JP2000505294A - Cytokines expressed by DIA / LIF-deficient embryonic stem cells for inhibition of differentiation - Google Patents

Abstract

  (57) [Summary] Novel cytokines are provided that have the ability to inhibit the differentiation of embryonic stem (ES) cells. This cytokine is referred to as ESRF and (i) in the absence of DIA / LIF, and (ii) in the absence of cytokines acting via gp130, and (iii) in the absence of interaction with gp130. And (iv) the ability to inhibit the differentiation of ES cells in the absence of interaction with the LIF receptor. The invention further provides methods for inducing and expanding ES cells, the ES cells themselves, and cell lines useful for inducing and assaying novel factors.

Description

DETAILED DESCRIPTION OF THE INVENTION    Cytokines expressed by DIA / LIF-deficient embryonic stem cells for inhibition of differentiationField of the invention   The present invention relates to a substance capable of inhibiting the differentiation of embryonic stem (ES) cells. The present invention Furthermore, methods of inducing and expanding ES cells, ES cells themselves, and novel factors Cell lines that are useful for inducing and assaying E. coli.Background of the Invention   Embryonic stem cells are archetypal stem cells that have the ability to renew indefinitely and to differentiate and grow. Has the ability to form all cell types found in somatic animals . Such stem cells are described as pluripotent if they can differentiate into many cell types Is done. These cells are fully involved in normal embryogenesis following reintroduction into the blastocyst And contribute functionally differentiated progeny to all somatic tissues and germline obtain. ES cells can also be induced to differentiate into a wide variety of cell types in culture . In summary, this is an in vitro process responsible for tissue diversification in the developing embryo. Is. Therefore, ES cells are used to analyze factors that control early embryo growth and differentiation. Provides a unique system for   ES cells are fully involved in normal embryogenesis following reintroduction into the host embryo, and Can contribute functionally differentiated progeny to all somatic tissues and the germline. It has the following characteristics. Their ability to form gametes is responsible for transmitting genetic alterations to animals. Enables ES cells to be used as a means of reaching. This is Gentra Gene discovery and mutation through strategy, and most importantly, heredity Used for accurate genetic changes through child targeting.   Generally, when needed for research or medical use, stem cells Must be isolated from the tissue sample by the Even after deep separation, these stem cell preparations consist of mixed cell types and Contains a high percentage of differentiated cells that are enriched for cells but are not classified as stem cells. No.   In addition, most stem cells cannot be easily expanded in culture and stem cells When attempts are made to culture and, more specifically, maintain established ES cell lines The cells to be cultured, which usually contain a mixed population of cell types, grow at different rates And stem cells become rapidly covered by non-stem cell types. Two exceptions Embryonic stem cells from specific lines of a number of mice (129 and Black 6) were cultured in vitro. Can be nourished. Therefore, the established lineage of embryonic stem cells is mouse strain 129 and Bla ck6, or the early (3¹/_TwoDay) culturing embryo cells Can be obtained by   To date, proven embryonic stem cells with the capacity for germline transmission have been Only established from inbred mice (especially strains 129 and C57BL / 6) . They are the feeder layer of embryonic fibroblasts and / or leukocytes, which are cytokines. Glycoprotein 130 (g), a disease inhibitory factor (LIF) or signal transducer Culture of early embryo cells in the presence of related cytokines acting via p130) (Yoshida et al., 1994). In the absence of a source of LIF, stem cells Cannot differentiate and multiply. In contrast, DIA / LIF or DIA / LIF producing cells ES cell cultures in the presence of a feeder cell layer retain their proliferative potential and Maintain stem cell morphology and maintain stem cell markers (eg, alkaline phosphatase) , Stage-specific embryonic antigen-1, and stem cell-specific transcription factor 0ct-3 / 4 ) Is expressed. ES cells expanded in the presence of LIF retain pluripotency, Eligible for generating germline chimeras when reintroduced into mouse blastocysts (Nichols et al., 1990; 1994).   Cytokines are important in maintaining the pluripotent phenotype of ES cells in vitro It is well known that it plays an important role. Therefore, it regulates the proliferation and differentiation of ES cells The study of the factors involved was based on an apparent molecular weight of 45k, termed "differentiation inhibitory activity" or DIA. The glycoprotein of Da was purified. It is a cytokine called "myeloid leukemia" Inhibitor "or LIF. DIA / LIF has self-renewal of undifferentiated ES cells And thereby allow their growth in vitro. ES cells maintained in the presence of this factor retain their full developmental potential. Sa In addition, ES cells are newly developed by direct culture of blastocysts in medium supplemented with DIA / LIF. Can be established. (The term "cytokine" as used herein refers to extracellular And affects cell survival and / or growth and / or differentiation Refers to any substance obtained. Primarily, this term refers to proteins that can inhibit ES cell differentiation. The like factor. )   In the absence of a source of DIA / LIF, stem cells cannot differentiate and proliferate. In contrast, ES cultured in the presence of a feeder layer of DIA / LIF or DIA / LIF producing cells Cells maintain their proliferative capacity, maintain their characteristic stem cell morphology, and Cell marker proteins such as alkaline phosphatase and stage-specific Gen-1 (SSEA-1)) (Williams et al., 1988; Smith et al., 1988). the most important In particular, ES cells expanded in the presence of DIA / LIF retain pluripotency, and If they are reintroduced into blastocysts, they are eligible to make chimeras (Nichols et al.) Pease et al., 1990).   Further studies suggest that undifferentiated ES cells may be matrix-located (matrix-loc differentiated cells express low levels of mRNA resulting in morphology Contrastingly high levels of soluble DIA / LIF and matrix-associated DI It reveals that it expresses both A / LIF. DIA with newly differentiated cells Enhanced production of LIF / LIF balances differentiation and self-renewal in stem cell populations It has been proposed that a mechanism for articulation can be provided. Physiological significance of DIA / LIF Sex was attributed to the inability of homozygous DIA / LIF-deficient female mice to support embryo implantation. Established by the finding that it is unfertile. However, DIA / LIF-/-embryos survive It is possible. This observation indicates that other factors compensate for the lack of DIA / LIF expression during early development. Suggest that you can. Many cytokines (DIA / LIF, interleukin-6 (IL -6), ciliary neurotrophic factor (CNTF), and ON (Including costatin M (OSM)) share the same effector molecule, gp130. And was recently shown. This is probably due to the biology reported for these events. Underlies considerable overlap in biological activity. Any of these cytokines Activation of the gp130 signal transduction process is sufficient to support the self-renewal of ES cells. Minutes. However, if any, any of these molecules will Play a role in maintaining early stem cell regeneration or other factors (single or It is not yet clear whether or not) are involved in this process.   Other mouse strains and more particularly other laboratory animals (eg, rats, rabbits, And guinea pigs), livestock (eg, sheep, goats, horses, cattle, and pigs) and Embryonic stem cells from other species, including primates, including humans and humans, and There is an urgent need to maintain. There is a double demand for this. First, gene targeting and other sophisticated genomic manipulation techniques (eg, Chromosome modifications (Smith A.J.H. et al., 1995) were selected for other species, particularly for the pharmaceutical industry. Genetic Adaptation for Use in Rats and Foreign Body Transplants Pigs that need it). The second need is a donor for transplant For the development of human ES cells as a universal source of cells. However, Established strains of true embryonic stem cells other than embryonic stem cells There are no known techniques for producing and maintaining. Also maintain ES cells The only known method is to use either DIA / LIF or related cytokines as described above. A feeder layer was used as a source of these or such cytokines. Public knowledge Further disadvantage of the ES induction method in Escherichia coli is that expression of gp130 by donor embryonic cells is essential That is.Description of prior art   `` Pluripotent Embryonic Stem Cell from the Rat Are Capable of Producin g Chimeras "(Iannaccone, P.M. et al., Dev. Biol. 163, 288- 292 (1994)) was described in the paper as consisting of "diploid rat embryonic stem cells." A cell line designated RESC-01 is described. In addition, the RESC-01 cell line was Can be used to make chimeras by injection into the blastocyst " I have. The article mentioned above did not characterize the RESC-01 cell line with reference to its karyotype, And without such characterization, the data presented is for the RESC-01 cell line. Is insufficient to support the claim that it consists of ES cells. This is RESC-01 Later disclosed information (personal information that the cell line has a mouse karyotype but not a rat). Communication, not published). Therefore, "rat-derived The ability to establish stem cell populations and use them to create chimeras Steps to provide a significant addition to the repertoire of genetic engineering in Japan The goal stated by Iannaccone et al. Has not been met until now. It is easy.   