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Use of Avian Cytokines in Mammalian Embryonic Stem Cell Culture1
IMMUNOLOGY Use of Avian Cytokines in Mammalian Embryonic Stem Cell Culture1 Z. YANG2 and J. N. PETITTE Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608 ABSTRACT Mouse blastocyst-derived embryonic stem (ES) cells are multipotent cells that can be used in vitro as models of differentiation and in vivo can contribute to all embryonic tissues including the germ line. The culture of ES cells requires a source of leukemia inhibitory factor (LIF), often provided by culture with a mouse fibroblast (STO) feeder layer, buffalo rat liver cellconditioned media (BRL-CM), or the addition of recombinant LIF. To date, all of the ES cell culture systems use mammalian sources of LIF. We found that mouse ES cells can be maintained for over 10 passages in an undifferentiated state with media conditioned by a chicken liver cell line (LMH-CM) or on a feeder layer made with primary chicken embryonic fibroblasts (CEF). These ES cells can undergo both spontaneous and induced differentiation, which is associated with the disappearance or reduction of the expression of alkaline phosphatase and SSEA-1, similar to that observed for ES cells cultured with BRL-CM or STO feeder layers. The ES cells cultured in LMH-CM did not express cytokeratin Endo-A antigen recognized by TROMA-1, but their differentiated progeny did express this antigen. In contrast to LMH-CM, Endo-A was expressed in ES cells cultured on CEF feeder layers and in differentiated progeny. These results indicate that avian cells can produce a LIF-like cytokine that is active in inhibiting the differentiation of mouse ES cells. This could provide a biological end point for the isolation and characterization of avian LIF. (Key words: cytokines, embryonic stem cells, chicken fibroblasts, chicken liver cell line, leukemia inhibitory factor) 1994 Poultry Science 73:965-974
INTRODUCTION Mouse embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst, are pluripotent, and are able to differentiate in vivo and in vitro. Models of differentiation in vitro include cardiomyocytes, lymphocyte precursors, and other hemopoietic lineages (Wiles and Keller, 1991; Wobus et al, 1991; Chen and Kosco,
Received for publication December 1, 1993. Accepted for publication March 3, 1994. 1 1he use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service, nor criticism of similar products not mentioned. 2 Present address: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX 77030.
1993). In addition, ES cells can spontaneously differentiate into embryoid bodies or undergo retinoic acid-induced differentiation (Evans and Kaufman, 1981; Martin, 1981; Robertson et al, 1983). In vivo applications of ES cells take advantage of their ability to develop somatic and germ line chimeras after injection into the blastocyst. This has given rise to the powerful technique of gene targeting through the use of homologous recombination, which has become the only means to develop transgenic mice with sitedirected changes to the genome (Hasty et al, 1991; Joyner, 1991; Reid et al, 1991). Embryonic stem cells can be maintained for long periods in vitro on mouse fibroblast feeder (STO) cells or in media conditioned by Buffalo rat liver (BRL) cells (Smith and Hooper, 1987). The differ-
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entiation-inhibiting substance in both culture systems has been identified as leukemia inhibitory factor (LIF) (Williams et ah, 1988), which is a pleiotropic cytokine with diverse effects in many cell types (Gough et ah, 1989). For example, LIF can induce differentiation of murine myeloid leukemia (Ml) cells, stimulate the proliferation of primary myoblasts, or inhibit differentiation of mouse ES cells. Mouse ES cells have been isolated successfully and maintained by the use of purified LIF, recombinant LIF, or feeder cells stably transfected with a LIF expression vector (Nichols et ah, 1990; McMahon and Bradley, 1990; Pease and Williams, 1990; Pease et ah, 1990). The successful use of mouse ES cells in studies designed to alter the genome in a site-specific manner has promoted considerable interest in the establishment of ES cells for other mammalian species. The technical scheme for the production of transgenic animals with targeted changes to the genome requires the ability to produce early embryonic chimeras that give rise to functional gametes, the culture of ES cells capable of producing germline chimeras, and the development of DNA constructs that undergo homologous recombination. The adaptation of these procedures to mammalian livestock species requires the establishment and culture of ES cells. Current attempts to develop culture conditions suitable for ES cell growth from agriculturally important species has been mixed. However, several groups have been able to culture cells with an ES cell phenotype from bovine, ovine, and porcine embryos and from mink (Piedrahita et ah, 1990; Notarianni et ah, 1990,1991; Saito et ah, 1992; Sukoyan et ah, 1992).
chimeras. Petitte et ah (1990) were the first to demonstrate that germline chimeras could be produced in domestic fowl by the transfer of early blastodermal cells from one embryo to another. Subsequent investigations have optimized the procedures so that avian germline chimeras can now be produced routinely (Carscience et ah, 1993; Petitte et ah, 1993). As with mammalian livestock species, the next required step is the establishment of ES cells. Therefore, as a prelude towards the development of an ES cell culture system for the avian embryo, we examined the ability of avian cells to support the maintenance of mouse ES cells and report that primary chicken embryonic fibroblasts (CEF) and media conditioned by a chicken liver cell line can successfully be used to culture mouse ES. MATERIALS AND METHODS Feeder Layers
The STO cells (American Type Cell Collection (ATTC), No. CRL 1503)3 w e r e cultured in 75-cm2 flasks in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), and were mitotically inactivated after a 2- to 3-h exposure of 10 /tg/mL Mitomycin C 4 Inactivated STO cells were seeded at a concentration of 1 x 105 cells/cm2 onto 60-mm or 35-mm diameter tissue culture dishes coated with .1% gelatin.4 Chick embryonic fibroblasts were obtained from 10-d-old chicken embryos. After removal of the head, viscera, legs, and wings, the remainder of the embryos were minced and incubated 15 min in a solution of .05% trypsin and .02% EDTA.5 Dissociated CEF cells were seeded into In contrast to the above mammalian 75-cm2 flasks at a concentration of 3 x 106 species, the application of these proce- cells per flask and cultured in DMEM with dures for manipulation of the avian ge- 10% FBS. After the 10th passage, cells were nome has been complicated by the discarded and new CEF cultures were peculiarities of the reproductive strategy prepared. The CEF feeder layers were in birds and efforts in this area have been prepared the same way as for STO feeder focused on the development of germline layers. Conditioned Media 3Rockville, MD 20850. 4Sigma Chemical Co., St. Louis, MO 63178-9916. s Boehringer Mannheim, Indianapolis, IN 46250.
