WO1997030151A1 - Cytokine exprimee par des cellules souches embryonnaires a deficience dia/lif pour l'inhibition de la differenciation - Google Patents

Cytokine exprimee par des cellules souches embryonnaires a deficience dia/lif pour l'inhibition de la differenciation Download PDF

Info

Publication number
WO1997030151A1
WO1997030151A1 PCT/GB1997/000426 GB9700426W WO9730151A1 WO 1997030151 A1 WO1997030151 A1 WO 1997030151A1 GB 9700426 W GB9700426 W GB 9700426W WO 9730151 A1 WO9730151 A1 WO 9730151A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
lif
dia
differentiation
esrf
Prior art date
Application number
PCT/GB1997/000426
Other languages
English (en)
Inventor
Christian Dani
Ian Paul Chambers
Mia Lydia Beuhr
Austin Gerard Smith
Original Assignee
The University Of Edinburgh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9603244.6A external-priority patent/GB9603244D0/en
Priority claimed from GBGB9611319.6A external-priority patent/GB9611319D0/en
Application filed by The University Of Edinburgh filed Critical The University Of Edinburgh
Priority to AU18027/97A priority Critical patent/AU1802797A/en
Priority to JP9529116A priority patent/JP2000505294A/ja
Priority to EP97903477A priority patent/EP0880584A1/fr
Publication of WO1997030151A1 publication Critical patent/WO1997030151A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines

Definitions

  • This invention relates to substances having the capacity to inhibit differentiation of embryonic stem (ES) ceils.
  • the invention further provides methods of deriving and propagating ES cells, ES cells per se and cell lines useful in deriving and assaying novel factors.
  • Embryonic stem cells are the archetypal stem cell, being capable both of unlimited self-renewal and of differentiating to form the whole gamut of cell types found in the adult animal. Such stem cells are described as pluripotential as they are capable of differentiating into many cell types. These cells will participate fully in normal embryogenesis following reintroduction into blastocysts and can contribute functional differentiated progeny to all somatic tissues and to the germ line. ES cells can also be induced to differentiate into a wide variety of cell types in culture, recapitulating in vitro processes responsible for tissue diversification in the developing embryo. ES cells therefore provide a unique system for the analysis of factors that control early embryonic growth and differentiation.
  • ES cells have the additional property of being able to participate fully in normal embryogenesis following reintroduction into host embryos and contribute functional differentiate progeny to all somatic tissues and to the germ line. Their ability to form gametes allows ES cells to be used as a means of transmitting genetic modifications into animals. This is exploited for gene discovery and mutation though gene trapping strategies and most importantly for precise gene alteration via gene targeting.
  • stem cells when required for research purposes or for medical use, stem cells have to be isolated from tissue samples by various fractionation procedures, but even after careful segregation of cell types, these stem cell preparations consist of mixed cell types and while enriched for stem cells, include high proportions of differentiated cells which are not categorised as stem cells.
  • stem cells cannot be grown readily in culture and when attempts are made to culture stem cells, and more particularly to maintaining established lines of ES cells, the cells being cultured (which ordinarily contain a mixed population of cell types) grow at different rates and stem cells rapidly become overgrown by non-stem cell types.
  • embryonic stem cells from two specific strains of mice (1 29 and Black 6) can be cultured in vitro.
  • established lines of embryonic stem cells can be obtained by culturing early (3 1 /2 day) embryonic cells from murine strain 1 29 and Black 6, or hybrids thereof.
  • Proven embryonic stem cells with the capacity for germ line transmission have to date only been established from specific inbred strains of mice (notably strains 1 29 and C57BL/6) . They are derived by culturing early embryonic cells in the presence of a feeder layer of embryonic fibroblasts and/or of cytokine leukaemia inhibitory factor (LIF) or related cytokines which act through the signal transducer glycoprotein 1 30 (gp1 30) (Yoshida et al., 1 994) . In the absence of a source of LIF the stem cells differentiate and cannot be propagated.
  • LIF cytokine leukaemia inhibitory factor
  • gp1 30 signal transducer glycoprotein 1 30
  • ES cells cultures in the presence of DIA/LIF or of a feeder layer of DIA/LIF- producing cells maintain their proliferative capacity, retain characteristic stem cell morphology, and express stem cell markers such as alkaline phosphatase, stage- specific embryonic antigen- 1 and the stem cell-specific transcription factor Oct-3/4.
  • stem cell markers such as alkaline phosphatase, stage- specific embryonic antigen- 1 and the stem cell-specific transcription factor Oct-3/4.