Furthermore, the title is “lsolation of a primate embryonic stem cell line”. The published paper (Thomson J.A. et al., Proc. Natl. Acad. Sci. U.S.A.) A cell line designated R278.5, which is stated to be, is described. But R278.5 has many Have the characteristics of a true ES cell according to at least one of the following criteria: Not shown to be a cell strain: (i) Broad contribution to chimeras without tumor formation (ii) Reconstitution of host tissue (iii) transfer of functional gametes to chimeras and generations of offspring GivingSummary of the Invention   Define the in vitro DIA / LIF contribution and examine factors that can regulate ES self-renewal. In order to establish an assay system for the release, we studied both DIA / LIF genes. ES cells with one copy deleted were generated. The present inventors describe herein that differentiation DIA / LIF-deficient ES cells synthesize a novel soluble factor that inhibits ES cell differentiation Report that. This factor differs from the previously characterized ES cell maintenance factor And, most importantly, acts independently of direct activation of gp130. LIF ES cells lacking the receptor were also generated. LIF negative and LIF receptor Negative cells (LIF (-) and rLIF (-)) form a further aspect of the invention Further useful in assay procedures.   As one aspect of the present invention, a novel site called ESRF (ES cell regeneration factor) Cain is provided and it is in the absence of DIA / LIF and directly with gp130 It is characterized by its ability to inhibit the differentiation of ES cells without interaction.   More particularly, a cytokine called ESRF is provided, which comprises (i) D In the absence of IA / LIF, and (ii) in the absence of cytokines acting via gp130 , And (iii) the ability to inhibit ES cell differentiation in the absence of interaction with gp130 It is characterized by (iv) ES cells in the absence of interaction with the LIF receptor The ability to inhibit differentiation is further used to characterize the cytokines of the invention. Can be   The cytokine of the invention is further characterized by a feature selected from: obtain:       -Distinguishable from DIA / LIF       -Distinguishable from IL-6 / sIL-6R       -Distinguishable from CNTF       -Distinguishable from oncostatin M       -Distinguishable from cardiotrophin-1 These are used individually or in combination. ("IL-6 / sIL-6R" "Interleukin-6 / soluble interleukin receptor").   Cytokines, referred to herein as ESRF, are used in experimental animals (ie, rodents). , Livestock and farm animals (eg, dogs, cats, sheep, horses, cows, pigs, Directly or from any mammalian species, including primates, including humans It is intended to include indirectly derived ESRF. Factor is internalized in the appropriate cell line. It can be obtained by isolation of a causally produced factor or by recombinant expression.   As shown, cytokines of the present invention differentiate by a different mechanism than gp130. And this feature further characterizes those features Can be used as   Furthermore, its ability to inhibit ES cell differentiation can neutralize anti-DIA / LIF antisera. Or neutralizing anti-IL-6 soluble receptor antiserum, or anti-CNTF antiserum Cannot be excluded by neutralizing   The novel cytokines of the present invention generate cell lines that are deficient in DIA / LIF and To identify the activity that inhibits the differentiation of ES cells in the supernatant from such cells More discovered. These cells in crude or partially purified form Supernatants from also form part of the invention. Therefore, the present invention further provides DIA / LI At least one polypeptide which can be obtained from the supernatant of F-deficient cells and has the following characteristics: Provide an at least partially purified composition comprising a tide:   -(I) in the absence of DIA / LIF, and (ii) cytokines acting through gp130. In the absence and (iii) in the absence of interaction with gp130, differentiate ES cells Ability to inhibit.   (iv) Inhibiting the differentiation of ES cells in the absence of interaction with the LIF receptor This ability can be further used to characterize a partially purified composition. These may, if desired, be further characterized by features selected from: RU:       -Distinguishable from DIA / LIF       -Distinguishable from IL-6 / sIL-6R       -Distinguishable from CNTF       -Distinguishable from oncostatin M       -Distinguishable from cardiotrophin-1. These are used individually or in combination.   The at least partially purified polypeptide component of the composition may also comprise the invention Is within the range.   Alternatively, the invention can be obtained by culturing cell line D7A3-PE, and       -Ability to inhibit ES cell differentiation       -Distinguishable from DIA / LIF       -Distinguishable from IL-6 / sIL-6R       -Distinguishable from CNTF       -Distinguishable from oncostatin M       -Distinguishable from cardiotrophin-1 Can be defined as a cytokine called ESRF.   The present invention further provides a method for producing a novel cytokine, ESRF, This involves the steps of culturing ESRF-producing cell lines and isolating ESRF from their supernatants. Process. Suitably, the ESRF producing cell line is cell line D7A3-PE or derived therefrom. Including incoming cells. The deposited cell line D7A3-PE constitutes a further aspect of the present invention. I do.   The present invention further provides a culture procedure utilizing ESRF. One proliferates ES cells This is a method of growing cells in the presence of a cytokine called ESRF. The step of causing The other is a method of establishing ES cells, which is called ESRF. Culturing the early embryo in the presence of the cytokine to be produced. These steps are , The only ES cell growth enhancer or other (eg, DIA / LIF or gp130 And other cytokines, including those that act via And use ESRF. The growth and / or establishment of ES cells according to the invention can be monitored by culturing cells containing a selectable marker such as t-4βgeo. You. Further, the method of the present invention provides for the expansion of somatic stem cells (eg, hematopoietic stem cells). Can be used to promote.   In certain embodiments of the invention, the cytokine of the invention is mediated by gp130. In combination with a second cytokine known to act, undifferentiated embryonic stem cells Used to induce, proliferate, and / or maintain vesicle culture. For example, a book Combinations of the cytokines of the invention with LIF can produce embryonic stem cells of rat or other species. Suitable for inducing, growing, and / or maintaining.   In this procedure for generating ES cells, cells expressing matrix-bound LIF Embryos, which preferably do not contain the zona pellucida, may be cultured on a feeder layer. It is profitable.   Suitable feeder layer cells are derived from fibroblasts. As an example, the matrix Mouse embryonic fibroblasts transfected to express synthetic LIF Can be A suitable stable transfected cell line is called DIA-M, and n Collection of Animal Cell Cultures (ECACC) with accession number 96053101 in 1996 Deposited on May 31, 2009.   DIA-M was prepared using the matrix-bound form of mouse LIF (Rathjen et al., 1990) and IRES. Stable transfection with expression vector containing cDNA for binding neo selectable marker It was prepared as a cloned derivative of the cloned C3H10T1 / 2 mouse embryo fibroblasts. (Departure See also WO 94/24301 for details of the current system).   Thus, in its more particular aspect, isolating embryonic stem cells from other species and A new methodology for propagation is provided. The approach involves soluble LIF and Matrix-bound isoforms of the embryonic stem cell regenerating factor (ESRF) and LIF, which are tocaines Of genetically modified fibroblasts overexpressing the form (Rathjen et al., 1990) Based on a new combination of feeder cell layers.   When using this protocol, rat embryonic stem cells are transformed into euploid rat karyotypes and Endogenous growth while retaining characteristic stem cell morphology and marker expression Can be In addition, a novel method for freezing and thawing ES cells has been described, The procedure for chimera production is presented.   According to a further aspect of the invention, comprising DIA / LIF and / or gp130 interaction Assay and / or detect growth factors that affect differentiation by a different mechanism A method for issuing is provided. This method determines the presence of the sample to be assayed. Culturing ES cells in the presence and absence of cells cultured in the absence of sample Detecting the change in proliferation or differentiation compared to the LIF negative (ESF) cells. Have LIF (-)) and / or LIF receptor negative (rLIF (-)) phenotype It is characterized by   Particular embodiments of the invention are cytokines having the following physicochemical properties: RU: An apparent molecular weight in saline or 4M urea equal to 100,000 daltons Or greater ・ Isoelectric point = 4.25 to 4.5 Stable above pH 3, but inactivated below pH 3 ・ Inactivated by 0.5M NaOH ・ Suitable for reduction with 10 mM dithiothreitol for 30 minutes at 37 ° C ・ Inactivated by incubation with trypsin ・ Inactivated by incubation at 50 ° C ・ Stable for long term (6 months) storage at 4 ℃ ・ Stable for freezing / thawing Stable on exposure to 4M urea ・ Stable in 1% CHAPS or CHAPSO ・ Inactivated by 0.01% SDS   The invention further provides at least five, and preferably at least, of the following features: 7, and furthermore, the cell line is non-mouse Provide an established line of embryonic stem cells characterized by having a karyotype: (i) The characteristic morphology of stem cells, which is a small dense cell with a high nucleus to cytoplasm ratio           Agglomerate growth as small tightly packed cells           including), (ii) (a) alkaline phosphatase, (b) stage-specific embryo antigen-1, (c) from Oct-3 / 4           Expression of one or more specific markers selected, (iii) non-expression of differentiation markers (eg, H19 RNA), (iv) considerable or unlimited growth potential, (v) stability against freezing and thawing, (vi) stable euploid karyotype, (vii) cytokine dependent growth, (viii) Cytokine withdrawal, aggregation, or chemical differentiation inducer           More inducible in vitro differentiation, (ix) Ability to form teratocarcinoma, including derivatives of endoderm, mesoderm, and ectoderm           Power, (x) colonies through the production of somatic stem cells and functionally differentiated progeny           The ability to colonize and / or reconstitute host tissue; (xi) forming the host embryo into a colony with the contribution of the functionally differentiated progeny to the chimera           Ability to (xii) produce functional gametes in chimeras and viable progeny           Ability to (xiii) Ability to incorporate exogenous DNA. Such cells preferably have at least one of the identified characteristics (i) to (viii). 