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Short notes 567 SHORT NOTE Pluripotent PETER ‘Murdoch Institute, Differentiation KOOPMAN’, Royal Children’s bourne, of Single F9 Embryonal *, * and RICHARD Hospital Parkville Carcinoma Cells G. H. COTTON’ and ‘Department of Paediatrics, 3052, Victoria, Australia University of Mel- F9 embryonal carcinoma cells were induced to form a variety of differentiated cell types in monolayer culture. Cells with the morphological, histochemical and immunocytochemical properties of parietal and visceral endoderm, neurones and adipocytes were identified. Cells expressing Thy-l antigen and large, multinucleated cells expressing cytoplasmic fibronectin were also observed. Various cell types were found together in colonies derived from individual F9 cells, allowing us to conclude that F9 cells are pluripotent in vitro. @ 1987 Academic Press, Inc. The differentiation of murine embryonal carcinoma (EC) cells in vitro is commonly used as a model of developmental events in the embryo [ 1, 21. The F9 EC cell line [3], one of the most extensively studied, shows minimal spontaneous differentiation [4], but can be induced to differentiate in vitro with agents such as retinoic acid (RA) [5, 61. However, a restricted range of differentiated cell lineages is generated in this way [5-91, which limits the validity and usefulness of these cells as a model for normal events. We report here that single F9 cells can be induced to form a variety of differentiated cell types together in low-density colonies in monolayer culture. Materials and Methods Cell culture. F9 cells [3] from Dr A. Levine, Princeton University, N. J., were grown as previously described [lo] in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (Flow Laboratories), in gelatin-coated culture vessels. Retinoic acid (BA) (Sigma, all-trans) was stored at -20°C as a lo-’ M stock in absolute ethanol, and diluted freshly into culture medium before use. ST0 fibroblast-conditioned medium was produced as previously described [IO]. Cell stocks were found free of mycoplasma by Hoechst 33258 staining [l 11. ZdentiJication of cell types. Various antigens were visualized by a modified peroxidase-antiperoxidase (PAP) technique [12]. Colonies were fixed in 5 % acetic acid/95% ethanol at -20°C for 10 min, thoroughly washed and incubated in 10 % normal swine serum (Flow) in phosphate-buffered saline for 30 min. Incubation for 1 h with primary antibody was followed by 30 min incubations with rabbit antimouse Ig (Dako 2109) at 1 : 200 where mouse or rat primary antibodies were used, or biotinylated rabbit anti-sheep Ig (Vector BA 6000) where sheep primary antibody was used, then swine anti-rabbit Ig (Dako 2196) at 1: 25, and finally rabbit PAP complex (Dako Zl13) at 1: 200. The chromogen was diaminobenzidine (Sigma). All incubations were at room temperature. In the case of A2B5 and its negative control antibody, fixation followed primary antibody incubation and washing. Primary antibodies were as follows: LE61 [13], a mouse monoclonal IgG, antibody supematant recognizing * To whom offprint requests should be sent. Present address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson Way, London NW1 2HE, England. Copyright @ 1987 by Academic Press, Inc. A0 rights of reproduction in any form reserved 00144827/87 $03.00 568 Short notes cytokeratin no. 18 of Mall et al. [14], provided by Dr E. B. Lane, used at 1 : 25; rabbit anti-mouse laminin antiserum (BRL 6265SA) used at 1 : 100; rat monoclonal IgM ascites antibody to stage-specific embryonic antigen 3 (SSEA-3), provided by Dr D. Solter, used at 1 : 100; sheep antibodies to human fibronectin (Serotec AHP 08X) used at 1 : 100; A2B5, a mouse monoclonal IgM ascites specific for GQ ganglioside [lS], provided by Dr P. Bartlett, used at 1 : 400; and 30 H12, a mouse monoclonal IgG antibody supematant recognizing Thy 1.2 antigen [16], provided by Dr P. Bartlett, used at 1 : 10. Negative controls used normal rabbit or sheep serum, or monoclonal antibodies of corresponding species and type but with irrelevant specificities. Colonies were stained to detect alkaline phosphatase as previously described [lo]. Neutral fats were stained with Oil Red 0 (Polysciences), using the supersaturated isopropanol method described by Lillie [ 171. Results and Discussion We have previously shown that a factor present in medium conditioned by ST0 fibroblasts [18] retards both the differentiation of F9 EC cells induced with lo-’ M retinoic acid (RA) and the spontaneous differentiation of NG2 EC cells [lo]. The present observations arose from further study of the competition between RA and SIG-conditioned medium. F9 cells were seeded at clonal density (100-500 cells/75 cm*), into either 25 % S’IDconditioned medium, lo-’ M RA, or both together. The extent of differentiation was examined after 22 days of culture. In the absence of RA, most colonies remained undifferentiated as judged by cell morphology and positive staining for alkaline phosphatase [ 191; minimal spontaneous differentiation to parietal endoderm-like cells was observed, as described by Sherman & Miller [4]. Colonies grown in the presence of IO-’ M RA had mostly differentiated entirely to parietal endoderm, as identified by immunohistochemical detection of cytokeratin no. 18 [14, 201 and laminin [21], but not SSEA-3 [20]. Colonies grown in the presence of lo-’ M RA and 25% SK&conditioned medium contained a variety of cell morphologies. Various differentiated cell types were identifiable within the same colony at day 35, in addition to alkaline phosphatase-positive cells (fig. 1 a). Parietal endoderm cells were identified by the above criteria (fig. 1 b, c); the presence of visceral endoderm cells was suggested by expression of SSEA-3 [27] (fig. 1 d>. Large, refractile droplets staining with the lipid stain Oil Red 0 [17] suggested the presence of adipocytes (fig. 1 e). Cells with refractile bodies and processes extending up to several millimetres were present; these bound the antibody A2B5 [14], suggesting that they were neurones (fig. lfl. Some cells were found to express Thy-l (fig. 1 g), an antigen present on various cell types including thymocytes 1231 and fibroblasts 1241, but not EC or parietal or visceral endoderm [25]. Large, multinucleated cells Fig. I. Markers of different cell types in F!9 colonies at day 35. Colonies were grown in 25 % S’IDconditioned medium and lo-’ M RA, and stained to reveal (a) cells expressing alkaline phosphatase; (b) parietal endoderm cells expressing cytokeratin; (c) laminin; (d) cells expressing SSEA3, a marker of visceral endoderm; (e) cells with large droplets of neutral fat; (t) neurone-like cells binding monoclonal antibody AZBS; (g) cells expressing Thy- 1; (h) tibronectin. Brightfield micrography. Bar, 100 urn. Exp Cell Res 168 (1987) Short Exp Cell notes 569 Res 168 (1987) 570 Short notes expressing fibronectin in their cytoplasm were observed (fig. 1 h); we were unable to determine whether these were trophoblast cells [26] or simply degenerating cells. Extracellular fibronectin was also present (fig. 1 h). Several other cell morphologies were seen which could not be identified using markers. Similar observations were made in two separate series of experiments. The inoculum for these cultures contained 88-92 % (n, 156-322) single cells, the remainder being doublets. In a separate experiment, single F9 cells were placed with finely drawn Pasteur pipettes into individual 2 cm*-culture dishes containing 25% SIG-conditioned medium and 10e7 M RA. Of 44 colonies arising, ten contained at least three of the cell types recognizable by morphology (parietal endoderm, neurones, and adipocytes). Clonal expansion of differentiated or determined cells present in groups in the inoculum can therefore not account for our observations. It is possible that generation of a number of different cell lineages in the colonies is mediated by time- or position-dependent cues acting on residual stem ceils, as is thought to occur in the embryo. Whilst differentiation of F9 cells relies on the presence of an inducer, the commonly used treatment of lo-’ M RA appears to induce so strongly that most stem cells form parietal endoderm before other cell types can be generated. The function of SIG-conditioned medium in our experiments may have been