Medium conditioned with BRL cells was obtained after incubating 13 mL of DMEM
AVIAN CYTOKINES AND STEM CELL CULTURE
with 10% FBS in a 75-cm2 flask of confluent BRL cells (BRL 3A, ATCC No. CRL 1442)3 and harvesting every 3 d for up to 12 d. Before use, the medium was filtersterilized, diluted to 80% with DMEM containing 15% FBS, supplemented with .1 mM /3-mercaptoethanol,4 and designated BRL-CM. A chicken liver cell line, LMH (Kawaguchi et ah, 1987) was cultured until confluent in 75-cm2 flasks in DMEM with 10% FBS. Medium conditioned with LMH cells was harvested as described above for BRL-CM, diluted to 80% with DMEM containing 15% FBS, supplemented with .1 mM /3-mercaptoethanol, and designated LMH-CM. Culture of Embryonic Stem Cells The ES cells (ES-E14TG2a, ATCC No. CRL 1821)3 initially grown on STO feeder layers were seeded onto 60-mm culture dishes with or without feeder layers at a concentration of 1 x 106 cells per 60-mm dish. The ES cells seeded onto STO or CEF feeder layers were cultured in DMEM supplemented with 15% FBS and .1 mM /3-mercaptoethanol. The ES cells cultured in BRL-CM or LMH-CM were seeded onto pregelatinized plates. All cultures were passed every 2 to 3 d. After 10 or more passages, ES cell stocks were frozen and thawed prior to testing their ability to undergo spontaneous or induced differentiation.
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glass at a concentration of 1 x 10 cells per dish and in DMEM with 10% FBS. Following a 6-h incubation, the media were replaced with DMEM with 10% FBS containing 1 fiM retinoic acid.4 Retinoic acidcontaining media were changed twice at 24-h intervals with freshly prepared retinoic acid-containing media. After 72 h, the cells were cultured further in DMEM with 10% FBS for 3 d. Alkaline Phosphatase The ES cells and their differentiated progeny were stained for alkaline phosphatase activity. Cells were fixed in acetonecitrate-formaldehyde solution, rinsed in deionized water, and incubated in a naphthol-fast red violet substrate solution according to the manufacturer's instructions (diagnostic kit No. 86).4 After rinsing in deionized water, the cells were counterstained with hematoxylin, dehydrated, and mounted. Immunocytochemical Staining
The ES cells and their differentiated derivatives were tested for the expression of SSEA-1 and Endo-A using specific monoclonal antibodies. Anti-SSEA-1 and TROMA-1, which recognizes Endo-A, were obtained from Developmental Studies Hybridoma Bank.6 All cultures for immunocytochemical staining were fixed in cold acetone at -20 C for 8 min. After incubation with each monoclonal antibody, the cells were incubated with a corresponding biotinylated Spontaneous Differentiation second antibody followed by a complex of The ES cells grown with feeder layers or avidin and biotinylated alkaline phosphaconditioned media were seeded at a con- tase according to the manufacturer's procentration of 1 x 105 cells per 35-mm tocol (Vectastain, ABC-AP Kit)7 Enpregelatinized dish containing a coverglass dogenous alkaline phosphatase activity and cultured in DMEM with 10% FBS for up was inhibited using levamisole.7 The cells to 7 d. were then counterstained with hematoxylin, dehydrated, and mounted. These antigens were visualized using an epifluoresInduced Differentiation cent microscope with a rhodamine filter. The ES cells grown with feeder layers or conditioned media were seeded onto RESULTS pregelatinized 35-mm dishes with a coverMorphology 6
Iowa City, IA 52242. Sector Laboratories, Burlingame, CA 94010.
When the ES cells were initially transferred from an STO feeder layer to LMH-
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CM, BRL-CM, or a CEF feeder layer, 10 to 15% of the cells spread and differentiated into endoderm-like cells. Once the ES cells were cultured for two or three passages, spontaneous differentiation diminished and remained at a constant rate of less than 5%. Regardless of the culture system used, the characteristic ES cell phenotype was maintained, i.e., small cells with a large nucleus and relatively little cytoplasm (Figure 1). Slight differences in colony phenotype were noted and appeared to be related to the use of feeder cells and conditioned media. The ES cells grown on a monolayer of CEF were arranged in ovalshaped colonies that were parallel to the long axes of the underlying fibroblasts (Figure 1C). On STO feeder cells where the fibroblasts spread in all directions, the colonies were more rounded (Figure 1A vs 1C). Compared to the colonies formed on feeder layers, the ES cells grown using conditioned media produced irregularly shaped, less tightly packed colonies
(Figures 1A, 1C vs IB, ID). No differences in ES cell growth rate were observed among the four culture systems even after 10 passages. When the ES cells maintained with CEF feeder layers or LMH-CM were cultured in DMEM with 10% FBS to allow spontaneous differentiation, they continued to form typical ES cell colonies and remained phenotypically undifferentiated for 2 d, similar to that observed for cells grown on STO cells or in BRL-CM. Subsequently, all ES cell cultures began to spread and form large, flattened cells. However, ES cells cultured with CEF feeder layers required an additional day to begin differentiation than did the ES cells kept with LMH-CM, BRLCM, or STO feeder layers. In all cases, spontaneous differentiation was relatively homogeneous and appeared to be completed after 7 d of culture. Retinoic acid-induced differentiation proceeded quickly, and within the first 24 h of culture the ES cells began to differentiate.
cJL FIGURE 1. Undifferentiated mouse embryonic stem cells cultured with a mouse fibroblast STO feeder layer (A), BRL-CM (B), a CEF feeder layer (C), and LMH-CM (D), respectively. Phase contrast, scale bar = 150 p. BRLCM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell lineconditioned media.
AVIAN CYTOKINES AND STEM CELL CULTURE
An assortment of derivatives was produced within 5 to 6 d, nearly 2 d earlier than spontaneous differentiation. The ES cells cultured under the four different culture conditions shared similar differentiated cell types consisting of large fibroblast-like cells, endoderm-like cells, and neuronal cells. Most endoderm-like cells formed sheets of different sizes. Dendrite or axonlike processes of neuronal cells often extended over a monolayer of fibroblastic or endoderm-like cells.