5 and preferably at least 7 and most preferably Further characterized by having at least one of the characteristics (ix) to (xiii) It is.   The established strain of the embryo cell provided by the present invention is not necessarily the above (i) to (xii It is understood that it is not necessary to show each and every feature of i). Therefore, For a given species, one or more features may not be present. For example, feature (ii) In rodent embryonic stem cells, all three specific markers described above (ie, , (A) alkaline phosphatase, (b) stage-specific embryonic antigen-1, and (c) Oct-3 / 4) I can do it. However, stage-specific embryonic antigen-1 (feature (ii) (b)) has been identified for other species, especially primates. Are not present or undetectable in embryonic stem cells of large mammals possible.   In addition, they may have a long gestation period and / or reach maturity after a long period It is impractical for mammals to indicate the presence of features (x) and (xi) Maybe. Also, due to ethical and legal constraints, certain species, such as humans, May not be able to demonstrate features (x) and (xi).   Description of the drawings   The invention will now be described in more detail, by way of example, with particular reference to the accompanying drawings, in which: Described in: Figure 1 shows the response of lif-r-/-ES cells to cytokines Figure 2 shows a phase contrast micrograph of rat ES cells Fig. 3 Spread of metaphase chromosomes from rat ES cells Figures 4A and 4B A. of rat ES cells. Alkaline phosphatase and B. Oc                       Shows marker expression in t-4 immunostaining Figures 5A, 5B, and 5C. Trophoblast, B. Body wall endoderm, and C.                       Shows differentiation to bipolar cells   The production, isolation and characterization of ESRF according to the present invention will be described in more detail by way of example. It is described here. Example 1 1.1 Generation of DIA / LIF-deficient ES cell lines   In order to disrupt DIA / LIF function in ES cells, both alleles of the gene must be It was deleted by the same recombination. ES cells in which one copy of the gene has been inactivated, The entire coding sequence of the DIA / LIF gene has been replaced by a selectable HPRT minigene. Using a replacement vector. Transfection into HPRT-deficient E14TG2a cells After the recombination event, HAT-resistant ES cell clones that have undergone a recombination event are placed outside the homologous 3 'arm. Identified by DNA hybridization analysis using the side probe. Expected DNA from positive clones with appropriate restriction enzymes Digested and hybridized to the 5 'external probe. A total of 11 exactly The clones targeted were identified from a primary screen of 89 colonies.   One germline eligible clone D6 was used for the second allele deletion . Insert the hygromycin resistance cassette into the HPRT marker in the targeting vector Was replaced with In parental cells, this construct is comparable to the frequency obtained with the HPRT vector. Homologous replacement events occurred at a comparable frequency (10%). This second construct in the D6 clone And transfectants into hygromycin Isolated by selection on B. Three classes of homologous recombination were screened. Identified in 162 colonies: recombination to previously targeted allele Occurred in 30% of the clones; 5 'or 3' homology regions of the wild-type allele. Area integration was detected in four cases; and only three clones were wild-type Received allele replacement. For these three clones, the DIA / LIF code Column deletion confirmed by absence of hybridization with full-length cDNA probe did. On the other hand, clones that have undergone recombination into only one homology arm retain the gene. I carried it. DIA / LIF mRNA loss using a sensitive ribonuclease protection assay And confirmed. After induction of differentiation, E14TG2a ES cells were previously described for other ES cell lines. As noted, both relatively high levels of matrix tie and diffusion types Expresses DIA / LIF mRNA. In contrast, protected fragments are undifferentiated or differentiated Is not detectable in RNA preparations from isolated double knockout ES cells. These fruits The experiment clearly confirms that the DIA / LIF deletion is a null mutation. Two independent ku Similar phenotypes of the loan, D1C2 and D7A3 (these are separate during drug selection) (Isolated from each plate) are reported in the following experiments. 1.2 Ability for in vivo differentiation of homozygous DIA / LIF negative ES cells   Chimeras were generated using DIA / LIF deficient ES cell clones. DIC2 and D7A3 ES Cells were injected into C57BL / 6 blastocysts and chimeric progeny were produced. Chimera in the sand High ES cell contribution, as judged by sandy coat colour did. This was confirmed by glucose phosphate isomerase isozyme analysis. Confirmed by determining the extent of ES cell contribution to blood and tail of 5 chimeric mice did. Both clones were cloned as indicated by the production of viable germline progeny. Contributed to gamete formation. These results generate a double knockout ES cell line That the two selections applied for did not destroy their normal viability Establish. 1.3 Stem cell regeneration can occur in the absence of DIA / LIF   ES cell differentiation in high-density monolayer cultures does not result in complete elimination of stem cells . The present inventors have previously proposed a model for feedback regulation of stem cell regeneration. This Here, the synthesis of DIA / LIF by newly differentiated ES cells is replaced by the remaining undifferentiated stem cells. Contributes to rescue. The validity of this hypothesis was determined by stem cell rescue after differentiation induction. Were tested by investigating the capacity of DIA / LIF-deficient ES cells for wild ES cells of the mutant, heterozygous, and homozygous mutants were Induced by exposure to xybenzamide (MBA) and differentiated at high cell density. After an additional 4 days in the basal medium (is), the number of undifferentiated ES colonies was determined by morphological examination and And alkaline phosphatase staining. Types of undifferentiated ES colonies Similar numbers were recovered from wild-type and two heterozygous ES cell cultures. Contrast In addition, the number of ES colonies was about 3-fold lower than the two DIA / LIF negative ES clones. won. This decrease is due to autocrine or paracrine production of DIA / LIF. Key components in the feedback regulation of stem cell regeneration in differentiating ES cell cultures Confirm that it is a component. Importantly, however, stem cell regeneration is It was not completely eliminated in the absence. This is because another regulator is acting in this system. Suggest dynamic. 1.4 Inhibition of ES cell differentiation by soluble factor (ESRF)   Excludes that culture conditions may have conferred selection for rare differentiation-deficient mutants The ability of DIA / LIF-deficient ES cells to rescue stem cells in co-culture assays. And further investigated. A convenient indicator cell line is the β-galactosidase reporter -Homologous recombination of DIA / LIF negative ES cells into the Oct-4 locus Generated. Β-gala in such targeted clones Cuctosidase expression is restricted to undifferentiated stem cells. The indicator cells are used for the MBA-induced Were plated on the layer of the wild type or mutant clone. Four days later, the index group The number of undifferentiated ES cell colonies of interest was stained for β-galactosidase activity. Decided. Again, the results indicate the ability of DIA / LIF-deficient differentiated cells to inhibit ES cell differentiation. Indicates that the force is reduced but not eliminated in relation to the parent cell line. Retinoid induction Similar results were obtained using the differentiated cells as feeder layers.   To determine whether this effect was due to a diffusive factor, a second type of co-culture was used. A nutrition experiment was conducted. Here, indicator cells were plated on a microporous insert on a differentiated cell layer. I did it. The insertion membrane prevents cell-cell contact between the two cell populations, Allows access to diffusible factors. Both parent and Oct-4-tagged ES cells As an index for staining for alkaline phosphatase and β-galactosidase Used. Similar results were obtained in both cases. Larger in wild-type culture Activity is significantly reduced but not eliminated in the presence of neutralizing DIA / LIF antiserum. This This indicates that wild-type cells also synthesize active factors other than DIA / LIF. Anti-DIA Residual activity in the presence of / LIF is comparable to the activity produced by DIA / LIF negative cells. It was similar. This indicates that response factor expression is significantly increased in these cells. Suggests that it is not regulated.   These data indicate that soluble factors other than DIA / LIF (single Or multiple) are synthesized by both wild-type and DIA / LIF-deficient differentiated cells. And confirm. However, mutant cells or wild-type cells in the presence of neutralizing anti-DIA / LIF A reduced number of colonies produced in response to vesicles produces a limited amount of this factor Indicates that 1.5 Isolation of differentiated cell lines expressing high levels of stem cell regeneration factor (ESRF)   In order to facilitate the characterization of stem cell regenerative activity, we have proposed a DIA / LIF negative A differentiated cell line was established from the embryoid body outgrowth of active ES cells. Several These cell lines have activity levels that are easily detectable in their culture supernatants. Occurred. One cell line (designated D7A3-PE) can grow indefinitely and undifferentiated E It can produce high levels of soluble factors that can maintain S cells. Their characteristic forms In addition, ES cells cultured in D7A3-PE conditioned medium contained alkaline phosphatase. And Rex-1 mRNA, and β-galacto retained in Oct-4 tagged cells Undifferentiated markers, such as sidase activity, continued to be expressed. This conditioned medium Was effective in several independent ES cell lines. Conditioned media or partial purification ES cells cultured in the presence of ESRF formed closely rounded colonies. This morphologically distinguishes both differentiated cells and ES cells maintained in DIA / LIF. obtain. ES cells are continuously passaged in the presence of ESRF and have the potential for multilineage differentiation. Hold power. The active agent can be concentrated at least 20-fold by centrifugation and Disrupted by incubation with lipsin. This is a proteolytic Is consistent with being a molecule. This may be due to heating to 50 ° C or to pH 3 Inactivated by less than acidification. 