Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state.

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.

Leukemia inhibitory factor (LIF), 1 a member of the interleukin (IL)-6 family, is a multifunctional cytokine that acts on a wide range of cell types including osteoblasts, hepatocytes, adipocytes, neurons, embryonic stem (ES) cells, and megakaryocytes (1,2). LIF was identified initially as an inducer of the differentiation of M1 leukemia cells (3,4) and re-discovered as an inhibitor of the differentiation of mouse ES cells (5). Mouse ES cells can be maintained without feeder cells in the presence of fetal calf serum and mouse LIF (mLIF) (6). Because the injection of ES cells into blastocysts can give rise to chimeric mice and the ES cells can contribute to all of the tissues including the germ cells, mutant mouse ES cells have been used to produce gene-disruptant mouse strains. The relative ease with which one can manipulate ES cells in vitro has made them a powerful tool for targeting endogenous genes, and this led to a dramatic increase in the number of targeted transgenic mice ("knock-out" mice) throughout the 1990s.
The challenge has been to isolate and characterize cells with similar properties from other species. It was a number of years before ES and/or embryonic germ (EG) cells were isolated from non-murine mammalian species including the medaka (7), rabbit (8), pig (9), monkey (10), and human (11) but germ line transmission has only been successfully demonstrated in the mouse. LIF is not required for self-renewal of the ES cells of primates including humans (10,11). Moreover, the maintenance of the undifferentiated state of other ES-like cells with the exception of murine is inadequate with only LIF (8,9). Thus, it has been thought that this cytokine is only able to inhibit differentiation of mouse ES cells.
The chicken is the only animal other than the mouse in which germ line transmission of transgenes has been achieved (12)(13)(14)(15)). Pain et al. (16) first reported that pluripotential avian stem cells could be produced and maintained by long term culture of stage X blastodermal cells with several cytokines: chicken stem cell factor, bovine basic fibroblast growth factor, mouse IL-6, human IL-11, and mLIF. Moreover, two chickens injected with 7-day cultured chicken embryonic cells were proven to be germ line chimeras. Park and Han (17) later reported the establishment of chicken EG cell lines from primordial germ cells in culture medium supplemented with human stem cell factor, bovine basic fibroblast growth factor, human IL-11, mLIF, and human insulin-like growth factor-1 and showed that the EG cells were incorporated into various chimeric tissues. However, there are no reports of chicken ES or EG cell lines contributing to the germ line after extended culture, although there have been promising developments in this regard. We suspect that mammalian cytokines are not fully effective on chicken ES or EG cells given the low identity between chicken and mammalian ILs (18). Only eight IL homologue genes (IL-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL- 17, have been cloned in the chicken, and these have only 20 -40% amino acid sequence identity with their mammalian homologues. Mammalian IL-1 fails to stimulate the division of chicken thymocytes in the presence of submitogenic levels of phytohemagglutinin (19), and mammalian IL-2 does not induce proliferation of chicken lymphocytes (20). Therefore, it seemed probable that chicken LIF (chLIF) would be more effective in maintaining chicken ES or EG cells in the undifferentiated state than its mammalian homologue.
LIF acts via a cell surface receptor complex composed of the low affinity LIF receptor ␤ and gp130, a common receptor subunit of the IL-6 cytokine family (1). LIF binds to LIF receptor ␤, which then forms a high affinity heterodimeric complex with gp130 (21,22). There are two major signaling pathways downstream of gp130 in M1 leukemia cells, the Jak-STAT and Shp2-ERK pathways (23). STAT1 and STAT3 are activated in ES cells, and activation of STAT3 by LIF is sufficient to maintain the undifferentiated state of mouse ES cells (24,25). In this study, we cloned the cDNA of the chicken homologue of mammalian LIF and compared the effect of recombinant chLIF (rchLIF) and mLIF on chicken ES-like cells (blastodermal cells).