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layers (Figure 4). No expression was found in STO or CEF feeder cells. Endo-A antigen was expressed at low levels on all of the spontaneously differentiated ES cells cultured with the four culture systems (Figure 4). However, after retinoic acid-induced differentiation, immunostaining of this antigen was heterogeneous and limited to endoderm-like cells, some fibroblastic cells, and other small, randomly distributed cells (Figure 4). DISCUSSION
Alkaline Phosphatase The undifferentiated ES cells cultured with CEF feeder layers or LMH-CM expressed high levels of alkaline phosphatase, similar to the ES cells cultured using STO feeder layers or BRL-CM (Figure 2). After spontaneous differentiation or retinoic acid-induced differentiation, the activity of this enzyme was totally lost in the ES cells from all cultures. No alkaline phosphatase activity was found in STO feeder cells or CEF feeder cells. SSEA-1 Antigen The undifferentiated ES cells cultured with CEF feeder layers or LMH-CM expressed high levels of SSEA-1 (Figure 2). After spontaneous differentiation, the expression of SSEA-1 was lost from all differentiated progeny of the ES cells maintained with LMH-CM, BRL-CM, or STO feeder layers (Figure 3). The expression of SSEA-1 by spontaneously differentiated ES cells cultured with CEF feeder layers was slightly different. A weak fluorescence still remained detectable on these cells (Figure 3). However, after retinoic acid-induced differentiation, ES cells from all four culture systems no longer expressed SSEA-1 (Figure 3). Endo-A Antigen Monoclonal antibody TROMA-1 was used to specifically recognize Endo-A antigen (Brulet et ah, 1980). There was no expression of Endo-A on the ES cells cultured with LMH-CM, STO feeder layers, or BRL-CM, but this antigen was expressed on the ES cells maintained on CEF feeder
This study has shown that CEF feeder layers and LMH-CM are capable of maintaining mouse ES cells in an undifferentiated state for over 10 passages. The cells retained typical morphologies of ES cells with a large nucleus and relatively little cytoplasm (Figure 1). After spontaneous differentiation, ES cell progeny consisted predominantly of large flattened, endoderm-like cells. The cell types observed after retinoic acid-induced differentiation were more heterogeneous than those observed after spontaneous differentiation and included fibroblast-like cells, endoderm-like cells, and neuronal cells. The same kinds of differentiated cells were obtained from the ES cells cultured with BRL-CM or STO feeder layers. The observation of flattened endodermlike cells obtained after spontaneous differentiation were consistent with previous studies (Robertson, 1987; Smith, 1991). In addition, Mummery et al. (1990) reported that when ES cells were induced to differentiate by retinoic acid in the absence of LIF, they appeared to form a mixed cell population consisting of endoderm-like cells but also of ill-defined mesoderm-like cell types. When ES cells were treated with retinoic acid in the presence of LIF, BRL-CM inhibited most types of differentiation but permitted the formation of parietal endoderm (Smith and Hooper, 1987; Mummery et al, 1990). Previous studies with the particular ES cell line used in the present study have consistently demonstrated the ability to differentiate into embryonic structures in the absence of feeder cells or conditioned medium and, when injected into a blastocyst, to contribute to the formation
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FIGURE 2. Alkaline phosphatase activity of undifferentiated embryonic stem cells (FC/CM), and after spontaneous differentiation with Dulbecco's Modified Eagle's Medium (DMEM), and retinoic acid-induced differentiation (DMEM + RA). Embryonic stem cells were cultured with mouse fibroblast STO feeder cells (1), BRL-CM (2), CEF feeder cells (3), or LMH-CM (4) for several passages. Spontaneous differentiation proceeded after culture in DMEM with 10% fetal bovine serum (FBS) for 7 d. For retinoic acid-induction, embryonic stem cells were treated with retinoic acid for 3 d followed by DMEM with 10% FBS for an additional 3 d. Alkaline phosphatase activity was detected with naphthol AS-B1 phosphate/fast red, counter-stained with hematoxylin. Scale bar = 12 /*. BRL-CM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell line-conditioned media.
of somatic a n d g e r m l i n e c h i m e r a s (Doetschman et ah, 1987; Hooper et ah, 1987; Smith and Hooper, 1987). Although the ES cells in this study were not tested for their ability to produce chimeric mice, the ability to differentiate spontaneously
or in response to a pulse of retinoic acid did not diminish with a change to a nonmammalian culture system. Embryonic stem cells share several functional characteristics with mouse embryonal carcinomas, particularly their abil-
AVIAN CYTOKINES AND STEM CELL CULTURE
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FIGURE 3. SSEA-1 immunofluorescent staining of undifferentiated embryonic stem cells (FC/CM) and after spontaneous differentiation with Dulbecco's Modified Eagle's Medium (DMEM) and retinoic acid-induced differentiation (DEME + RA). Embryonic stem cells were cultured with mouse fibroblast STO feeder cells (1), BRL-CM (2), CEF feeder cells (3), or LMH-CM (4) for several passages. Spontaneous differentiation proceeded after culture in DMEM with 10% fetal bovine serum (FBS) for 7 d. For retinoic acid induction, embryonic stem cells were treated with retinoic acid for 3 d followed by DMEM with 10% FBS for an additional 3 d. In each case corresponding bright and fluorescent fields are shown. Scale bar = 25 /t. BRL-CM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell line-conditioned media.
ity to differentiate in vivo and in vitro (Papaioannou and Rossant, 1983; Smith and Hooper, 1987) and the expression of various antigenic determinants (Mummery et al, 1990; Kimber et al, 1993). Evans (1972) reported that the mouse embryonal carcinoma cell line, SIKR, was maintained for approximately 30 cell generations by a CEF feeder layer. When reinoculated into mice, the SIKR cells produced teratomas containing at least 10 types of tissues (Evans, 1972). Though similarities exist between embryonal carcinomas and ES cells, the usefulness of an avian feeder layer or conditioned media in ES cell culture remained untested until now. High levels of alkaline phosphatase activity are characteristic of embryonal
carcinomas and are lost after spontaneous or induced differentiation (Damjanov et al, 1971; Bernstine et al, 1973; Strickland et al, 1980). Because this enzyme was highly active in ES cells grown in the four different culture systems and subsequently diminished upon differentiation (Figure 2), the ES cells were further characterized using immunological markers. The SSEA-1 antigen, a stage-specific mouse embryonic antigen (Solter and Knowles, 1978), is a widely used marker for ES cells in culture. This antigen is expressed on the cell surface of undifferentiated embryonal carcinoma and ES cells but not on their differentiated progeny (Magnuson et al, 1982; Mummery et al, 1990). In the present study, the
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FIGURE 4. TROMA-1 immunofluorescent staining of undifferentiated embryonic stem cells (FC/CM) and after spontaneous differentiation with Dulbecco's Modified Eagle's Medium (DMEM) and retinoic acid-induced differentiation (DMEM + RA). Embryonic stem cells were cultured with a mouse fibroblast STO feeder layer (1), BRL-CM (2), a CEF feeder layer (3), or LMH-CM (4) for several passages. Spontaneous differentiation proceeded after culture in DMEM with 10% FBS for 7 d. For retinoic acid induction, embryonic stem cells were treated with retinoic acid for 3 d followed by DMEM with 10% FBS for an additional 3 d. In each case corresponding bright and fluorescent fields are shown. Scale bar = 25 ft. BRL-CM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell line-conditioned media.