1.6 Purification and biochemical properties   For ESRF purification, conditioned media is prepared from D7A3-PE cells in the absence of serum . 150 cm cells containing normal growth medium<sup>Two</sup>Inoculate into tissue culture flasks and Grow to near fluence. The culture is then washed with PBS and G MEM and 100 μM 2-mercaptoethanol, 1 μg / ml insulin, 5 μg / ml 5 of Ham F12 basal medium supplemented with transferrin and 10 nM sodium selenite Transfer to defined medium consisting of a 0:50 mixture. Incubate the culture for 4 hours, then Discard the medium and replace with fresh defined medium (50 ml / flask). this Media is conditioned by incubating with D7A3-PE cells at 37 ° C for 96 hours I do. The medium is then harvested and replaced. Three consecutive collections each It can be obtained from a culture. Clarifying the harvested medium by centrifugation, and Destruction Pass the bacteria through a 0.2 μm filter. The ability to inhibit the differentiation of ES cells is 1/10 It can be routinely detected in unfractionated, conditioned medium. However, for four days of full activity The assay requires less than 25% dilution. The activity in the conditioned medium was determined by freezing and thawing. Stable on digestion, but incubated at 50 ° C for 30 minutes, acidic to pH 2 Upon incubation or with trypsin. 100,000 dal Limitations in Amicon cells using membranes with tons of apparent molecular weight cutoff The activity can be concentrated by filtration. Biological activity can be recovered quantitatively, Suggest that the ESRF has a molecular weight greater than 100,000 daltons.   ESRF was quantitatively recovered from the conditioned medium by serial dilution with saturated ammonium sulfate. Take it. ESRF activity is recovered in the 25-35% fraction. This fraction contains 20-25% of total Contains Parkin. Upon reconstitution in 1/100 volume of starting medium, a viscous solution is obtained. This It splits into a fluid phase and a gel phase during cryopreservation. Both phases are biologically active Including certain ESRFs. The gel material forms a highly glycosylated extracellular matrix. It seems to consist of minutes. ESRF, ink with salt buffer, more effectively 0.1% CHAPS Can be extracted from the gel by evaporation. This finding indicates that ESRF has an affinity for ECM components Is suggested. Consistent with this, significant levels of ESRF activity were Incubate from A3-PE cell monolayer with basal medium containing 0.1% CHAPS for 2 hours (Under such conditions, the cells were significantly lysed) Can survive without evidence). Isoforms in which ESRF specifically associates with extracellular matrix May be present in To obtain ESRF-containing fractions by ammonium sulfate precipitation Another protocol is shown in Appendix 9.   Ultracentrifugation studies (see Appendix 10) show that ESRF activity is Does not significantly associate with organs, but is a soluble protein or protein complex And suggest.   The maximum concentration required for biological activity is 1.5 nM (urea resolubilized ammonium sulfate Based on the protein content of the um precipitated material (see Appendix 9)) and Estimate that the child mass is equal to 100,000 daltons.   Further fractionation of the ESRF can be achieved with a lectin column. ESRF is a soy lectin And lentil lectin, but not wheat embryo agglutinin (Attachment 11). Biological activity is not affected by the addition of heparin to the assay medium. No.   Size exclusion chromatography of the ammonium sulfate fraction (50 mM sodium phosphate) ESRF is better than BSA when using TSK G3000SWXL HPLC column in pH buffer (pH 7.2). Moved as one peak with a short retention time. It has a molecular weight of 100-150 , 000 daltons.   ESRF can be further purified by liquid phase isoelectric focusing of ammonium sulfate fraction material. You. Electrophoresis was performed in the presence of 1% CHAPS and 2% ampholytes in BioRad Rotofor cells Can be done below. ESRF is recovered quantitatively in 2-4 fractions at pH 4.25-4.5. ESR F is insoluble in these fractions, but cultured for ES cell bioassay Redissolve upon direct addition to medium or upon adjusting buffer pH. SDS game Electrophoresis has two apparent molecular weights of 115,000 and 180,000 daltons. Indicates that major proteins are specific to the active fraction.   For further purification of ESRF, heparin-agarose affinity chromatography Can be achieved by Samples in 20 mM phosphate buffer (pH 3.5) containing 0.1% CHAPS To load. Under these conditions, most substances bind to the column and ESRF can be significantly purified. ESRF does not elute at 100 mM salt, Elution over high salt concentrations. A miscellaneous elution profile is associated with altered glycosylation. Suggest movement. (Similarly, the miscible elution profile also Torography). Very low levels of biologically active fraction (Not detectable at 280 nm). Protein is also known as Kumashi -10 times greater than required for blue staining, conventional silver staining, or full biological activity Periodate pre-incubation of SDS PAGE gels loaded with high amounts of material Silver staining (in combination with improved staining of glycoproteins) It is not detectable. The presence of low amounts of protein indicates that ESRF is complete at nanomolar concentrations. Indicates that it is fully active.   Biotinylated and collected fractions of active material after SDS PAGE in the presence of reducing agents And immunoblotting of heteroduplexes with apparent molecular weights of 70,000-80,000. Showed presence. No other bands were seen. This observation indicates that these fractions Confirm that it has been purified. In addition, natural ESRF is disulfide-bonded. Indicates that it is likely to be a dimer.   The concentration of IL-6 in the conditioned media was determined using a sensitive B9 cell proliferation assay. The conditioned medium induced B9 cell proliferation, but the mitogenic response was less than 5 pg / ml IL-6. Equilibrated in concentration. Neutralizing IL-6 soluble receptor It was confirmed by using the putter antibody RS13. Biological activity is completely inhibited by the presence of RS13 Was. In contrast, inhibition of ES cell differentiation by D7A3-PE cell conditioned medium Was not modified by This is because this low level of IL-6 affects the effectiveness of this medium Indicates not to. The results further indicate that the conditioned medium is mitotic for BAF-mgp130 cells. Demonstrated by the finding that it was not mitogenic. The latter is soluble IL-6 receptor Response to IL-6 only in the presence of the A positive result is that D7A3-PE cell conditioned media does not contain any IL-6 soluble receptor. And   Potential improvement of CNTF can be achieved by specific immunodepletion of conditioned media containing 4-68 anti-CNTF mAB and And the use of neutralizing anti-CNTF antisera. Both treatments were D7A3-PE cell-conditioned. Did not affect the ability of the activated media to inhibit ES cell differentiation. In addition, the activity Retained by the addition of RS13 anti-IL-6R antiserum to anti-CNTF-depleted medium.   These results indicate that both IL-6 / sIL-6R and CNTF had differentiated DIA / LIF-deficient ES Confirm that they are not responsible for the inhibition of ES cell differentiation by cells. 1.7 Self-renewal factors are mouse oncostatin M and cardiotrophin-1 different   Mouse cardiotrophin-1 has a molecular weight of 22,000 daltons. mouse Oncostatin M has a molecular weight of 30-40,000 daltons. ESRF, in contrast, It has an apparent molecular weight of more than 100,000 daltons. It is the presence of 4M urea Even below, it is determined by an ultrafiltration membrane with a cutoff of 100,000 daltons. Size exclusion stored quantitatively and corresponding to molecular weights above 100,000 daltons Has chromatographic mobility. 1.8 ESRF is active in LIF receptor-deficient ES cells   LIF and related cytokines are derived from low-affinity LIF receptor and gp130 Act through a heterodimeric receptor. ES lacking the LIF receptor Cells were generated by two rounds of gene targeting. These cells are transformed into IL-6 and LI Soluble IL-6 receptor acts via gp130 homodimer not involved in F receptor Can be used to grow. LIF receptor negative ES cells Is pluripotent and contributes to multiple tissues in chimeras. These are LIF, C Self-renewal response to NTF, Oncostatin M, or Cardiotrophin-1 Not shown. However, as shown in FIG. 1, LIF receptor-deficient ES cells were To maintain responsiveness. This finding indicates that ESRF differs from the cytokine LIF group. Confirm that it becomes. 1.9 Self-renewal factor (ESRF) does not act through direct interaction with gp130   All cytokines that can maintain ES cell self-renewal described to date are gp It works through a receptor complex containing 130 signal transducers. Mo A noclonal antibody is raised against mouse gp130, which is gp130-mediated May block sexual signaling (Saito et al., Unpublished data). Of these antibodies In the presence, ES cell responses to DIA / LIF, IL-6 / sIL-6R, CNTF, and human OSM Are inhibited, and the cells differentiate (Saito et al., Unpublished data). About ES cells The activity of all WEHI-3B cell conditioned media (the source of mouse cardiotrophin-1) It is completely eliminated by anti-mouse gp130.   Two different neutralizing anti-mouse gp130 antibodies maintain ES cell self-renewal by ESRF The impact on the system was investigated. In the presence of ESRF, both antibodies can be used by wild-type ES cells. Alkaline phosphatase positive undifferentiated colonies or Oct-4 Β-galactosidase positive G418 resistant undifferentiated cells by the targeted cells None of Ronnie's production was inhibited (Mountford et al., 1994). Assay Antibody integrity at the endpoint was determined by IL-6 / sIL-6R in mitogenic stimulation of BAF-mgp130 cells. Confirmed by retention of ability to block intense.   