to retard the effect of RA sufficiently to promote the correct balance between stimulation of differentiation and retention of a stem cell population. We tested this hypothesis by subjecting F9 cells to two other treatments expected to have a similar effect. We have previously shown, using NG2 EC cells, that delaying the addition of lop7 M RA until day 4 of clonal growth resulted in retention of stem cells, rather than the complete differentiation to parietal endoderm seen when RA is present from day 0 [271. Further, RA can be used at 10V8 M to induce differentiation less efficiently than at 10e7 M [5, 281. Both these strategies were applied to clonal-density F9 colonies, and in both cases, cells with the properties of parietal and visceral endoderm, adipocytes, and neurones, as well as Thy-l-positive cells and tibronectin-positive cells were detected as above, after 35 days of growth. These observations are consistent with our hypothesis. Neither treatment appeared to be as efficient as 25% STGconditioned medium/10e7 M RA in inducing multiple differentiation. Previous studies have described conditions for the culture of F9 cells which yield parietal [5] or visceral [6] endoderm, but not both together. Other studies have reported neurone-like cells in bulk cultures of F9 treated with RA and (dbCAMP) [7], or when RA-db-CAMP treatment was followed by nerve growth factor (NGF) [8]. Our results indicate that added NGF or db-CAMP is not necessary for neurone formation. Formation of lipid cells in RA-treated F9 cultures has also been mentioned [9]. Despite these reports, F9 is still usually referred to as nullipotent. We have shown that single F9 cells can reproducibly yield multiple cell types in the same colony and must therefore be considered pluripotent in Exp Cell Res 168 (1987) Short notes 571 vitro. It is possible that other ‘nullipotent’ EC lines may be able to differentiate in a complex manner in vitro, given the correct conditions, and that the varied behaviour amongst EC cell lines is attributable to regulation of, rather than potential for, differentiation. We thank Drs Birgitte Lane, Perry Bartlett and Davor Solter for the gifts of monoclonal antibodies. References 1. Martin, G R & Evans, M J, Cell 6 (1975) 467. 2. Strickland, S, Cell 24 (1981) 277. 3. Artzt, K, Dubois, P, Bennett, D, Condamine, H, Babinet, C &Jacob, F, Proc natl acad sci US 70 (1973) 2988. 4. Sherman, M I & Miller, R A, Dev biol 63 (1978) 27. 5. Strickland, S & Mahdavi, V, Cell 15 (1978) 393. 6. Hogan, B L M, Taylor, A & Adamson, E, Nature 291 (1981) 235. 7. Kuff, E L & Fewell, J W, Dev biol 77 (1980) 103. 8. Liesi, P, Rechardt, L & Wartiovaara, J, Nature 306 (1983) 265. 9. Linder, S, Krondahl, U, Sennerstam, R & Ringertz, N R, Exp cell res 132 (1981) 453. 10. Koopman, P & Cotton, R G H, Exp cell res 154 (1984) 233. 11. Chen, T R, Exp cell res 104 (1977) 255. 12. Stemberger, L A, Immunocytochemistry, 2nd edn. Wiley, New York (1979). 13. Lane, E B, J cell biol 92 (1982) 665. 14. Moll, R, Francke, W W, Schiller, D L, Geiger, B & Krepler, R, Cell 31 (1982) 11. 15. Eisenbarth, G S, Walsh, F S & Nirenberg, M, Proc natl acad sci US 76 (1979) 4913. 16. Ledbetter, J A & Herzenberg, L A, Immunol rev 47 (1979) 63. 17. Lillie, R D, Histopathologic technic. Blakiston, Philadelphia (1948). 18. Martin, G R & Evans, M J, Proc natl acad sci US 72 (1975) 1441. 19. Bemstine, E G, Hooper, M L Grandchamp, S & Ephrussi, B, Proc natl acad sci US 70 (1973) 3899. 20. 21. 22. 23. 24. 25. 26. 27. 28. Lane, E B, Hogan, B L M, Ku&men, M & Garrels, J I, Nature 303 (1983) 701. Hogan, B L M, Cooper, A R & Kurkinen, M, Dev biol80 (1980) 289. Shevinsky, L H, Knowles, B B, Damjanov, I & Solter, D, Cell 30 (1982) 697. Reif, A E & Allen, J M V, J exp med 120 (1964) 413. Stem, P L, Nature new biol 246 (1973) 76. Stem, P L, Martin, G R & Evans, M J, Cell 6 (1975) 455. Wartiovaara, J, Leivo, I & Vaheri, A, Dev biol 69 (1979) 247. Koopman, P & Cotton, R G H, Differentiation 31 (1986) 55. Jetten, A M, Jetten, M E R & Sherman, M I, Exp cell res 124 (1979) 381. Received July 25, 1986 Revised version received October 13. 1986 F’rintcd in Sweden 37-878332 Exp CdRes 168 (1987)