Animals, Embryos, and Cell Lines-Partially inbred chickens (H-B15
White Leghorn), originally supplied by Dr. Vainio (Turk University, Turku, Finland), were bred in our animal facilities and provided with feed and chlorinated water ad libitum. Chicken tissues were prepared from 7-week-old chickens. Fertilized laid eggs of White Leghorn chickens were purchased from Akita Co. (Fukuyama, Japan).
Lipopolysaccharide Treatment-For suppression subtractive hybridization (SSH), cultured IN24 cell lines were exposed to serum-free IMDM containing lipopolysaccharide (LPS, Escherichia coli O127:B8, Difco Laboratories). They were grown to 80% confluence in 60-mm culture dishes (BD Biosciences) in 10% FBS-IMDM, washed three times with serum-free IMDM, and resuspended in 5 ml of serum-free IMDM or serum-free IMDM with 10 g/ml LPS. The cells were cultured for 24 h in this condition, and poly(A) ϩ RNA was isolated every 4 h.
Cloning of Chicken LIF-To clone chLIF cDNA, total RNA was isolated from IN24 cells with ISOGEN-LS (Takara, Japan) and poly(A) ϩ RNA was purified with Oligotex-dT30 Super (Nippon Roche, Tokyo, Japan). SSH was performed with the Clontech PCR-Select TM cDNA subtraction kit (BD Biosciences Clontech) as described by the manufacturer with some modifications. The starting material consisted of 4 g of poly(A) ϩ RNA from LPS-treated or control cells (tester RNA and driver RNA, respectively). The subtracted cDNA was diluted 1:400 prior to PCR. Primary PCR was performed for 30 cycles, and secondary PCR was performed for 15 cycles. Three microliters of secondary PCR product were cloned into pGEM-T Easy vector (Promega).
This subtraction cDNA library was screened by the method of differential display using the Clontech PCR-Select differential screening kit as described by the manufacturer with some modifications. Random clones were amplified by PCR for 23 cycles, and five microliters of product was separated on a 1.5% agarose gel and stained with EtBr. The PCR products including the cDNA insert were denatured with NaOH, and transferred to a nylon membrane. This cDNA dot blot membrane was then baked for 2 h at 70°C. Probes for hybridization were prepared from the subtracted cDNA and labeled with digoxigenin using a digoxigenin DNA-labeling kit (Roche Diagnostics). The membranes were hybridized to digoxigenin-labeled probes according to the supplier's directions (Roche Diagnostics). The selected positive clones were sequenced with an automated ABI PRISM 3100 genetic analyzer (Applied Biosystems Japan Ltd.) using the ABI PRISM BigDye Terminator (Applied Biosystems Japan Ltd.).
To complete the 3Ј-and 5Ј-ends of the chLIF cDNA, rapid amplification of cDNA ends (RACE) was performed using the BD SMART RACE cDNA amplification kit (BD Biosciences, Clontech) according to the manufacturer's instructions.
Reverse Transcription (RT)-PCR-For expression analysis, 0.5 g of poly(A) ϩ RNA purified as described above was reverse-transcribed at 42°C with the SuperScript preamplification system (Invitrogen) in a 20-l reaction mixture containing oligo(dT) [12][13][14][15][16][17][18] primer as described by the manufacturer. The cDNA was extracted with phenol-chloroform, ethanol-precipitated, and resuspended in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Semi-quantitative PCR was carried out with the ABI PRISM 7700 sequence-detection system (Applied Biosystems Japan Ltd.) using SYBR Green PCR core reagents (Applied Biosystems Japan Ltd.). The cDNAs were normalized using chicken ␤-actin SYBR Green PCR according to the manufacturer's instructions. Equal quantities of normalized cDNA were used as template in PCR reactions with chLIF-specific primers as follows: forward (5Ј-TCCTCAACGCCTCAC-TGG-3Ј) and reverse (5Ј-GCCCTGCTGCTTCTTCTT-3Ј). The primers used for amplification of ␤-actin were as follows: forward (5Ј-CACCTT-CCAGCAGATGTGGAT-3Ј) and reverse (5Ј-GCAAATGCTTCTAAACC-GGACT-3Ј). PCR amplification was carried out with 10 M of primers, 0.2 mM of each dNTP and 1ϫ TaqDNA polymerase buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 ), and 1.5 units of Ampli-Taq-Gold (PerkinElmer Life Sciences) in a final volume of 50 l in a GeneAmp PCR System 9700 (Applied Biosystems Japan Ltd.) by first heating the mixture to 95°C for 10 min and then holding it for 1 min at 94°C (denaturation), 1 min at 60°C (annealing), and 2 min at 72°C (polymerization). After 25 amplification cycles in the linear range, the reaction mixture was held for 10 min at 72°C as a final extension step. The PCR products were analyzed on 1.5% of agarose gel and visualized with EtBr.