The results of these experiments with neutralizing antibodies to mouse gp130 have been shown to Effect of ESRF in contrast to the effects of all ES cell self-renewal factors described above Show that it is not mediated by direct activation of gp130.   Addition of cytokines acting on ES cells through gp130 activates STAT3 and Immediately leads to the induction of the early gene tisll. However, under the same conditions, ESRF addition , A clear increase in STAT3 activity as determined by gel shift analysis, And does not cause induction of tisll transcript by RNase protection assay. This is ES RF action is based on a unique intracellular signaling pathway (single or multiple) from gp130 signaling. Number).   The biological response of ES cells to ESRF also affects their LIF or related It is distinguishable from the response to Itokine. ES cells are characteristic in the presence of ESRF Are more compact and very closely rounded colonies (this is the culture surface (Having a tendency to peel off from). In the transition to LIF-containing media, These colonies are slightly flatter and thinner, characteristic of ES cells cultured in LIF. Take a look. Undifferentiated colonies are maintained for at least 11 days using ESRF alone I can do it. However, colony size is dependent on LIF's effectiveness in promoting continued stem cell expansion. In contrast to the fruits, it does not seem to increase after the first four days. Against stem cell proliferation The additive or synergistic effect is the sub-optimal amount of LIF and ESRF It is clear in the combination of. These data represent differences in the self-renewal process. Matches the effect. 1.10 Mouse ES cells maintained using ESRF retain pluripotency   ESRF maintains the undifferentiated phenotype of ES cells in vitro. This effect is less Both last for 11 days. However, growth was limited and the cultures spread after about 4 days. Stop. This is because cultures grown in the presence of LIF (which In contrast to a continuous increase). ES cells maintained in ESRF are chimeras Cells to determine whether they can contribute to Cultured for 4 days in the presence. These were then placed in a medium containing LIF prior to blastocyst injection. Transfer to promote the spread of stem cells. This protocol was applied to the ES cell line Zin40 . It carries a constitutively expressed nuclear-localized β-galactosidase marker You You.   Zin40 cells plated in control medium undergo complete differentiation and Did not give rise to any ES cell colonies when re-plated in the presence of LIF Was. However, cells plated during ESRF retain undifferentiation as described above. Many alkaline phosphatase positives during re-plating Stem cell colonies resulted. From the replated and expanded culture Cells contribute to the widespread presence of β-galactosidase positive cells in the middle-mature embryo Thus, it contributed extensively to the chimeras as determined. These chimeric embryos Was morphologically normal. The level of Zin40 contribution is the same as that maintained in LIF alone. Comparable to levels obtained from co-injection of vesicles. 1.11 Use of ESRF   Regulation of the equilibrium between stem cell regeneration and differentiation is a consequence of tissue diversification and embryonic development during embryogenesis. It is important for tissue regeneration and repair in somatic mammals. Generation of DIA / LIF-deficient ES cells Has studied the role of this cytokine in stem cell regeneration and differentiation, And allowed the characterization of other factors involved in the regulation of this process. This result The result is that DIA / LIF-deficient differentiated cultures retain a particular ability to maintain a population of undifferentiated cells. Make sure you have. This finding has factors and factors other than those characterized to date. And signaling pathways have the ability to maintain self-renewal of pluripotent ES cells. Show. This is of fundamental importance for the isolation and expansion of stem cells from other species Can be proved.   Proliferation and differentiation of mouse pluripotent embryonic stem (ES) cells by specific cytokines Control. In vitro proliferation of ES cells is related to the signaling factor gp130 Maintained through activation of intracellular processes. Different sites in ES cell culture In an attempt to define the relative contribution of Cain to self-renewal, we found that Generating ES cells lacking a functional gene for Itokine differentiation inhibitory activity (DIA / LIF) Was. These cells are significantly reduced in stem cell regeneration when induced to differentiate. Demonstrated ability. This is the primary regulation of DIA / LIF present in normal ES cell cultures. Indicates a nodal cytokine. However, undifferentiated ES cell colonies still show DIA / It is also produced in the complete absence of LIF. This is due to differentiated ES cell progeny, Due to the secretion of soluble, macromolecular activity. In addition to DIA / LIF, cytokines Ciliary neurotrophic factor (CNTF), soluble interleukin-6 receptor -Interleukin-6 and cardiotrophin in combination with (IL-6 / sIL-6R) 1, and Oncostatin M each activate gp130 and expand ES cells I support. The involvement of either CNTF or IL-6 / sIL-6R in our system, It has been prevented through the use of neutralizing antisera against these factors. The most important thing That is, the effect of all the above cytokines on ES cells is In the presence of neutralizing antibodies, the activity is abolished, while the activity in D7A3-PE conditioned medium has no effect. It is not to receive. These findings suggest that ES cell self-renewal is a gp130-independent signal. And differentiated ES cell derivatives activate this pathway Confirm that the secretion factor is secreted.   The cell line designated D7A3-PE is the European Collection of Animal Cell Cultures (ECCAC) and deposited on November 18, 1994 under deposit number 94111845. Example 2-Induction of non-mouse ES cells   Despite serious efforts in some laboratories, it is called ES-like cells from rats. To date, only one report has been established (Iannacone et al. [1994] Dev. Biol .)Met. However, as mentioned above, Iannacone cells later developed a mouse karyotype. And no rat karyotype. Therefore, ESRF rat ES cells Potential applications for the induction of spores were being investigated. 2.1 Induction and proliferation of rat embryonic stem cells in the presence of ESRF   Rat blastocysts were transformed into C3H10T1 in standard ES cell culture medium containing LIF and ESRF. / 2-derived feeder cells were cultured on the irradiated feeder cell layer. Pick up inner cell clumps , Trypsin, and replated in the same conditions. occured Colonies of small, morphologically undifferentiated cells are individually picked, separated, and And plated again. In this manner, stem cell cultures are harvested with approximately 50% of Fischer And Sprague Dawley rat blastocysts and low percentages of BDIX and DA strains of It started reproducibly from blastocysts. Maintain undifferentiated cells for an extended period Can be widely spread by normal passage. Freeze them by the usual procedure, and Can be recovered from storage in liquid nitrogen.   More specifically, the procedure is as follows:   2.2 Protocol for rat ES cell induction   2.2.1 Female rats are mated with males and checked every morning for the presence of sperm in the vagina. You. Pregnant blastocysts in the morning of the fifth day (the day when spermatozoa were found = day 1) were added to Medium 1 (Appendix 1). ) To flush from the uterus. If present, clear the zona pellucida with acid T Removed by brief exposure to yrode medium.   2.2.2 Feeder layer is prepared from DIA-M fibroblasts. DIA-M cells are mouse L Matrix-related morphology from clonal derivatives of IF (Rathjen et al., 1990) Stable transfection with an expression vector containing cDNA and IRES-linked neo selectable marker This is a cloned derivative of C3H10T1 / 2 mouse embryo fibroblasts. these The cells express high levels of the recombinant matrix LIF. Feeder layer, 15m in diameter Approximately 75,000 gamma-irradiated DIA-M cells into a 4 m cell culture plate (from Nunclon). It is made by distributing the cells. Add 0.5 ml of Medium 2 (Appendix 1) to each well Add.   2.2.3. The zona-free embryos are cultured in these wells for 5 days. Embryo supported After adhering to the bearing layer (after 2-3 days), the medium (medium 2) is changed daily.   2.2.4 After 4 days of culture, large clumps of cells were individually picked from the feeder layer and Roughly disrupted by trituration with a Pasteur pipette, and the DIA-M feeder layer (top Wells containing 0.5 ml of Medium 3 (Appendix 1) per well Individually.   Dish with 7% CO in air<sub>Two</sub>Incubate at 37 ° C in I can. These culture conditions are used in all further steps.   2.2.5 Small, clear, small colonies of morphologically undifferentiated cells It can be found in the wells 1-7 days later. If large enough, try these Pick up for processing or crushing. (See step 5). Small for trypsinization If so, transfer the colony to a fresh support layer intect. Or If the wells contain less differentiated tissue, remove the colonies from the monolayer. And may allow for redeposition.   2.2.6 Colonies for trypsinization were briefly digested with phosphate buffered saline. Rinse only three times, and then add 0.05% trypsin and 0.5% EDTA containing 0.5 mM EDTA. Incubate for 45-60 seconds in chicken serum. Then add them to a small amount of medium 3 to break up into single cells and small clumps by grinding Add to wells (described in step 3 above). Alternatively, colonies are trypsinized Without treatment, cut them apart with a glass needle and finely draw a pipette ( finelyd rawn pipette to physically aggregate the fragments by grinding them. May be broken into agglomerates.   2.2.7 The cell line is maintained by repeating the passage of the above colonies. 1 As the number of colonies per well increases, transfer the culture to several wells. Can be separated and / or transferred to large wells.   2.2.8 Cells were karyotyped at intervals of 3-4 passages and euploids were detailed in Appendix 2. Ensure that it is maintained according to the protocols described.   2.2.9 Cells were separated by oct-4 protein (Appendix 3), oct-4 mRNA (Appendix 4), alkaline phosphatase (Appendix 5), and SSEA-1 (Appendix 6) Is tested for expression.   