expression of SSEA-1 antigen was found on undifferentiated ES cells cultured with LMH-CM or CEF feeder layers but not on their differentiated derivatives except after spontaneous differentiation of ES cells cultured with CEF feeder layers. The weak fluorescence remaining on these cells may indicate delayed differentiation and corresponded with a delay in colony spreading after transfer from CEF feeder layers to feeder layer-free dishes. Monoclonal antibody TROMA-1 reacts specifically with cytokeratin Endo-A (Brulet et ah, 1980). Endo-A is not expressed in the inner cell mass but appears after the formation of endoderm (Oshima, 1982; Duprey et a\., 1985). Additionally, this antigen is not expressed in undifferentiated ES cells but is highly expressed after
spontaneous and retinoic acid-induced differentiation (Mummery et ah, 1990). In the present experiment, Endo-A was not expressed on ES cells maintained with LMH-CM, BRL-CM, or STO feeder layers, but was expressed after spontaneous differentiation. After retinoic acid-induced differentiation, the expression was limited to endoderm-like cells and some of the fibroblastic cells. The ES cells cultured on a CEF feeder layer expressed a high level of Endo-A, suggesting that some changes or modifications had already occurred or that Endo-A may not be a reliable indicator of differentiation. Nevertheless, after retinoic acid-induced differentiation, TROMA-1 staining was similar to that observed from the other three culture systems.
AVIAN CYTOKINES AND STEM CELL CULTURE
The LIF has a potent differentiationinhibiting activity on ES cells (Smith, et al, 1988; Williams et al, 1988) and seems to be biologically active across several mammalian species. For example, human LIF improves the viability of cultured ovine embryos (Fry et al, 1992) and stimulates the proliferation of mouse myelocytic leukemia cells (Ohno and Abe, 1991). Most significantly, the recent cloning and sequencing of the ovine and porcine LIF genes indicate that LIF is one of the most conserved hematopoietic cytokines and that ovine LIF is biologically active in the mouse (Willson et al, 1992). We have shown that media conditioned by LMH cells or feeder layers made with primary CEF can replace a mammalian feeder layer or BRL-CM in mouse ES cell culture. After maintenance with the chicken culture system for over 10 passages, the ES cells still remained pluripotent and were capable of undergoing spontaneous or retinoic acid-induced differentiation that was associated with the disappearance or reduction of the ES cell-specific markers alkaline phosphatase and SSEA-1. Hence, it appears that avian cells can produce a LIFlike cytokine that is active in inhibiting the differentiation of mouse ES cells. The isolation and characterization of a pleiotropic cytokine such as LIF would be useful in several areas, including studies of the avian immune system, hematopoiesis, and the establishment of an avian ES cell line. The culture of mouse ES cells using an avian cell culture system could provide an essential bioassay. ACKNOWLEDGMENTS
The authors wish to acknowledge Beth Kegelmeyer for technical assistance and Carolle Bolnet for her advice. Timely discussions with Muquarrab Qureshi were greatly appreciated. This work was supported by grants from the National Research Initiative Competitive Grants Program/USDA (No. 91-37205-6320) and North Carolina Biotechnology Center (No. 9113-ARG-0412). REFERENCES Bernsteine, E. G., M. L. Hooper, S. Grandchamp, and B. Ephrussi, 1973. Alkaline phosphatase activity in mouse teratoma. Proc. Natl. Acad. Sci. USA 70:3899-3903.
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conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78:7634-7638. Magnuson, X, C. T. Epstein, L. M. Silver, and G. R. Martin, 1982. Pluripotent embryonic stem cell lines can be derived from t w5 /t w5 blastocysts. Nature 298:750-753. McMahon, A. P., and A. Bradley, 1990. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62:1073-1085. Mummery, C. L., A. Feyen, E. Freund, and S. Shen, 1990. Characteristics of embryonic stem cell differentiation: a comparison with two embryonal carcinoma cell lines. Cell Differ. Dev. 30:195-206. Nichols, J., E. P. Evans, and A. G. Smith, 1990. Establishment of germline-competent embryonic stem (ES) cells using differentiationinhibiting activity/LIF. Development 110: 1341-1348. Notarianni, E., C. Galli, S. Laurie, R. M. Moor, and M. J. Evans, 1991. Deviation of pluripotent embryonic cell lines from pig and sheep. J. Reprod. Fertil. S43:255-260. Notarianni, E., S. Laurie, R. M. Moor, and M. J. Evans, 1990. Maintenance and differentiation in culture of pluripotential embryonic cell lines from pig blastocysts. J. Reprod. Fertil. S41:51-56. Ohno, M., and T. Abe, 1991. Rapid calorimetric assay for the quantification of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). J. Immunol. Methods 145:199-203. Oshima, R. G., 1982. Developmental expression of murine extra-embryonic endodermal cytoskeletal proteins. J. Biol. Chem. 257:3414-3421. Papaioannou, V. E., and J. Rossant, 1983. Effect of the embryonic environment on proliferation and differentiation of embryonal carcinoma cells. Cancer Surv. 2:165-183. Pease, S., P. Braghetta, D. Gearing, D. Grail, and R. L. Williams, 1990. Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF). Dev. Biol. 141: 344-352. Pease, S., and R. L. Williams, 1990. Formation of germ-line chimeras from embryonic stem cells maintained with recombinant leukemia inhibitory factor. Exp. Cell Res. 190:209-211. Petitte, J. N , C. L. Brazolot, M. E. Clark, G. Liu, A.M.V. Gibbins, and R. J. Etches, 1993. Accessing the genome of the chicken using germline chimeras. Pages 81-101 in: R. J. Etches and A.M.V. Gibbins, ed., Manipulation of the Avian Genome. CRC Press, Boca Raton, FL. Petitte, J. N , M. E. Clark, G. Liu, A. M. VerrinderGibbins, and R. J. Etches, 1990. Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108:185-189. Piedrahita, J. A., G. B. Anderson, and R. H. Bon Durant, 1990. On the isolation of embryonic stem cells: comparative behavior of murine, porcine, and ovine embryos. Theriogenology 34: 879-901. Reid, L. H., E. G. Shesely, H.-S. Kim, and O. Smithies, 1991. Cotransformation and gene targeting in