Expression of Chicken LIF in E. coli-The coding region of chLIF was amplified with the following primers: forward primer (5Ј-CGGGATCC-CGGGCGCTGCTGGGGACGAG-3Ј) and reverse primer (5Ј-CCCTCGA-GTTATCAGGCGGGGCTGAGGTGAGGTA-3Ј). The PCR product was digested with BamHI and XhoI and subcloned into the pGEX-6P-1 plasmid (Amersham Biosciences) harboring glutathione S-transferase. E. coli BL21 transformed with the recombinant plasmid was grown at 20°C to A 600 ϭ 0.5 and induced with 0.1 mM isopropyl-␤-D-thiogalactopyranoside for 15 h. After sonication, the recombinant protein was bound to glutathione-Sepharose 4B (Amersham Biosciences) and Pre-Scission protease (Amersham Biosciences) was added to remove the glutathione S-transferase affinity tail and elute the rchLIF. This was purified with a Superdex 200 HR column (Amersham Biosciences) using the ⌬KTAexplorer 10S system (Amersham Biosciences). The recombinant protein was analyzed by electrophoresis on a 15% polyacrylamide gel and visualized by Coomassie Brilliant Blue R-250 staining.
Culture of Blastodermal Cells-Freshly laid unincubated White Leghorn eggs were used. Incubation and cultivation of blastodermal cells prepared from stage X chicken embryos were carried out essentially as described previously (27), and embryonic stages were defined according to Eyal-Giladi and Kochav (28). Stage X blastoderms were separated from the yolk, and the area pellucida was removed by gentle aspiration with a micropipette into PBS at room temperature and dissociated by pipetting with a Pasteur pipette. The blastodermal cells were centrifuged twice at 400 ϫ g, and the cell pellet was resuspended at 7 ϫ 10 4 cells/ml in Dulbecco's modified Eagle's medium high glucose (Invitrogen) containing 10% FBS (HyClone, lot number AJL11137), 2% of chicken serum (Dainippon Pharmaceutical), 1 mM sodium pyruvate (Invitrogen), 1% of non-essential amino acid (Invitrogen), 1 M of each of adenosine, guanosine, cytidine, uridine, and thymidine (Sigma), 100 units/ml penicillin (Invitrogen), and 100 g/ml streptomycin (Invitrogen). This medium is referred to as cytokine-free chicken embryonic stem cell medium (CESM). The blastodermal cells were cultured in the cytokine-free CESM with or without recombinant mouse LIF (Chemicon) or rchLIF in 24-well culture plates at 38.5°C in 5% CO 2 .
Alkaline Phosphatase (ALP) Reaction-Cultured blastodermal cells were gently washed with PBS and fixed in cold 4% paraformaldehyde for 15 min at 4°C. After further washing with PBS, they were resuspended in ALP-staining mixture containing 100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 5 mM MgCl 2 , 1 mg/ml nitro blue tetrazolium (Sigma), and 0.1 mg/ml BCIP (Sigma) and incubated for 20 min at 37°C. The reaction was stopped by adding 10 mM EDTA, and the wells were washed with PBS. The stained cells were observed with an inverted fluorescence microscope (BX51, Olympus, Japan).