2.2.10 Cultures with penicillin and streptomycin or gentamicin In the presence of any of the Change the type of antibiotic used every 3-4 weeks Additionally, the risk of developing antibiotic resistant strains of the microorganism is minimized.   2.2.11 To produce a chimeric animal, cells are isolated as described in step 5 above. Dissociate in trypsin and introduce into host embryo. For chimera production Three examples of the protocol are described below:   a) In the early fibroblast stage, host embryos are placed on the morning of the fifth day of pregnancy (early blastocyst stage). The female rat uterus is flushed with medium 1. And put them in Medium 4 The cells are cultured for 24 hours and the separated cells are injected into the blastocyst cavity the next morning. Embryo Allow to recover for 2 hours in incubator, then transfer to rat uterus on day 5 of pseudopregnancy Move.   b) Collect the host embryo at the early blastocyst stage in the same manner as in 2.2.11.a. Apart them Immediately injected with these cells and immediately transferred to rat uterus on day 4 of pseudopregnancy Move.   c) Host embryos were transferred from the oviducts of rats on the fourth day of gestation (8-cell stage) using Medium 1 Let me rush. Using a micromanipulator, cut the transparent zone into narrow strips (sl it) and introduce some cells into the sub-zona region. Then The embryos are returned to the female uterus on day 5 of pseudopregnancy.   2.2.12 Cells and host blastocysts are strains with different coat colors And chimeric animals can be identified by color approximately one week after birth. is there Alternatively, microsatellite markers can be used to distinguish donor cell and host-derived populations. Can be used to   2.2.13 Cells can be transfected by the procedure described in Appendix 7.   2.2.14 Cells may be frozen by the procedure described in Appendix 8.   2.3 Conclusion   Both LIF and ESRF are required for the maintenance of undifferentiated cells. Either When these components are removed, the culture is fully differentiated into trophoblast cells.   The status of ES cells in undifferentiated cells after an extended culture period can be one or more of the following: (I) maintenance of apparent ES cell morphology; (ii) cytokine cessation (Iii) expression of early embryo and ES cell-specific antigen SSEA-1, (Iv) Expression of pluripotent cell-specific transcription factor Oct-3 / 4, (v) alkaline phosphatase And (vi) non-expression of H19.   The characteristic stem cell morphology of the rat ES cell culture is shown in FIG. These cells are Morphologically indistinguishable from mouse ES cells cultured under the same conditions. Culture The chromosome complement is shown in FIG. Marker expression is shown in FIG. 4; Rephosphatase, lower panel shows Oct-4 immunostaining. Figure 5 shows an example of rat ES cell differentiation Showing: a. Trophoblast, b. Body wall endoderm, c. Unidentified bipolar cells.   These findings indicate that ESRF uses LIF (or gp130) to enable the establishment of ES cells. Provide other direct examples that can cooperate with other cytokines acting through the   2.4 Further characterization of rat ES cells   Using the protocol we presented in Section 2.2, we used rat embryos. Cell lines can be reproducibly obtained from blastocysts. This is mouse ES cells grown under the same conditions And morphologically identical. This cell is small, has a high nucleus to cytoplasm ratio, It grows closely packed together into clumps with a round, smooth, transparent appearance. These colonies are morphologically distinct from mouse ES cell clumps under the same conditions. I cannot separate. The majority of the cells in these cultures will maintain the undifferentiated phenotype. Nevertheless, some degree of differentiation occurs. Cells consist of endoderm and giant cells Most often differentiate as vesicles or other trophoblast-like cells, but other cell morphologies Seen occasionally.   In addition to having the characteristic morphology of mouse ES cells, rat cells Express Kerr. Alkaline phosphatase is found in rat and mouse primordial germ cells. Expressed by vesicles, as well as mouse ES cells. Rat cell line obtained by the present inventors Is also strongly positive for alkaline phosphatase, but If so, the expression of this enzyme is stopped. Rat cells are SSEA-1 (stage-specific embryonic antigen) Positive staining with antibodies against 1), which also Expressed by ES cells. Finally, immunohistochemical staining was performed using oct-4 (primitive Totipotent cells such as germ cells, blastocyst inner cell mass, and mouse ES cells Is a transcription factor that is only expressed by the rat cells) This is illustrated.   We maintain a cell line with these morphological characteristics, Oct-3 / 4, SSEA-1, and oct-3 / 4 for more than 25 passages in vitro (over 3 months). And alkaline phosphatase.   We believe that these are truly rat by testing their karyotype. I have confirmed that it is a cell. These are 42 modal chromosome numbers And is characteristic of rats and is not found in normal mouse strains It has a middle centromeric chromosome and a submetacentric chromosome. cell Strains remain euploid at high passage numbers.   If cells are frozen according to the protocol in Appendix 8, colonies will be grown in liquid nitrogen Store in and then thaw and culture without increasing the incidence of differentiation or ploidy changes Can be maintained in.   The maintenance of the undifferentiated phenotype in these strains depends on three factors. Factors are these if the cell line survives for more than a few passages and remains undifferentiated. Should be present in the system. Medium must contain soluble DIA / LIF and ESRF (See section 2.1), and the cells are preferably divided (m Itotically) proliferate in feeder layers of inactive DIA-M cells (Section 2.2.2). LIF Or if any of the ESRFs are removed from the system, will the cells all eventually differentiate? (Mostly as trophoblast-like cells) or die (Table 1, below). Once established The rat cells are then placed on a separate feeder cell layer or gelatin-coated plastic. Even on a rack, it can be propagated. Late passage cells can also grow in the absence of ESRF. You.                                                     Maximum passage number With DIA / LIF, ESRF, and DIA-M support layers> 25 Without DIA / LIF 1-2 No ESRF 4 No DIA / M support layer 4-5   The chimera is introduced into the host embryo using the procedure described in Section 2.2.11, It can be produced from these cells.   Genetically modified rats can be used for transgene integration or for rat ES cells. Gene targeting followed by chimera production and germline transmission Can be born.   2.5 ESRF activity in human teratocarcinoma cells   Conditioned media and ammonium sulfate for the pluripotent human teratocarcinoma cell line GCT 27X1 The effect of fractionated ESRF was tested. Assays were performed on feeder layers in serum-containing media. Includes growth of cell colonies from single cells in the presence for 10-14 days. Cells Observe daily using a phase contrast microscope. Under the conditions of this assay, control Cell viability and colony formation in the medium is low. ESRF preparations have consistently The positive effect of stem cell survival in these assays was illustrated. Cell viability Enhanced early in the assay, and final colony counts are substantially increased did. Therefore, ESRF or its related activities may be in vitro in human pluripotent cells. Active in promoting the survival of shaped cancer stem cells. Appendix 1: Medium Medium 1: Flash medium. PB1 containing 10% fetal calf serum and antibiotics (Whitt ingham, D., 1971. Culture of mouse ova. J Reprod Fert (Suppl. 14): 7-21 See). Medium 2: ESRF medium. 80% GMEM, 20% fetal calf serum, 2000 units human DIA / ml, And 50 units / ml penicillin, 50 mg / ml streptomycin, or 50 mg / ml One of gentamicin. Semi-purified ESRF was determined by bioassay of mouse ES cells. Add at a defined concentration (see Appendix 9). Medium 3: Gardner G1 or G2 (Barnes et al., 1995, Human Reproduction 10: 3243-3). 247). Appendix 2: Cell karyotype analysis 1. Colonies of cells for karyotyping were picked from the dishes and harvested from medium in medium 3. Incubate for 2 hours in a dish containing a feeder layer of DIA-M cells in 8 mg / ml Colcemid . 2. Remove cell clumps and in PBS (Dulbecco's phosphate buffered saline) several times Rinse easily. 3. The aggregates are treated with hypotonic saline (1% sodium citrate for more than one week). Rinse briefly, transfer to fresh saline and add 8-10 Leave for a minute. 4. Transfer the clumps to a hard watch glass and remove excess hypotonic saline. 5. The dishes were then prepared freshly in 3: 1 fixative (1 part per 3 parts of absolute ethanol). Of glacial acetic acid) and leave for 1 hour. 6. Transfer the cell clumps to a clean microscope slide and allow the fixative to almost completely evaporate. Observe under a microscope until it develops. 7. Then, 2-4 drops of 60% acetic acid are added to the cells to break up the tissue. 8. Continue to move the drops around the slide by blowing until they are completely evaporated I can. 9. Slides are stained in 2% Giemsa stain in Giemsa buffer (from Gurr) . They can be attached or tested without attachment. Appendix 3: Immunohistochemical staining for oct-4 protein 1. The medium is removed from the wells and the cells are washed with complete PBS (1.5 mM MgCl2 and 1 mM C Wash twice with Dulbecco's phosphate buffered saline containing aCl2. 2. Cells are fixed in 3.7 formalin in complete PBS for 10 minutes. 3. Cells are permeabilized with 0.2% Triton in complete PBS for 15 minutes. 4. Cells were harvested using the Vectastain Elite ABC kit (supplied by Vector Laboratories). Block for 20 minutes in goat serum at the dilution indicated in the protocol described in (1). 5. Primary antibody (affinity-purified anti-oct-4 antiserum from rabbit (Palmieri 1995)) is diluted 1: 5000 in PBS and added to the cells for 30 minutes. 6. Wash cells for 10 minutes in PBS. 7. Biotinylated secondary antibody (goat anti-rabbit) was prepared using Vectastain kit (Vector Labora). tories) and add to the cells for 30 minutes. 8. Wash cells for 10 minutes in PBS. 9. `` ABC reagent '' was prepared according to the protocol of Vectastain kit (Vector Laboratories). And allowed to stand for 30 minutes, and added to the cells for 30 minutes. Ten. Wash cells for 10 minutes in PBS. 11． The peroxidase substrate was replaced with the VIP key supplied by Vector Laboratories. And prepare according to the instructions. 