mouse embryonic stem cells. Mol. Cell Biol. 11: 2769-2777. Robertson, E. J., 1987. Embryo-derived stem cell lines. Pages 71-112 in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. IRL Press, Oxford, England. Robertson, E. J., M. J. Evans, and M. H. Kaufman, 1983. X-chromosome instability in pluripotential stem cell lines derived from parthenogenetic embryos. J. Embryol. Exp. Morphol. 74:295-309. Saito, S., N. Strelchnko, and H. Nieman, 1992. Bovine embryonic stem cell-like cell lines culture over several passages. Roux's Arch. Dev. Biol. 201: 134-141. Smith, A. G., 1991. Culture and differentiation of embryonic stem cells. J. Tiss. Cult. Meth. 13: 89-94. Smith, A. G., J. K. Heath, D. D. Donaldson, G. G. Wong, J. Moreau, M. Stahl, and D. Rogers, 1988. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688-690. Smith, A. G., and M. L. Hooper, 1987. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev. Biol. 121:1-9. Solter, D., and B. B. Knowles, 1978. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc. Natl. Acad. Sci. USA 75:5565-5569. Strickland, S., K. K. Smith, and K. R. Marotti, 1980. Hormonal induction of differentiation in teratocarcinoma stem cells: Generation of parietal endoderm by retinoic acid and dibutyryl cAMP. Cell 21:347-355. Sukoyan, M. A., A. N. Golubitsa, A. I. Zhelezova, A. G. Shilov, S. Y. Vatoin, L. P. Maximovsky, L. E. Andreeva. J. McWhir, S. D. Pack, S. I. Bayborodin, A. Y. Kerkis, H. I. Kizolva, and O. L. Serov, 1992. Isolation and cultivation of blastocyst-derived stem cell lines from American mink (Mustek vison). Mol. Reprod. Dev. 33: 418-431. Wiles, M. V., and G. Keller, 1991. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 111:259. Williams, R. L., D. J. Hilton, S. Pease, T. A. Willson, C. L. Stewart, D. P. Gearing, E. F. Wagner, D. Metcalf, N. A. Nicola, and N. M. Gough, 1988. Myeloid leukemia inhibitory factor (LIF) maintains the developmental potential of embryonic stem cells. Nature 336:684-687. Willson, T. A., D. Metcalf, and N. M. Gough, 1992. Cross-species comparison of the sequence of leukemia inhibiting factor gene and its protein. Eur. J. Biochem. 204:21-30. Wobus, A. M., G. Wallukat, and J. Hescheler, 1991. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 48:173.
INTRODUCTION Mouse embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst, are pluripotent, and are able to differentiate in vivo and in vitro. Models of differentiation in vitro include cardiomyocytes, lymphocyte precursors, and other hemopoietic lineages (Wiles and Keller, 1991; Wobus et al, 1991; Chen and Kosco,
Received for publication December 1, 1993. Accepted for publication March 3, 1994. 1 1he use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service, nor criticism of similar products not mentioned. 2 Present address: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX 77030.
1993). In addition, ES cells can spontaneously differentiate into embryoid bodies or undergo retinoic acid-induced differentiation (Evans and Kaufman, 1981; Martin, 1981; Robertson et al, 1983). In vivo applications of ES cells take advantage of their ability to develop somatic and germ line chimeras after injection into the blastocyst. This has given rise to the powerful technique of gene targeting through the use of homologous recombination, which has become the only means to develop transgenic mice with sitedirected changes to the genome (Hasty et al, 1991; Joyner, 1991; Reid et al, 1991). Embryonic stem cells can be maintained for long periods in vitro on mouse fibroblast feeder (STO) cells or in media conditioned by Buffalo rat liver (BRL) cells (Smith and Hooper, 1987). The differ-
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entiation-inhibiting substance in both culture systems has been identified as leukemia inhibitory factor (LIF) (Williams et ah, 1988), which is a pleiotropic cytokine with diverse effects in many cell types (Gough et ah, 1989). For example, LIF can induce differentiation of murine myeloid leukemia (Ml) cells, stimulate the proliferation of primary myoblasts, or inhibit differentiation of mouse ES cells. Mouse ES cells have been isolated successfully and maintained by the use of purified LIF, recombinant LIF, or feeder cells stably transfected with a LIF expression vector (Nichols et ah, 1990; McMahon and Bradley, 1990; Pease and Williams, 1990; Pease et ah, 1990). The successful use of mouse ES cells in studies designed to alter the genome in a site-specific manner has promoted considerable interest in the establishment of ES cells for other mammalian species. The technical scheme for the production of transgenic animals with targeted changes to the genome requires the ability to produce early embryonic chimeras that give rise to functional gametes, the culture of ES cells capable of producing germline chimeras, and the development of DNA constructs that undergo homologous recombination. The adaptation of these procedures to mammalian livestock species requires the establishment and culture of ES cells. Current attempts to develop culture conditions suitable for ES cell growth from agriculturally important species has been mixed. However, several groups have been able to culture cells with an ES cell phenotype from bovine, ovine, and porcine embryos and from mink (Piedrahita et ah, 1990; Notarianni et ah, 1990,1991; Saito et ah, 1992; Sukoyan et ah, 1992).
chimeras. Petitte et ah (1990) were the first to demonstrate that germline chimeras could be produced in domestic fowl by the transfer of early blastodermal cells from one embryo to another. Subsequent investigations have optimized the procedures so that avian germline chimeras can now be produced routinely (Carscience et ah, 1993; Petitte et ah, 1993). As with mammalian livestock species, the next required step is the establishment of ES cells. Therefore, as a prelude towards the development of an ES cell culture system for the avian embryo, we examined the ability of avian cells to support the maintenance of mouse ES cells and report that primary chicken embryonic fibroblasts (CEF) and media conditioned by a chicken liver cell line can successfully be used to culture mouse ES. MATERIALS AND METHODS Feeder Layers
The STO cells (American Type Cell Collection (ATTC), No. CRL 1503)3 w e r e cultured in 75-cm2 flasks in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), and were mitotically inactivated after a 2- to 3-h exposure of 10 /tg/mL Mitomycin C 4 Inactivated STO cells were seeded at a concentration of 1 x 105 cells/cm2 onto 60-mm or 35-mm diameter tissue culture dishes coated with .1% gelatin.4 Chick embryonic fibroblasts were obtained from 10-d-old chicken embryos. After removal of the head, viscera, legs, and wings, the remainder of the embryos were minced and incubated 15 min in a solution of .05% trypsin and .02% EDTA.5 Dissociated CEF cells were seeded into In contrast to the above mammalian 75-cm2 flasks at a concentration of 3 x 106 species, the application of these proce- cells per flask and cultured in DMEM with dures for manipulation of the avian ge- 10% FBS. After the 10th passage, cells were nome has been complicated by the discarded and new CEF cultures were peculiarities of the reproductive strategy prepared. The CEF feeder layers were in birds and efforts in this area have been prepared the same way as for STO feeder focused on the development of germline layers. Conditioned Media 3Rockville, MD 20850. 4Sigma Chemical Co., St. Louis, MO 63178-9916. s Boehringer Mannheim, Indianapolis, IN 46250.
