantly of macrophage-like cells and haematopoietic blast cells. These colony types (and occasionally BFU-E) were the only of many tested to give rise to secondary colonies upon re-plating.27 For convenience, colony types (a) and (b) will be collectively referred to as mixed colonies in this report. The pre-multi-CFC assay for the detection of haematopoietic multi-CFC precursor cells was performed as described previously.27 Bone marrow cells (5104) in IMDM/10% FCS were incubated with and without cytokines in 24 well tissue culture plates. After 6 days, measured aliquots of viable cells (1–3104) obtained by harvesting the entire contents of each well were added to soft agar cultures and stimulated with 10 U/ml rov SCF, 10 U/ml rovIL-3, and 1.5 U/ml epo. This combination of cytokines stimulated the maximum number of total and mixed colonies, which were enumerated on day 14. Colonies did not develop in unstimulated, medium control cultures. For ease of comparison between groups, results were expressed as the number of total and mixed colonies/105 cultured cells seeded at the start of the soft agar cell clonogenic assay.

Statistics Student’s t-test was applied where appropriate to data normalized by log10 transformation.

Acknowledgements The authors wish to thank: the Scottish Office Agriculture, Environment and Fisheries Department for financial support of this work; Celltech Ltd (UK) for the pEE14 expression system; Heng-Fong Seow

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