Development 107, 113-122(1989) Printed in Great Britain ©The Company of Biologists Limited 1989 113 Expression of multiple heparin-binding growth factor species by murine embryonal carcinoma and embryonic stem cells JOHN K. HEATH 1 *, GARY D. PATERNO 12 , A. CATHERINE LINDON 1 and DYLAN R. EDWARDS 13 ^Cancer Research Campaign Laboratories, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK 2 ICRF Developmental Biology Unit, Department of Zoology, University of Oxford, South Parks Road, Oxford OX] 3PS, UK 3 Present Address: University of Calgary, Health Sciences Centre, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 1N4 * Corresponding author Summary Culture medium conditioned by P19 embryonal carci- noma (EC) cells contains potent mitogenic activity which is markedly potentiated when the medium is conditioned in the presence of heparin. Fractionation of P19 medium conditioned in the presence of heparin reveals the existence of two biochemically distinct growth factor species both of which exhibit high affinity for immobi- lised heparin and significant activity as amphibian mesoderm-inducing agents. One of the species is re- covered as a single polypeptide of apparent M T = 15 000. This molecule is immunologically related to the protein product of the human K-FGF proto-oncogene. Tran- scripts derived from the murine K-FGF gene are also expressed by both differentiated and undifferentiated EC cells and embryonic stem cells. The second heparin- binding growth factor is recovered as a complex of four polypeptides, the largest of which has an apparent M r = 17 000. This agent is immunologically and bio- chemically distinct from both acidic and basic fibroblast growth factor as well as K-FGF, and represents the predominant mitogenic activity in EC-cell-conditioned medium. Key words: oncogene, growth factor, heparin, embryonal carcinoma cells, embryonic stem cells. Introduction It has been well established in the last few years that murine embryo-derived embryonal carcinoma (EC) cells express specific growth and differentiation regulat- ory factors (reviewed Heath & Rees; 1985, Rizzino, 1987; Heath & Smith, 1988; Mummery et al. 1989). The identity of these agents is of interest since they are strong candidates for important regulatory factors in the control of cell proliferation and differentiation in early mammalian development. The principal mitogenic activity expressed by EC cells is characterised by a strong affinity for immobilised heparin. Heath and Isacke (1984) purified a growth factor from PC13 EC-cell-conditioned medium, embry- onal carcinoma derived growth factor (ECDGF), which was purified as a M T = 17 500 polypeptide with potent mitogenic effects on a variety of cell types in vitro. The expression of ECDGF was developmental^ regulated, since the activity was no longer present in conditioned medium when the cells were induced to differentiate in vitro by exposure to retinoic acid (RA) (Isacke & Deller, 1983). Further optimisation of ECDGF purifi- cation techniques revealed that it exhibited strong affinity to immobilised heparin (Heath, 1987) and was therefore a candidate member of the heparin-binding family of growth factors (HBGFs), of which the proto- type members are basic (bFGF) and acidic (aFGF) fibroblast growth factor (reviewed Gospodarawicz et al. 1987). Van Veggel et al. (1987) and Van Zoelen et al. (1989) have also reported the presence of a heparin- binding growth factor activity in EC-cell-conditioned medium which strongly resembles ECDGF in bio- chemical and biological properties. Further important evidence for a functional relationship between ECDGF and HBGFs came from the finding that, like aFGF and bFGF, ECDGF was a potent inducer of mesodermal differentiation in isolated animal pole explants of Xeno- pus laevis embryos (Slack et al. 1987). This property has been found to be a distinctive feature of other members of the HBGF family (Paterno et al. 1989). Direct structural characterisation of ECDGF by amino acid sequencing has, however, proved problema- tic, largely due to the difficulties in obtaining sufficient quantities of the factor from PC13 EC-cell-conditioned medium. Several lines of evidence have shown, how- ever, that ECDGF differs significantly in structure from either aFGF or bFGF (reviewed Heath & Smith, 1988). These include failure to react with antibodies directed against either aFGF or bFGF, distinct tryptic peptide