Medium conditioned with BRL cells was obtained after incubating 13 mL of DMEM
AVIAN CYTOKINES AND STEM CELL CULTURE
with 10% FBS in a 75-cm2 flask of confluent BRL cells (BRL 3A, ATCC No. CRL 1442)3 and harvesting every 3 d for up to 12 d. Before use, the medium was filtersterilized, diluted to 80% with DMEM containing 15% FBS, supplemented with .1 mM /3-mercaptoethanol,4 and designated BRL-CM. A chicken liver cell line, LMH (Kawaguchi et ah, 1987) was cultured until confluent in 75-cm2 flasks in DMEM with 10% FBS. Medium conditioned with LMH cells was harvested as described above for BRL-CM, diluted to 80% with DMEM containing 15% FBS, supplemented with .1 mM /3-mercaptoethanol, and designated LMH-CM. Culture of Embryonic Stem Cells The ES cells (ES-E14TG2a, ATCC No. CRL 1821)3 initially grown on STO feeder layers were seeded onto 60-mm culture dishes with or without feeder layers at a concentration of 1 x 106 cells per 60-mm dish. The ES cells seeded onto STO or CEF feeder layers were cultured in DMEM supplemented with 15% FBS and .1 mM /3-mercaptoethanol. The ES cells cultured in BRL-CM or LMH-CM were seeded onto pregelatinized plates. All cultures were passed every 2 to 3 d. After 10 or more passages, ES cell stocks were frozen and thawed prior to testing their ability to undergo spontaneous or induced differentiation.
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glass at a concentration of 1 x 10 cells per dish and in DMEM with 10% FBS. Following a 6-h incubation, the media were replaced with DMEM with 10% FBS containing 1 fiM retinoic acid.4 Retinoic acidcontaining media were changed twice at 24-h intervals with freshly prepared retinoic acid-containing media. After 72 h, the cells were cultured further in DMEM with 10% FBS for 3 d. Alkaline Phosphatase The ES cells and their differentiated progeny were stained for alkaline phosphatase activity. Cells were fixed in acetonecitrate-formaldehyde solution, rinsed in deionized water, and incubated in a naphthol-fast red violet substrate solution according to the manufacturer's instructions (diagnostic kit No. 86).4 After rinsing in deionized water, the cells were counterstained with hematoxylin, dehydrated, and mounted. Immunocytochemical Staining
The ES cells and their differentiated derivatives were tested for the expression of SSEA-1 and Endo-A using specific monoclonal antibodies. Anti-SSEA-1 and TROMA-1, which recognizes Endo-A, were obtained from Developmental Studies Hybridoma Bank.6 All cultures for immunocytochemical staining were fixed in cold acetone at -20 C for 8 min. After incubation with each monoclonal antibody, the cells were incubated with a corresponding biotinylated Spontaneous Differentiation second antibody followed by a complex of The ES cells grown with feeder layers or avidin and biotinylated alkaline phosphaconditioned media were seeded at a con- tase according to the manufacturer's procentration of 1 x 105 cells per 35-mm tocol (Vectastain, ABC-AP Kit)7 Enpregelatinized dish containing a coverglass dogenous alkaline phosphatase activity and cultured in DMEM with 10% FBS for up was inhibited using levamisole.7 The cells to 7 d. were then counterstained with hematoxylin, dehydrated, and mounted. These antigens were visualized using an epifluoresInduced Differentiation cent microscope with a rhodamine filter. The ES cells grown with feeder layers or conditioned media were seeded onto RESULTS pregelatinized 35-mm dishes with a coverMorphology 6
Iowa City, IA 52242. Sector Laboratories, Burlingame, CA 94010.
When the ES cells were initially transferred from an STO feeder layer to LMH-
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CM, BRL-CM, or a CEF feeder layer, 10 to 15% of the cells spread and differentiated into endoderm-like cells. Once the ES cells were cultured for two or three passages, spontaneous differentiation diminished and remained at a constant rate of less than 5%. Regardless of the culture system used, the characteristic ES cell phenotype was maintained, i.e., small cells with a large nucleus and relatively little cytoplasm (Figure 1). Slight differences in colony phenotype were noted and appeared to be related to the use of feeder cells and conditioned media. The ES cells grown on a monolayer of CEF were arranged in ovalshaped colonies that were parallel to the long axes of the underlying fibroblasts (Figure 1C). On STO feeder cells where the fibroblasts spread in all directions, the colonies were more rounded (Figure 1A vs 1C). Compared to the colonies formed on feeder layers, the ES cells grown using conditioned media produced irregularly shaped, less tightly packed colonies
(Figures 1A, 1C vs IB, ID). No differences in ES cell growth rate were observed among the four culture systems even after 10 passages. When the ES cells maintained with CEF feeder layers or LMH-CM were cultured in DMEM with 10% FBS to allow spontaneous differentiation, they continued to form typical ES cell colonies and remained phenotypically undifferentiated for 2 d, similar to that observed for cells grown on STO cells or in BRL-CM. Subsequently, all ES cell cultures began to spread and form large, flattened cells. However, ES cells cultured with CEF feeder layers required an additional day to begin differentiation than did the ES cells kept with LMH-CM, BRLCM, or STO feeder layers. In all cases, spontaneous differentiation was relatively homogeneous and appeared to be completed after 7 d of culture. Retinoic acid-induced differentiation proceeded quickly, and within the first 24 h of culture the ES cells began to differentiate.
cJL FIGURE 1. Undifferentiated mouse embryonic stem cells cultured with a mouse fibroblast STO feeder layer (A), BRL-CM (B), a CEF feeder layer (C), and LMH-CM (D), respectively. Phase contrast, scale bar = 150 p. BRLCM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell lineconditioned media.
AVIAN CYTOKINES AND STEM CELL CULTURE
An assortment of derivatives was produced within 5 to 6 d, nearly 2 d earlier than spontaneous differentiation. The ES cells cultured under the four different culture conditions shared similar differentiated cell types consisting of large fibroblast-like cells, endoderm-like cells, and neuronal cells. Most endoderm-like cells formed sheets of different sizes. Dendrite or axonlike processes of neuronal cells often extended over a monolayer of fibroblastic or endoderm-like cells.