  • ES cells expanded in the presence of LIF remain pluripotential and competent to produce germline chimaeras when reintroduced into mouse blastocysts (Nichols e al., 1 990; 1 994) . It is known that cytokines play an important role in the maintenance of the piuripotential phenotype of ES cells in vitro.
  • DIA glycoprotein of apparent molecular weight 45 kDa named "Differentiation inhibiting activity" or DIA.
  • This is identical to the cytokine "Myeloid leukaemia inhibitory factor” or LIF.
  • DIA/LIF acts to sustain the self-renewal of undifferentiated ES cells and thereby allows their propagation in vitro.
  • ES cells maintained in the presence of this factor retain their full development potential.
  • ES cells can be established de novo by direct culture of blastocysts in medium supplemented with DIA/LIF.
  • cytokine refers to any substance which acts from outside a cell and is capable of affecting cell survival and/or growth and/or differentiation. Primarily the term denotes a proteinaceous factor capable of inhibiting differentiation of ES cells) .
  • stem cells In the absence of a source of DIA/LIF, the stem cells differentiate and cannot be propagated. By contrast, ES cells cultured in the presence of DIA/LIF or of a feeder layer of DIA/LIF-producing cells maintain their proliferative capacity, retain characteristic stem cell morphology and express stem cell marker proteins such as alkaline phosphatase and stage-specific embryonic antigen-1 (SSEA-1 ) (Williams et al., 1 988; Smith et al., 1 988).
  • SSEA-1 stage-specific embryonic antigen-1
  • ES cells expanded in the presence of DIA/LIF remain piuripotential and competent to produce chimeras when reintroduced into mouse blastocysts (Nichols et al., 1 990; Pease et al., 1 990) .
  • DIA/LIF undifferentiated ES cells express low levels of mRNA which gives rise to the matrix-localised form of DIA/LIF, whereas differentiated cells express relatively high levels of both soluble and matrix- associated DIA/LIF. It has been proposed that the enhanced production of DIA/LIF by newly differentiated cells may provide a mechanism for regulating the balance between differentiation and self-renewal in stem cell populations.
  • the physiological importance of DIA/LIF has been established by the finding that homozygous DIA/LIF-deficient female mice are sterile due to an incapacity to support embryo implantation. However, DIA/LIF -/- embryos are viable. This observation suggests that other factors may compensate for the absence of DIA/LIF expression during early development.
  • cytokines including DIA/LIF, interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF) and oncostatin M (OSM), share the same effector molecule gp 1 30. This presumably underlies the considerable overlap in biological activities reported for these factors. Activation of gp1 30 signalling processes by any of these cytokines is sufficient to support ES cell self-renewal. However, it is not yet clear which, if any, of these molecules plays a role in the maintenance of stem cell renewal early during embryogenesis or whether other factor(s) are involved in this process.
  • DIA/LIF interleukin-6
  • CNTF ciliary neurotrophic factor
  • OSM oncostatin M
  • a paper entitled “Pluripotent Embryonic Stem Cells from the Rat Are Capable of Producing Chimeras", lannaccone, P.M. et al., Dev. Biol. 1 63, 288-292 ( 1 994) describes a cell line designated RESC-01 which is stated in the paper to consist of "diploid rat embryonic stem cells” . Further the RESC-01 cell line is stated to be capable of being “used to produce chimeras by injection into rat blastocysts” .
  • DIA/LIF in vitro and establish an assay system for detecting factors which can regulate ES self-renewal
  • ES cells in which both copies of DIA/LIF gene have been deleted .
  • differentiated DIA/LIF-deficient ES cells synthesize a novel soluble factor which inhibits ES cell differentiation. This factor is distinct from previously characterised ES cell maintenance factors and most significantly acts independently of direct activation of gp 1 30.
  • ES cells have also been generated which lack LIF receptor.
  • the LIF-negative and LIF receptor-negative cells (LIF(-) and rLIF(-)) are additionally useful in assay procedures which form further aspects of the invention.
  • ESRF ES cell Renewal Factor
  • a cytokine designated ESRF and characterised by the capacity to inhibit differentiation of ES cells (i) in the absence of DIA/LIF and (ii) in the absence of cytokines which act through gp1 30 and (Ui) in the absence of interaction with gp1 30.
  • the capacity to inhibit differentiation of ES cells (iv) in the absence of interaction with LIF-receptor may further be used to characterise the cytokines of the invention.
  • IL-6/slL-6R is an abbreviation of "interleukin-6/solubie interleukin-6 receptor" .
  • the cytokine designated ESRF herein is intended to include ESRF derived directly or indirectly from any mammalian species, including laboratory animals (i.e. rodents), domestic and farmyard animals (e.g. dogs, cats, sheep, horses, cows, pigs etc.) as well as primates including humans.