Immunofluorescence Staining-Cultured blastodermal cells were pelleted, washed three times with PBS, and fixed in cold 4% paraformaldehyde for 30 min at 4°C. After washing with PBS, they were incubated with anti-EMA-1 or anti-SSEA-1-monoclonal antibodies (MC-480) from the Development Studies Hybridoma Bank (University of Iowa) in the presence of 10% BlockAce (Snow Brand Milk Products) for 1 h at 4°C. After washing, the cells were exposed to fluorescein isothiocyanate-conjugated sheep anti-mouse Ig F(abЈ) 2 fragment (Silenus, Victoria, Australia) for 30 min at 4°C and washed again. The stained cells were observed with the inverted fluorescence microscope.

RESULTS
Cloning of Chicken LIF-Human LIF mRNA is strongly expressed in activated monocytes (29). We used SSH and differential display to isolate chicken LIF cDNA from the genes differentially expressed in a chicken monocytic cell line (IN24) stimulated with LPS. Using the cDNA from LPS-treated IN24 cells as tester and the cDNA from non-treated cell as a driver, we generated a subtracted cDNA population by SSH as described under "Experimental Procedures." Approximately 1,000 clones of the resulting cDNA library were screened by differential display, and we eventually identified 122 cDNA fragments that appeared to originate from differentially expressed genes. The nucleotide sequences of these cDNA fragments were analyzed by a homology search in DDBJ/EMBL/ GenBank TM databases using the BLASTN program (30). One cDNA fragment showed approximately 30% identity to a part of mammalian LIF.
The complete chLIF open reading frame from which this fragment was derived was obtained by the RACE cDNA amplification method, and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence are shown in Fig. 1. The sequence is 789-bp long and contains a complete 633-bp open reading frame. The deduced amino acid sequence contains 211 amino acids, and computer-assisted analysis (www.cbs.dtu.dk/services/SignalP-2.0/) and sequence comparison with human and mouse LIF sequences revealed the presence of a signal peptide of 24 amino acids (underlined) at its N terminus (Figs. 1 and 2A). The chLIF protein contains six asparagine-linked glycosylation sites (Asn-X-Ser or Asn-X-Thr, boxed).
Comparison of Chicken and Mammalian LIF Sequences-The deduced amino acid sequence of chicken LIF was compared with that of human LIF (Swiss Prot accession number P15018) and mLIF (Swiss Prot accession number P09056) using the Pileup command (GCG program, GCG sequence analysis software, version 9.0, GCG Inc.). An alignment of the sequences is shown in Fig. 2A. The putative chLIF protein includes the six cysteine residues conserved in mammalian LIFs. The number of asparagine-linked glycosylation sites was the same, but the position of the three of them differed between chLIF and the mammalian LIFs.
The chicken LIF precursor protein has a 42.7 and 39.3% identity with human and mouse LIF, respectively (Fig. 2B). Thus, the degree of identity between chicken and mammalian LIFs is low as with other interleukins.
Expression Analysis-To determine the expression profile of chLIF, we performed semi-quantitative RT-PCR with cDNAs prepared from various tissue and in vitro stimulated IN24 cell lines as described under "Experimental Procedures." To detect changes in chLIF mRNA expression, identical RT-PCR reactions were performed with mRNAs obtained from LPS-treated and control IN24 cells. Control cells contained low levels of chLIF mRNA, and LPS-treatment induced a gradual increase to a maximum at 20 h (Fig. 3A). The expression profile of chLIF in various tissues is shown in Fig. 3B. The chLIF amplification product (211-bp) was detected in all of the tissues and cell lines and was most abundant in the liver and thymus.
Bioactivity of rchLIF-A cDNA fragment encoding the putative mature form of chLIF was amplified by PCR and purified as described under "Experimental Procedures." The purified product had an apparent molecular mass of ϳ19 kDa on SDS-PAGE, which was expected from its calculated molecular mass. rchLIF was Ͼ90% pure (Fig. 4).