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layers (Figure 4). No expression was found in STO or CEF feeder cells. Endo-A antigen was expressed at low levels on all of the spontaneously differentiated ES cells cultured with the four culture systems (Figure 4). However, after retinoic acid-induced differentiation, immunostaining of this antigen was heterogeneous and limited to endoderm-like cells, some fibroblastic cells, and other small, randomly distributed cells (Figure 4). DISCUSSION
Alkaline Phosphatase The undifferentiated ES cells cultured with CEF feeder layers or LMH-CM expressed high levels of alkaline phosphatase, similar to the ES cells cultured using STO feeder layers or BRL-CM (Figure 2). After spontaneous differentiation or retinoic acid-induced differentiation, the activity of this enzyme was totally lost in the ES cells from all cultures. No alkaline phosphatase activity was found in STO feeder cells or CEF feeder cells. SSEA-1 Antigen The undifferentiated ES cells cultured with CEF feeder layers or LMH-CM expressed high levels of SSEA-1 (Figure 2). After spontaneous differentiation, the expression of SSEA-1 was lost from all differentiated progeny of the ES cells maintained with LMH-CM, BRL-CM, or STO feeder layers (Figure 3). The expression of SSEA-1 by spontaneously differentiated ES cells cultured with CEF feeder layers was slightly different. A weak fluorescence still remained detectable on these cells (Figure 3). However, after retinoic acid-induced differentiation, ES cells from all four culture systems no longer expressed SSEA-1 (Figure 3). Endo-A Antigen Monoclonal antibody TROMA-1 was used to specifically recognize Endo-A antigen (Brulet et ah, 1980). There was no expression of Endo-A on the ES cells cultured with LMH-CM, STO feeder layers, or BRL-CM, but this antigen was expressed on the ES cells maintained on CEF feeder
This study has shown that CEF feeder layers and LMH-CM are capable of maintaining mouse ES cells in an undifferentiated state for over 10 passages. The cells retained typical morphologies of ES cells with a large nucleus and relatively little cytoplasm (Figure 1). After spontaneous differentiation, ES cell progeny consisted predominantly of large flattened, endoderm-like cells. The cell types observed after retinoic acid-induced differentiation were more heterogeneous than those observed after spontaneous differentiation and included fibroblast-like cells, endoderm-like cells, and neuronal cells. The same kinds of differentiated cells were obtained from the ES cells cultured with BRL-CM or STO feeder layers. The observation of flattened endodermlike cells obtained after spontaneous differentiation were consistent with previous studies (Robertson, 1987; Smith, 1991). In addition, Mummery et al. (1990) reported that when ES cells were induced to differentiate by retinoic acid in the absence of LIF, they appeared to form a mixed cell population consisting of endoderm-like cells but also of ill-defined mesoderm-like cell types. When ES cells were treated with retinoic acid in the presence of LIF, BRL-CM inhibited most types of differentiation but permitted the formation of parietal endoderm (Smith and Hooper, 1987; Mummery et al, 1990). Previous studies with the particular ES cell line used in the present study have consistently demonstrated the ability to differentiate into embryonic structures in the absence of feeder cells or conditioned medium and, when injected into a blastocyst, to contribute to the formation
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FIGURE 2. Alkaline phosphatase activity of undifferentiated embryonic stem cells (FC/CM), and after spontaneous differentiation with Dulbecco's Modified Eagle's Medium (DMEM), and retinoic acid-induced differentiation (DMEM + RA). Embryonic stem cells were cultured with mouse fibroblast STO feeder cells (1), BRL-CM (2), CEF feeder cells (3), or LMH-CM (4) for several passages. Spontaneous differentiation proceeded after culture in DMEM with 10% fetal bovine serum (FBS) for 7 d. For retinoic acid-induction, embryonic stem cells were treated with retinoic acid for 3 d followed by DMEM with 10% FBS for an additional 3 d. Alkaline phosphatase activity was detected with naphthol AS-B1 phosphate/fast red, counter-stained with hematoxylin. Scale bar = 12 /*. BRL-CM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell line-conditioned media.
of somatic a n d g e r m l i n e c h i m e r a s (Doetschman et ah, 1987; Hooper et ah, 1987; Smith and Hooper, 1987). Although the ES cells in this study were not tested for their ability to produce chimeric mice, the ability to differentiate spontaneously
or in response to a pulse of retinoic acid did not diminish with a change to a nonmammalian culture system. Embryonic stem cells share several functional characteristics with mouse embryonal carcinomas, particularly their abil-
AVIAN CYTOKINES AND STEM CELL CULTURE
FC/CM
DMEM
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DMEM + RA
FIGURE 3. SSEA-1 immunofluorescent staining of undifferentiated embryonic stem cells (FC/CM) and after spontaneous differentiation with Dulbecco's Modified Eagle's Medium (DMEM) and retinoic acid-induced differentiation (DEME + RA). Embryonic stem cells were cultured with mouse fibroblast STO feeder cells (1), BRL-CM (2), CEF feeder cells (3), or LMH-CM (4) for several passages. Spontaneous differentiation proceeded after culture in DMEM with 10% fetal bovine serum (FBS) for 7 d. For retinoic acid induction, embryonic stem cells were treated with retinoic acid for 3 d followed by DMEM with 10% FBS for an additional 3 d. In each case corresponding bright and fluorescent fields are shown. Scale bar = 25 /t. BRL-CM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell line-conditioned media.
ity to differentiate in vivo and in vitro (Papaioannou and Rossant, 1983; Smith and Hooper, 1987) and the expression of various antigenic determinants (Mummery et al, 1990; Kimber et al, 1993). Evans (1972) reported that the mouse embryonal carcinoma cell line, SIKR, was maintained for approximately 30 cell generations by a CEF feeder layer. When reinoculated into mice, the SIKR cells produced teratomas containing at least 10 types of tissues (Evans, 1972). Though similarities exist between embryonal carcinomas and ES cells, the usefulness of an avian feeder layer or conditioned media in ES cell culture remained untested until now. High levels of alkaline phosphatase activity are characteristic of embryonal
carcinomas and are lost after spontaneous or induced differentiation (Damjanov et al, 1971; Bernstine et al, 1973; Strickland et al, 1980). Because this enzyme was highly active in ES cells grown in the four different culture systems and subsequently diminished upon differentiation (Figure 2), the ES cells were further characterized using immunological markers. The SSEA-1 antigen, a stage-specific mouse embryonic antigen (Solter and Knowles, 1978), is a widely used marker for ES cells in culture. This antigen is expressed on the cell surface of undifferentiated embryonal carcinoma and ES cells but not on their differentiated progeny (Magnuson et al, 1982; Mummery et al, 1990). In the present study, the
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FIGURE 4. TROMA-1 immunofluorescent staining of undifferentiated embryonic stem cells (FC/CM) and after spontaneous differentiation with Dulbecco's Modified Eagle's Medium (DMEM) and retinoic acid-induced differentiation (DMEM + RA). Embryonic stem cells were cultured with a mouse fibroblast STO feeder layer (1), BRL-CM (2), a CEF feeder layer (3), or LMH-CM (4) for several passages. Spontaneous differentiation proceeded after culture in DMEM with 10% FBS for 7 d. For retinoic acid induction, embryonic stem cells were treated with retinoic acid for 3 d followed by DMEM with 10% FBS for an additional 3 d. In each case corresponding bright and fluorescent fields are shown. Scale bar = 25 ft. BRL-CM = buffalo rat liver-conditioned media; CEF = chick embryonic fibroblasts; LMH-CM = LMH cell line-conditioned media.