  • the factor may be obtained by isolation of endogenously produced factor in an appropriate cell line, or by recombinant expression.
  • the cytokine of the invention is believed to inhibit differentiation by a mechanism which is distinct from gp 1 30 and this feature may be used as a further characterising feature thereof.
  • the novel cytokine of the invention was discovered by generating cell lines that were DIA/LIF deficient and identifying in supernatants from such cell lines an activity that inhibited differentiation of ES cells.
  • the supernatants from these cells in crude or partially purified form also form part of the present invention.
  • the present invention further provides an at least partially purified composition obtainable from supernatants of DIA/LIF deficient cells and comprising at least one polypeptide having the following characteristics
  • the capacity to inhibit differentiation of ES cells (iv) in the absence of interaction with LIF-receptor may further be used to characterise the partially purified compositions. These may optionally further be characterised by features selected from the following:
  • cardiotrophin-1 taken individually or in combination.
  • At least partially purified polypeptide components of said compositions also fall within the present invention.
  • the invention may alternatively be defined as a cytokine designated ESRF which is obtainable by culturing cell line D7A3-PE and is characterised by
  • the invention further provides a method of producing the novel cytokine ESRF which comprises culturing a ESRF-producing cell line and isolating ESRF from the supernatant therefrom.
  • the ESRF-producing cell line comprises the cell line D7A3-PE or a cell derived therefrom.
  • the deposited cell line D7A3-PE constitutes a further aspect of the present invention.
  • the invention further provides culture procedures which utilise ESRF.
  • One is a method of propagating ES cells which comprises propagating the cells in the presence of the cytokine designated ESRF.
  • Another is a method of establishing ES cells which comprises culturing early embryos in the presence of the cytokine designated ESRF.
  • These procedures can utilise ESRF as the sole ES cell- propagation enhancing agent, or others may be included in the culture medium, for example DIA/LIF or other cytokines including ones which act through gp 1 30.
  • Propagation and/or establishment of ES cells in accordance with the invention may be monitored by culturing cells which include a selectable marker such as Oct- ⁇ geo. Further the method of the invention may be utilised to promote the propagation of somatic stem cells such as haematopoietic stem cells.
  • the cytokine of the invention in combination with a second cytokine known to act via gp 1 30, is used to derive, propagate and/or maintain a culture of embryonic stem cells in an undifferentiated state.
  • a combination of the cytokine of the invention with LIF is suitable for deriving, propagating and/or maintaining embryonic stem cells of rats or other species.
  • Suitable feeder layer cells are derived from fibroblasts. Examples include mouse embryo fibroblasts that have been transfected so as to express matrix- associated LIF. A suitable stably transfected cell line is designated DIA-M and has been deposited at the European Collection of Animal Cell Cultures (ECACC) on 31 May 1 996 under Accession No. 96053101 .
  • ECACC European Collection of Animal Cell Cultures
  • DIA-M was produced as a clonal derivative of C3H1 OT1 /2 mouse embryo fibroblasts stably transfected with an expression vector containing a cDNA for the matrix-associated form of mouse LIF (Rathjen et al., 1 990) and an IRES-linked neo selectable marker. (See also WO 94/24301 for details of the expression system) .
  • a new methodology for isolating and propagating embryonic stem cells from other species is based on a novel combination of soluble LIF plus the cytokine Embryonic Stem Cell Renewal Factor (ESRF) plus a feeder layer of genetically modified fibroblasts which overexpress the matrix-associated isoform of LIF (Rathjen et al., 1 990) .
  • ESRF cytokine Embryonic Stem Cell Renewal Factor
  • rat embryonic stem cells can be propagated indefinitely whilst retaining a euploid rat karyotype and characteristic stem cell morphology and marker expression. Furthermore a novel method for freezing and thawing the ES cells is described and procedures are given for chimaera production.
  • a method for assaying and/or detecting growth factors that affect differentiation by a mechanism that is distinct from mechanisms involving DIA/LIF and/or gp 1 30 interactions which comprises culturing ES cells in the presence of a sample to be assayed, and detecting variation in growth or differentiation compared to cells cultured in the absence of the sample, characterised in that the ES cells have an LIF-negative (LIF(-)) and/or LIF receptor-negative (rLIF(-)) phenotype.