ALP has been shown previously to be a marker of mouse primordial germ cell, teratocarcinoma cells (31), and chicken and quail embryonic cells (16). To define the effect of rchLIF on the proliferation of ALP-positive cells, chicken blastodermal cells were cultured with or without rchLIF or mLIF for 9 days. As shown in Fig. 5A, rchLIF had good activity and the rate of formation of ALP-positive cells was high compared with mLIF over 6 days of incubation. However, mLIF did increase the number of positive cells compared with no LIF. After 9 days of culture, almost all of the blastodermal cells formed cystic-like EBs in the presence of mLIF and in the absence of LIF but very few appeared in the presence of rchLIF.
The chicken embryonic cells were characterized by the presence of surface antigens (EMA-1 and SSEA-1), which are known to be specific to ES cells (16). The EMA-1 antibody recognizes epitopes specific to primordial germ cell in both mouse and chicken (32,33). To define the ability of rchLIF to maintain the chicken blastodermal cells in the undifferentiated state, the cells were cultured with or without rchLIF or mLIF for 9 days. As shown in Fig. 5B, the rate of EMA-1-positive cells was high with rchLIF compared with mLIF in all of the conditions of cultivation. In the presence of rchLIF, EMA-1-positive cells increased gradually up to 5 days of culture, whereas they decreased in the presence of mLIF. After 7 days of culture, the chicken blastodermal cells cultured with rchLIF formed a large colony as they did with mLIF but many strongly EMA-1-positive cells were observed in the rchLIF colonies. The same result was obtained with SSEA-1 antibody (data not shown).
Activation of STAT3 and Mitogen-activated Protein Kinase-There are two major pathways of intracellular signaling from LIF in ES cells, the Jak-STAT3 pathway and the Shp2-ERK pathway. The LIF signal is mainly transmitted to the nucleus by the Jak-STAT3 pathway, and the Shp2-ERK pathway does not contribute directly to stem cell self-renewal (34). To determine whether rchLIF activates STAT3, we examined tyrosine phosphorylation of STAT3 and phosphorylation of MEK1/2 in the Shp2-ERK pathway. As shown in Fig. 6, treatment with rchLIF led to phosphorylation of STAT3 but treatment with mLIF did not. Phosphorylation of STAT3 by rchLIF was greatest with 50 ng/ml rchLIF and fell at a higher concentration of rchLIF (200 ng/ml). It was maximal with 20 -50 ng/ml rchLIF (data not shown). On the other hand, both rchLIF and mLIF induced phosphorylation of MEK1/2 (Fig. 6). 150 ng/ml mLIF and 50 ng/ml rchLIF were optimal for inducing MEK1/2 phosphorylation. These results indicate that the signal pathways activated by chLIF and mLIF in chicken blastodermal cells differ. DISCUSSION LIF plays a vital role in maintaining the proliferation and pluripotentiality of mouse ES cells in vitro (35). Pain et al. (16) characterize ES cells derived from cultures of chicken and quail embryos and reported that LIF also permitted the long term multiplication of cells with markers and morphogentic potential similar to mouse ES cells. However, there are no reports of chicken ES cell lines contributing to the germ line after extensive culture. We suspected that mammalian cytokines might not function correctly in chicken ES cells because of the low degree of identity between avian and mammalian ILs. Therefore, we cloned the cDNA of the chicken homologue of mammalian LIF and compared the biologically effect of recombinant chLIF and mLIF on chicken blastodermal cells. We confirmed that the LIF gene we isolated is encoded by the chicken genome by genomic PCR and Southern hybridization (data not shown). However, we did not identify the typical polyadenylation signal and poly(A) tail.