expression of SSEA-1 antigen was found on undifferentiated ES cells cultured with LMH-CM or CEF feeder layers but not on their differentiated derivatives except after spontaneous differentiation of ES cells cultured with CEF feeder layers. The weak fluorescence remaining on these cells may indicate delayed differentiation and corresponded with a delay in colony spreading after transfer from CEF feeder layers to feeder layer-free dishes. Monoclonal antibody TROMA-1 reacts specifically with cytokeratin Endo-A (Brulet et ah, 1980). Endo-A is not expressed in the inner cell mass but appears after the formation of endoderm (Oshima, 1982; Duprey et a\., 1985). Additionally, this antigen is not expressed in undifferentiated ES cells but is highly expressed after
spontaneous and retinoic acid-induced differentiation (Mummery et ah, 1990). In the present experiment, Endo-A was not expressed on ES cells maintained with LMH-CM, BRL-CM, or STO feeder layers, but was expressed after spontaneous differentiation. After retinoic acid-induced differentiation, the expression was limited to endoderm-like cells and some of the fibroblastic cells. The ES cells cultured on a CEF feeder layer expressed a high level of Endo-A, suggesting that some changes or modifications had already occurred or that Endo-A may not be a reliable indicator of differentiation. Nevertheless, after retinoic acid-induced differentiation, TROMA-1 staining was similar to that observed from the other three culture systems.
AVIAN CYTOKINES AND STEM CELL CULTURE
The LIF has a potent differentiationinhibiting activity on ES cells (Smith, et al, 1988; Williams et al, 1988) and seems to be biologically active across several mammalian species. For example, human LIF improves the viability of cultured ovine embryos (Fry et al, 1992) and stimulates the proliferation of mouse myelocytic leukemia cells (Ohno and Abe, 1991). Most significantly, the recent cloning and sequencing of the ovine and porcine LIF genes indicate that LIF is one of the most conserved hematopoietic cytokines and that ovine LIF is biologically active in the mouse (Willson et al, 1992). We have shown that media conditioned by LMH cells or feeder layers made with primary CEF can replace a mammalian feeder layer or BRL-CM in mouse ES cell culture. After maintenance with the chicken culture system for over 10 passages, the ES cells still remained pluripotent and were capable of undergoing spontaneous or retinoic acid-induced differentiation that was associated with the disappearance or reduction of the ES cell-specific markers alkaline phosphatase and SSEA-1. Hence, it appears that avian cells can produce a LIFlike cytokine that is active in inhibiting the differentiation of mouse ES cells. The isolation and characterization of a pleiotropic cytokine such as LIF would be useful in several areas, including studies of the avian immune system, hematopoiesis, and the establishment of an avian ES cell line. The culture of mouse ES cells using an avian cell culture system could provide an essential bioassay. ACKNOWLEDGMENTS
The authors wish to acknowledge Beth Kegelmeyer for technical assistance and Carolle Bolnet for her advice. Timely discussions with Muquarrab Qureshi were greatly appreciated. This work was supported by grants from the National Research Initiative Competitive Grants Program/USDA (No. 91-37205-6320) and North Carolina Biotechnology Center (No. 9113-ARG-0412). REFERENCES Bernsteine, E. G., M. L. Hooper, S. Grandchamp, and B. Ephrussi, 1973. Alkaline phosphatase activity in mouse teratoma. Proc. Natl. Acad. Sci. USA 70:3899-3903.
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Brulet, P., C. Babinet, R. Kemle, and F. Jacob, 1980. Monoclonal antibodies against trophectodermspecific markers during mouse blastocyst formation. Proc. Natl. Acad. Sci. USA 77:4113-4117. Carscience, R. S., M. E. Clark, A. M. Verrinder Gibbins, and R. J. Etches, 1993. Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 117:669-675. Chen, U., and M. Kosco, 1993. Differentiation of mouse embryonic stem cells in vitro: HI. Morphological evaluation of tissues developed after implantation of differentiated mouse embryoid bodies. Dev. Dyn. 197:217-226. Damjanov, I., D. Solter, and N. Skreb, 1971. Enzyme histochemistry of experimental embryo derived teratocarcinomas. Z. Krebsforsch. 76:249-256. Doetschman, T., R. G. Gregg, N. Maeda, M. L. Hooper, D. W. Melton, S. Thompson, and O. Smithies, 1987. Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330:576-578. Duprey, P., D. Morello, M. Vasseur, C. Babinet, H. Londamine, P. Brulet, and F. Jacob, 1985. Expression of the cytokeratin endo-A gene during early mouse embryogenesis. Proc. Natl. Acad. Sci. USA 82:8535-8539. Evans, M. J., 1972. The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells. J. Embryol. Exp. Morphol. 28: 163-176. Evans, M. J., and M. H. Kaufman, 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-156. Fry, R. G, P. A. Batt, R. J. Fairclough, and R. A. Parr, 1992. Human leukemia inhibitory factor improves the viability of cultured ovine embryos. Biol. Rep. 46:470-474. Gough, N. M., R. L. Williams, D. J. Hilton, S. Pease, T. A. Willson, H. Stahl, D. P. Gearing, N. A. Nocola, and D. Metcalf, 1989. LIF: a molecule with divergent actions on myeloid leukaemic cells and embryonic stem cells. Reprod. Fertil. Dev. 1:281-288. Hasty, P., J. Rivera-Perez, C. Chang, and A. Bradeley, 1991. Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells. Mol. Cell. Biol. 11:4509-4517. Hooper, M., K. Hardy, A. Handyside, S. Hunter, and M. Monk, 1987. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326:292-295. Joyner, A. L., 1991. Gene targeting and gene trap screens using embryonic stem cells: New approaches to mammalian development. BioEssays 13:649-656. Kawaguchi, T., K. Nomura, Y. Hirayama, and T. Kitagawa, 1987. Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res. 47:4460-4464. Kimber, S. J., D. G. Brown, P. Pahlsson, and B. Nilsson, 1993. Carbohydrate antigen expression in murine embryonic stem cells and embryos. II. Sialyated antigens and glycolipid analysis. Histochem. J. 25:628-641. Martin, G. R., 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium
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