  • a specific embodiment of the invention is a cytokine having the following physicochemical properties:-
  • the invention further provides an established line of embryonic stem cells characterised by possessing at least five and preferably at least seven of the following features:
  • the characteristic morphology of stem cells including growth in clumps as small tightly packed cells with a high nuclear to cytoplasmic ratio, (ii) expression of one or more specific markers selected from (a) alkaline phosphatase, (b) stage-specific embryonic antigen-1 , (c) Oct-3/4, (iii) non-expression of differentiation markers, for example H 1 9 RNA, (iv) substantial or unlimited propagation potential, (v) stability to freezing and thawing, (vi) stable euploid karyotype, (vii) propagation dependent on cytokines, (viii) in vitro differentiation inducible by withdrawal of cytokine(s), aggregation or chemical differentiation inducers, (ix) ability to form teratocarcinomas comprising derivatives of endoderm, mesoderm and ectoderm, (x) ability to colonise and/or reconstitute host tissues through the production of somatic stem cells and functionally differentiated progeny, (xi) ability
  • Such cells preferably possess at least five and preferably at least seven of the specified features (i) to (viii), and most preferably are further characterised by possessing at least one of the specified features (ix) to (xiii) .
  • established lines of embryonic cells provided according to the invention need not necessarily exhibit each and every one of the features (i) to (xiii) listed above. Thus for certain species, one or more characteristics may be absent.
  • rodent embryonic stem cells may display all three of the specific markers mentionad above, i.e. (a) alkaline phosphatase, (b) stage-specific embryonic antigen-1 and (c) Oct-3/4,.
  • stage-specific embryonic antigen-1 feature (ii)(b)
  • Figure 1 illustrates the response of lif-r -/- ES cells to cytokines
  • Figure 2 shows a phase contrast photomicrograph of rat ES cells
  • Figure 3 shows a metaphase chromosome spread from rat ES cells
  • Figures 4A show marker expression in rat ES cells of A. Alkaline & 4B phosphatase and B. Oct-4 immunostaining
  • Figures 5A show differentiation of rat ES cells into A. Trophoblast, 5B & 5C B. Parietal endoderm and C. Bipolar cells
  • both alleles of the gene were deleted via two rounds of homologous recombination.
  • ES cells in which one copy of the gene is inactivated were generated using a replacement vector in which the entire coding sequence of the DIA/LIF gene is replaced by a selectable HPRT mini- gene.
  • HAT-resistant ES cell clones which had undergone the recombination event were identified by DNA hybridisation analysis using a probe extemal to the 3' arm of homology.
  • DNAs of positive clones were restricted with the appropriate restriction enzyme and hybridized to a 5' external probe. In total, 1 1 correctly targeted clones were identified from a primary screen of 89 colonies.
  • D6 One germline competent clone, D6, was used for the deletion of the second allele.
  • a hygromycin resistance cassette was substituted for the HPRT marker in the targeting vector.
  • this construct gave rise to homologous replacement events at a comparable frequency ( 10%) to that obtained with the HPRT vector.
  • This second construct was electroporated into the D6 clone and transfectants were isolated by selection in hygromycin B.
  • Three classes of homologous recombinant were identified amongst 1 62 colonies screened: recombination into the previously targeted allele had occurred in 30% of clones; integration into the 5' or 3' homology region of the wild-type allele was detected in 4 cases; and only three clones had undergone replacement of the wild-type allele.
  • DIA/LIF-deficient ES cell clones were used to generate chimaeras.
  • DIC2 and D7A3 ES cells were injected into C57BL/6 blastocysts and chimaeric offspring were produced.
  • Chimaeras showed high ES cell contribution as judged by the sandy coat colour. This was confirmed by the determination of the degree of ES cell contribution to blood and tail of five chimaeric mice by glucose phosphate isomerase isozyme analysis. Both clones contributed to gametogenesis as indicated by the production of viable germ-line offspring.
  • the number of undifferentiated ES cells colonies derived from the indicator population was determined by staining for ⁇ -galactosidase activity.
  • the results indicate that the capacity of DIA/LIF-deficient differentiated cells to inhibit ES cell differentiation is reduced relative to the parental cell line, but is not abolished. Similar results were obtained using retinoid-induced differentiated cells as the feeder layer.
  • a second kind of co-culture experiment was carried out in which the indicator cells were plated on a microporous insert above the layer of differentiated cells.
  • the insert membrane prevents cell-cell contact between the two cell populations but allows the access of diffusible factors.
  • Both parental and Oct-4-tagged ES cells were used as indicators, staining for alkaline phosphatase and ⁇ -galactosidase respectively. Similar results were obtained in both cases.
  • the greater activity in wild-type cultures is significantly reduced in the presence of neutralizing DIA/LIF antiserum, but is not eliminated. This indicates that wild-type cells also synthesize active factors other than DIA/LIF.