LIF is a member of the cytokine family that includes IL-6, IL-11, oncostatin M, and cardiotrophin. These proteins share a common helical structure with four antiparallel ␣ helices and act through a common signal-transducing receptor (gp130). In chickens, only an IL-6 homologue has been cloned (36). Chicken IL-6 is 35% identical to human IL-6, and chLIF is 40% identical to mLIF. These low identities are similar to those of other IL proteins. However, the position of six cysteine residues and the number of asparagine-linked glycosylation sites (six sites) were conserved between chicken and mammalian LIF, implying a similar tertiary structure.
In mammals, LIF is produced among other things by heart cells (37), hepatocytes (5), activated monocytes (29), and thymic epithelial cells (38). We found that expression of chLIF was highest in liver and thymus cells and in activated-IN24 cells (chicken monocytic cell line) treated with LPS for 20 h. These features of the expression profile suggest that the biological activity of chLIF may be similar to that of mammalian LIF.
The purpose of this work was to compare the biological effects of chicken and mouse LIFs on chicken ES-like cells (blastodermal cells). We expressed rchLIF linked to glutathione S-transferase and purified it to greater than 90% purity by two-step chromatography (Fig. 4). We sequenced 35 amino acid residues from the N terminus to confirm that the protein was correctly translated (data not shown). SSEA-1 and EMA-1 and ALP activity were used markers of undifferentiated ES cells (5,16,39,40), and rchLIF proved more effective than mLIF in maintaining the blastodermal cells in the undifferentiated state (Fig. 5A). Mouse ES cells detach and form EBs that can be induced to attach again and to differentiate into various cell types including muscle, hematopoietic, and nerve cells (41)(42)(43)(44). When seeded in non-tissue culture dishes without LIF, chicken embryonic cells develop organized floating structures resembling EBs (16). In the monkey, EBs are produced by culturing ES cell aggregates in Petri dishes and extended culture of the EBs results in the formation of cystic EBs (45). In this study, the chicken blastodermal cells cultured with or without mLIF formed aggregates after 7 days of incubation (data not shown) and gave rise to structures resembling cyst-like EBs after 9 days (Fig. 5A). On the other hand, when the chicken blastodermal cells were cultured with rchLIF, they did not form these structures (Fig. 5A). In addition, in the presence of rchLIF but not mLIF, EMA-1-positive cells gradually increased during 5 days of culture (Fig. 5B). These results show that chLIF is able to maintain the chicken ES cells in the undifferentiated state, whereas mLIF is not.
ALP activity and the differentiation antigens (EMA-1 and SSEA-1) are useful markers of ES cells, but they are not actual regulators of the pluripotent state. Recently, it was shown that  expression of oct3/4 and nanog is limited to pluripotent cells and that these proteins have regulatory roles (46 -48). Hence, they would be the best markers of undifferentiated ES cells. However, because the corresponding genes have not been cloned in chickens, we used the phosphorylation of STAT3 as an indicator of undifferentiated ES cells. This has two advantages. First, it is a direct reflection of the action of rchLIF because LIF is responsible for this phosphorylation in ES cells. Second, some commercial antibodies against STAT3 and phosphorylated STAT3 cross-react with chicken STAT3 (49). rchLIF but not mLIF activated STAT3 in the blastodermal cells (Fig.  6). Interestingly, mLIF did induce a high level of phosphorylation of MEK1/2. Burdon et al. (50) have reported that activation of the Shp2-ERK pathway actively impairs the pluripotency of mouse ES cells and that the pluripotent signal from gp130 is a finely tuned balance of positive and negative effectors (50). Our results and other evidence indicate that chicken LIF is indispensable for maintaining the undifferentiated state of chicken blastodermal cells in culture. An analysis of quail LIF is in progress.
The production of transgenic birds is an important technology in both fundamental and applied avian biology. Different methods have been employed to generate transgenic chickens including microinjection, retroviruses, and transfection of primordial germ or EG cells. However, transgenic chicken technology must be developed if complete chicken ES or EG cell lines are to be established. Our finding should contribute to the development of this technology.