12． Peroxidase substrate is applied to cells for 2-10 minutes until color of the appropriate intensity develops. During the addition. 13. Wash cells in tap water for 5 minutes. 14. Cells should be examined and photographed at once. Appendix 4: In situ staining for oct-4ml mRNA Note: Make all solutions with DEPC-treated water 1. The probe was combined with 1.75 mM digoxigenin-11-UTP in the synthesis mixture and oct-4 Stul fragment of Bluescript plasmid carrying the POU homeodomain of the gene Synthesize from 2. The cells conjugated to the cell culture plate are transferred to PBS (Dulbecco's phosphate buffered saline). Fix overnight at 4 ° C. in 4% paraformaldehyde in brine). 3. Rinse them with PBT (PBS + 0.1% Tween) and a series of graded (grad) ed series) dehydrate in methanol (25%, 50%, 75% and 100%) and Store at -20 ° C until needed. 4. The cells are rehydrated through a series of methanol, rinsed with PBT, and RIPA (1 1% NP-40, 0.5% NaD0C, 0.1% SDS, 1 mM EDTA, and 50 mM T in 50 mM NaCl ris) 3 times. 5. Cells are first purified with 1: 1 hybridization buffer: PBT (hybridase Solution buffer: 0.05% of a stock solution of 100 mg / ml heparin, and 0.1% Tween  20 in 50% formaldehyde in 10 × SSC (pH 4.5) Wash with% hybridization buffer. 6. Hybridization buffer and enzyme containing herring sperm DNA (100 μg / ml) Add mother transfer RNA (10 mg / ml) to the cells and incubate the dish at 70 ° C overnight. Lab. 7. The probe was denatured, added to the wells at a dilution of 1: 100 to 1: 200, and the dishes were removed. Incubate at 70 ° C overnight. 8. After hybridization of cells, washing buffer (2 × SSC containing 0.1% Tween 20) Wash briefly at 65 ° C with 50% formaldehyde in water). After this wash, Wash three more times for 20 minutes each. 9. The cells are cooled to room temperature and TBST (0.8% NaCl, 0.02% KCl, 2.5% (ris-HCl (pH 7.5)) three times. Ten. Sheep serum was protected by heating at 70 ° C for 30 minutes with regular shaking. Activate. 10% inactivated sheep serum in TBST is added to the cells and the plate is Let warm for 1 hour. 11． Serum is removed and in 1% sheep serum diluted to a concentration of 1: 2000 in TBST With an anti-digoxigenin alkaline phosphatase-binding Fab fragment. Store dishes at 4 ° C. overnight. 12． The cells are briefly washed three times with TBST at room temperature and then exposed for a total of at least 2 hours. Wash three times. 13. Cells are washed with alkaline phosphatase buffer (100 mM NaCl, 50 mM MgCl2, 0.1% Tw een 20, 100 mM Tris (pH 9)) three times. 14. The wash was removed and cells were harvested at 4.5 / ml of alkaline phosphatase buffer. Stain in the dark in a solution of μl NBT and 3.5 μl BCIP (x-phosphatase) . 15． When the appropriate intensity of staining is observed, the reaction is repeated three times in PBT containing 1 mM EDTA. To stop. Cells can be stored in this solution for a short time at 4 ° C. You. Appendix 5: Alkaline phosphatase staining of cultured cells 1. Grow cells in tissue culture dishes for at least 1 hour in cold 80% ethanol. Fix for a while. They can be stored at 20 ° C. in this fixation for several weeks. 2. Cells were digested with 50% absolute ethanol, 30% absolute ethanol, and two changes of steam. Rehydrate by washing for 20 minutes each in a solution of running water. 3. Cells were harvested with 0.05% Fast Red TR Salt. 0.01% alpha naphthyl phosphate, 0.03 % MgCl_TwoAnd 0.25% borax for 5-10 minutes. 4. The cells are washed in distilled water and observed immediately. If necessary, wash cells with water May be stored in 50% glycerin. Appendix 6: Immunohistochemical staining of SSEA-1 1. Handle cells growing on the feeder layer attached to the glass cover glass. Place in well for ease. These are treated with PBS (Dulbecco's phosphate buffered Wash twice in saline). 2. Primary antibody (monoclonal anti-SSEA-1 from J. Ansell) was added to 0.15% BS Dilute 1: 1000 with serum-free medium containing A and buffer with Hepes. This is a cell And incubate the dishes at 4 ° C. for 45 minutes. 3. The cells are washed three times with cooling medium. 4. The secondary antibody (FITC-labeled anti-mouse IgM) was diluted 1:10 in medium and incubated at 4 ° C for 30 minutes. Add to cells. 5. The cells are washed three times with cooling medium. 6. The cells are washed three times with cold PBS. 7. Cells are fixed in cold 95% methanol / 5% acetic acid for 3 minutes. 8. Rinse cells with PBS, remove coverslips, and place on glass slides Flip over a few drops of 10% PBS: 90% glycerin. 9. The slides are examined under a fluorescence microscope. Appendix 7: Transfection of cells   Procedure 1: Calcium phosphate co-precipitation. 1. Cells are trypsinized and freshly loaded with feeder layer and normal medium Transfer to well. 2. Dish at 37 ° C with 2.5% CO_TwoAnd put it in the incubator. 3. Transfection mixture (50μl / ml BES, 0.012M CaCl_Two, And 1 μg / (to the final concentration of ml DNA) to the wells and incubate the cells overnight You. 4. The transfection mixture is removed the next morning and replaced with normal medium. 5. Transfected cells into a method determined by integrated DNA Therefore, select and / or identify. Step 2: Lipofection 1. Lipids and Gibco from the PerFect transfection kit from Invitrogen? A transfection solution is prepared using Optimem et al.   a. Add lipid to Optimem (12μl / ml)   b. Add DNA to Optimem (2μl / ml)   c. Mix lipid / Optimem with DNA / Optimem   d. Incubate at 37 ° C for about 15 minutes. 2. Wash the cell colonies three times with PBS. 3. Transfer colonies to trypsin for 1 minute. 4. Transfer the colonies to a small amount of Optimem and pull with a Pasteur pipette (pul led) Make powder. 5. The medium with the feeder layer is removed from the wells and the Optimem plus lipids and And replace with DNA. 6. Add trypsinized cells to wells and incubate for 5 hours You. 7. Remove Optimem and replace with cell culture medium (medium 2). Step 3: Electroporation 1. Wash colonies three times with PBS. 2. Trypsinize for 2-3 minutes. 3. Transfer colonies to 50 μl of PBS and grind until cells are disaggregated. 4. Electroporation containing 650 μl of PBS and 100 μg of DNA (in 100 μl of PBS) Transfer PBS with cells to the solution cuvette. 5. Leave the cuvette on ice for 10 minutes. 6. Electroporate at 0.8 kV, 3 μF, time constant = 0.1. 7. Leave the cuvette on ice for 10 minutes. 8. The contents of the cuvette were divided into 4 wells (200 μl each) and 30 Add 0 μl of Medium 2 to each well. 9. The cells are left for 3 hours and the medium is changed (using medium 2). 10. Leave overnight before handling further. Appendix 8: Freezing and thawing cells 1. Remove colonies of cells with a drawn pipette. 2. Quickly transfer colonies to a small volume of PBS, then to a small volume of 0.25 ml frozen mixture . 3. Quickly transfer the drops of the frozen mixture to a cryotube containing 0.4 ml of the frozen mixture. 4. Transfer to -70 ° C freezer for overnight freezing, then add liquid N_TwoTo the freezer Move.   Thaw: 1. Put 4.5-9.5 ml of medium into 25 ml universal. 2. Thaw frozen vials in a water bath, then transfer vial contents to universal Pipette in. 3. Centrifuge at <1000 rpm (600-800 is optimal) for 3-5 minutes. 4. The medium is aspirated from the DIA-M feeder layer. 5. The medium is aspirated from the universal, leaving clumps of cells. 6. Universally add 1 ml of Medium 2 and slowly resuspend. 7. Pipette cells (0.5 ml per well) onto feeder layer and culture Return to nutrition.   Frozen mixture:   42% FCS   48% medium 3   10% DMSO Appendix 9: ESRF purification and assays 9.1 Preparation of conditioned medium containing ammonium sulfate cut-off containing ESRF 1. ESRF-producing adherent cell line (D7A3-PE) near confluency in GMEM / 10% FCS Propagate to growth. Currently, about 30-40 large (175cm^Two) A flask is used. 2. The medium is removed, the cells are rinsed twice with PBS, then 10 mM HEPES, 3 μM Supplemented with sodium renate, 1 μg / ml transferrin, and 1 μg / ml insulin Return to filled serum-free GMEM / Ham F12 (1: 1). Gas the cells, and Return to incubator in medium for a minimum of 3-4 hours, or more conveniently overnight. This This is an adaptation and cleaning process. 3. Remove the medium and use fresh serum-free medium for 3-5 days of acclimation replace. 35 ml of medium per flask. 4. The conditioned medium is collected, centrifuged to remove dissociated cells, and sterile filtered. You. Collect about 1000 ml of medium before proceeding to step 5. 5. The conditioned medium is 35% saturated with ammonium sulfate. 5 saturated ammonium sulfate Add in cold room with stirring at ml / min and leave for another 60 minutes. Settled The protein is pelleted by centrifugation, which is Make up about 20% of the protein (Bradford assay). 6. The precipitate is solubilized in 2M urea. Centrifuge (Step 5) to polypropylene bottle Perform in a jar and the precipitate gives a viscous smear down the bottle length. This Taking this into account, the solubilization is performed for 1 hour at room temperature using a vibrating roller device. 1 in 6 bottles 5-20 ml. 7. The solubilized material is finally applied to tissue culture grade PBS (in a cold room). ) Dialyze overnight, then freeze in aliquots. 9.2 Assay   A number of embryonic stem cell lines were used to assay ESRF activity. Most commonly :   CP1 for general morphology and histochemistry of alkaline phosphatase   COKO18 general morphology and oct4 expression (detected by lacZ reporter activity )for The basic protocol is the same in each case. 1. ES cells were placed on a gelatinized 24-well plate in 0.5 ml GMEM / 10% FCS, Seed at a density of 00 cells / well. Use antibiotics at appropriate concentrations (ESRF sump If the filter is not sterile filtered). After 2-3 hours, test samples are added to the wells and the plates are incubated for 4 days. Return to incubator. 3.4 days later, cells were washed with alkaline phosphatase activity (Sigma Alkaline Phosphatase).  Leukocyte kit, Catalog No. 86-R) or β-galactosidase activity (X-gal Staining). 4. Test cells and colonies for a representative ES cell phenotype:   -Small round cells   − Compact and round colonies   -High expression of alkaline phosphatase or β-galactosidase activity Expression of oct4 reflected.   One unit of ES cell activity was transferred at day 4 above in a 0.5 ml assay. Defined as the minimal amount that produces a tabular ES cell phenotype (at which concentration most cells Note that it does not have an ES cell phenotype). Appendix 10: Ultracentrifugation of PE conditioned media 1. PE-CM was prepared as described above (Appendix 9). 2. PE-CM was centrifuged at 100,000 × g for 75 minutes at 4 ° C .; (S = 106). 3. The supernatant (S1) is removed and the pellet (P1) is solubilized in 0.5 ml of 2M urea Was. 4. The supernatant (S1) was centrifuged again at 265,000 × g for 18 hours; (S = 3.3). 5. Remove the supernatant (S2) and solubilize the pellet in 0.5 ml of 2M urea (P2) did. 6. Replace S1, S2, P1, and P2 with Pharmacia PD10 (S) or BioGel P6-DG (P) Desalted / exchanged to PBS on a system. 7. Samples were assayed for ESRF activity.   result   Activity similar to S1 PE-CM   No S2 activity detected   P1 Very low activity (smaller than PE-CM)   P2 Very strong activity Appendix 11: Lectin affinity chromatography of ESRF 1. Liquid phase isoelectric focusing of 25-35% saturated ammonium sulfate cut-off PE conditioned medium (Rotofor). 2. The precipitated material (pI = 4.25-4.5) was solubilized in 2M urea. 3. The solubilized material is transferred to a lectin buffer on a Pharmacia PD-10 column.^*Was replaced. 4. Transfer 200 μl of the exchanged material to soy lectin, lentil lectin or Load onto a column containing wheat germ agglutinin (equilibrated with lectin buffer) and allow 30 minutes And then add another 200 μl and leave the column in the cold room overnight. Was. 5. The column was washed with 5 × column volume of lectin buffer; the wash was collected. 6. Sugar in lectin buffer^**Elute with 5 × column volumes containing The eluted material was collected. 7. Exchange washed and sugar-eluted material for 20 mM sodium phosphate, pH 7.3 And assayed for ESRF activity.   ^*Lectin buffer 20mM sodium phosphate (pH7.3)                          1M NaCl                          0.1mM CaCl_Two                          0.1mM MnCl_Two ^**Sugar soybean-galactose                          Lentil-α-methyl mannoside                          Wheat germ-N-acetylglucosamine   result   Active retention and specific elution on soybean and lentil lectin columns However, the activity was not retained on wheat germ agglutinin and was not specifically eluted.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 15/09 C12R 1:91) (C12N 5/10 C12R 1:91) (81) Designated country EP ( AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ) , TM), AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, I , IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventors Chambers, Ian Paul United Kingdom Road (no address), Kings Buildings, The University of Edinburgh, Center for Genome Research (72) Inventor Viewer, Mia Lydia United Kingdom E9 93 Jewish Edinburgh, West Mains Road (no address), Kings Building Ngth, The University of Edinburgh, Center for Geno Li in the search (72) inventor Smith, Austin Gerald UK Ieichi 9 3 Jeikyu over Edinburgh, West Mains Road (no address), Kings Birudi Holdings, the University of Ede Inbara, Center for genome Li in the search

Claims (1)

[Claims] 1. Called ESRF and (i) produced in the absence of DIA / LIF, and (ii) produced via gp130 In the absence of the cytokines used and (iii) no interaction with gp130. And cytokines characterized by the ability to inhibit the differentiation of ES cells. 2. Can inhibit ES cell differentiation in the absence of interaction with the LIF receptor The cytokine of claim 1. 3. The method of claim 1, wherein the inhibition of differentiation is provided by a mechanism different from gp130. Is a cytokine according to claim 2. 4. By culturing cell line D7A3-PE and recovering conditioned medium from them A cytokine according to any of the preceding claims, which can be obtained. 5. Can be obtained by culturing cell line D7A3-PE, and       -Ability to inhibit ES cell differentiation       -Distinguishable from DIA / LIF       -Distinguishable from IL-6 / sIL-6R       -Distinguishable from CNTF       -Distinguishable from oncostatin M       -Distinguishable from cardiotrophin-1 A cytokine called ESRF, characterized by: 6. 6. The method of claim 5, wherein the inhibition of differentiation is caused by a mechanism different from gp130. Listed cytokine. 7. Its ability to inhibit ES cell differentiation depends on neutralizing anti-DIA / LIF antisera. The size according to claim 5 or 6, characterized in that the size cannot be excluded. Tocaine. 8. Its ability to inhibit ES cell differentiation neutralizes anti-IL-6 soluble receptor antiserum 8. The method according to claim 5, characterized in that it cannot be excluded by performing Or a cytokine described in 9. Its ability to inhibit ES cell differentiation is eliminated by neutralizing anti-CNTF antisera. Cytokine according to any of the preceding claims, characterized in that it cannot be removed. N. 10. Its ability to inhibit ES cell differentiation depends on neutralizing anti-gp-130 antiserum. A site according to any of the preceding claims, characterized in that it cannot be excluded. Cain. 11. Can be obtained from the supernatant of DIA / LIF-deficient cells, and (i) in the absence of DIA / LIF, And (ii) in the absence of cytokines acting via gp130, and (iii) gp130 and At least have the ability to inhibit the differentiation of ES cells in the absence of the interaction of A partially purified composition or a component thereof, also comprising one polypeptide. 12. Can inhibit differentiation of ES cells in the absence of interaction with LIF receptor 13. The partially purified composition according to claim 12, wherein 13. At least one of the same as specified in any one of claims 2 to 10 13. The partially purified according to claim 11 or claim 12, further having certain characteristics. Compositions or their ingredients. 14． A purified polypeptide of the composition according to any of claims 11 to 13. Do ingredients. 15. (i) cytokines acting in the absence of DIA / LIF, and (ii) gp130 Cells in the absence of and (iii) in the absence of interaction with gp130 Or a portion comprising such a polypeptide A method for producing a partially purified composition, comprising purifying the supernatant of DIA / LIF-deficient cells. Subject to a production procedure, thereby isolating the polypeptide. 16. Including growing cells in the presence of a cytokine called ESRF How to grow ES cells. 17． The ES cells may act via ESRF and DIA or other gp130 cytokines 17. The method of claim 16, wherein the method is grown in the presence of a combination with 18. Establishing ES cells, including culturing embryo-derived cells in the presence of ESRF Way. 19. The cells are capable of interacting with ESRF and DIA or other cytokines that act via gp130. 19. The method of claim 18, wherein the method is cultivated in the presence of a combination with the enzyme. 20. Establishing ES cells, including culturing primordial germ cells in the presence of ESRF Way. 21. The cells are capable of interacting with ESRF and DIA or other cytokines that act via gp130. 19. The method of claim 18, wherein the method is cultivated in the presence of a combination with the enzyme. 22. 22. The method according to any one of claims 16 to 21, wherein the cells are rodent cells. Law. 23. 22. The method according to any one of claims 16 to 21, wherein the cells are derived from a livestock species. Law. 24. 22. The method according to claim 16, wherein the cells are derived from a primate species. Method. 25. The expanded cell is a stem cell selection marker such as 0ct-4neo or 0ct-4βgeo. 23. A method according to any one of claims 14 to 22, which owns a car. 26. A method of proliferating somatic stem cells, such as hematopoietic stem cells, which Culturing the cells in the presence. 27. A cell line called D7A3-PE (ECCAC94111845). 28. An established line of embryonic stem cells having at least five of the following characteristics, and more preferably: Preferably, the cell line is characterized by having at least seven. Established lineage of embryonic stem cells characterized by having a karyotype other than mouse: (i) The characteristic morphology of stem cells, which is a small dense cell with a high nucleus to cytoplasm ratio           Agglomerated growth as directly packed cells), (ii) (a) alkaline phosphatase, (b) stage-specific embryo antigen-1, (c) from 0ct-3 / 4           Expression of at least one specific marker selected, (iii) non-expression of a differentiation marker (eg, H19 RNA), (iv) considerable or unlimited growth potential, (v) stability against freezing and thawing, (vi) stable euploid karyotype, (vii) cytokine dependent growth, (viii) Induced by cytokine withdrawal, aggregation, or chemical differentiation inducer           In vitro differentiation, (ix) Ability to form teratocarcinoma, including derivatives of endoderm, mesoderm, and ectoderm           Power, (x) colonies through the production of somatic stem cells and functionally differentiated progeny           The ability to form and / or reconstitute host tissues; (xi) forming the host embryo into a colony with the contribution of the functionally differentiated progeny to the chimera           Ability to (xii) produce functional gametes in chimeras and viable progeny           Ability to (xiii) Ability to incorporate exogenous DNA. 29. At least five of the above identified features (i) to (viii), and preferably 29. The embryonic stem of claim 28, wherein the embryonic stem is characterized by having at least seven. Established cell line. 30. By having at least one of the above-mentioned characteristics (ix) to (xiii) 30. The established line of embryonic stem cells according to claim 29, which is further characterized. 31. By having at least two of the above identified features (ix) to (xiii) 30. The established line of embryonic stem cells according to claim 29, which is further characterized. 32. 32. An embryo according to claim 28 or claim 31 having a proven rat karyotype. Established stem cell line. 33. Distinguishing by mechanisms different from those involving DIA / LIF and / or gp130 interactions A method for assaying and / or detecting a growth factor that affects activation, comprising: Culturing ES cells in the presence of the sample to be assayed; Detect changes in proliferation or differentiation compared to cells cultured in the absence of Wherein the ES cells have a LIF negative and / or rLIF negative table. A method characterized by having a present type.