  • the residual activity in the presence of anti-DIA/LIF was similar to that produced by DIA/LIF-negative cells which suggests that expression of the responsible factor is not significantly up-regulated in these cells.
  • D7A3-PE differentiated cell lines from embryoid body outgrowths of the DIA/LIF- negative ES cells.
  • Several of these cell lines produced levels of activity which were readily detectable in their culture supernatants.
  • One cell line, named D7A3-PE could be propagated indefinitely and produced high levels of a soluble factor which could maintain undifferentiated ES cells.
  • ES cells cultured in D7A3-PE conditioned medium continued to express markers of the undifferentiated state such as alkaline phosphatase and Rex-1 mRNA and in the case of Oct-4 tagged cells retained ⁇ -galactosidase activity. This conditioned media was effective on several independent ES cell lines.
  • ES cells cultured in the presence of conditioned media or partially purified ESRF formed tight, rounded up colonies, morphologically distinguishable from both differentiated cells and ES cells maintained in DIA/LIF.
  • the active factor could be concentrated at least 20-fold by ultrafiltration and was destroyed by incubation with trypsin, consistent with a proteinaceous macromolecule. The factor was inactivated by heating to 50°C or by acidification below pH3. 1 .6 Purification and biochemical properties
  • conditioned medium is prepared from D7A3-PE cells in the absence of serum.
  • Cells are inoculated into 1 50cm 2 tissue culture flasks in normal growth media and grown to near confluence. Cultures are then rinsed with PBS and transferred into defined medium consisting of a 50:50 mix of GMEM and Ham's F1 2 basal media supplemented with 1 00 ⁇ M 2-mercaptoethanol, 1 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin and 10nM sodium selenite. Cultures are incubated for 4 hours then the medium is discarded and replaced with fresh defined medium (50ml/flask) .
  • This medium is conditioned by incubation with the D7A3-PE cells at 37°C for 96 hours.
  • the medium is then harvested and replaced. Three successive harvests can be obtained from each culture.
  • the harvested medium is clarified by centrifugation and passed through a sterile 0.2 ⁇ m filter.
  • the ability to inhibit ES cell differentiation is routinely detectable at a 1 in 1 0 dilution of the unfractionated conditioned medium though full activity over a four day assay requires no greater than 25% dilution.
  • Activity in the conditioned media is stable upon freezing and thawing, but is lost upon incubation at 50°C for 30 min, acidification to pH2 or incubation with trypsin.
  • Activity can be concentrated by ultrafiltration in an Amicon cell using a membrane with a nominal molecular weight cut-off of 100,000 Daltons. Quantitative recovery of biological activity is obtained, indicating that native ESRF has a molecular weight in excess of 100,000 Daltons.
  • ESRF is quantitatively recovered from conditioned medium by stepwise precipitation with saturated ammonium sulphate.
  • ESRF activity is recovered in the 25-35% fraction. This fraction contains 20-25% of total protein.
  • Upon reconstitution in 1 /100 volume of starting medium a viscous solution is obtained which partitions into fluid and gel phases upon cold storage. Both phases contain biologically active ESRF.
  • the gel material is likely to consist of highly glycosylated extracellular matrix components.
  • ESRF can be extracted from the gel by incubation with salt buffers or more efficiently with 0.1 % CHAPS. This finding suggests that ESRF has avidity for ECM components.
  • ESRF activity can be extracted directly from monolayers of D7A3-PE cells by incubation for 2 hours with basal medium containing 0.1 % CHAPS. (Under such conditions the cells remain viable with no evidence of significant lysis) . It is possible that ESRF may exist in isoforms which associate differentially with extracellular matrix. Alternative protocols for obtaining an ESRF-containing fraction by ammonium sulphate precipitation are given in Appendix 9.
  • the maximum concentration required for biological activity is 1 .5nM, based on the protein content of urea resolubilised ammonium sulphate precipitated material (see Appendix 9) and assuming a molecular weight equal to 1 00,000 Daltons.
  • ESRF binds to soya bean lectin and to lentil lectin, but not to wheat germ agglutinin (appendix 1 1 ) . Bioactivity is not affected by the addition of heparin to the assay media.
  • ESRF can be further purified by solution phase electrofocussing of the ammonium sulphate fractionated material. Focussing may be performed in the presence of 1 % CHAPS and 2% ampholytes in a BioRad Rotofor cell. ESRF is recovered quantitatively in 2-4 fractions of pH 4.25-4.5. ESRF is insoluble in these fractions, but resolubilizes upon direct addition to culture medium for the ES cell bioassay or upon adjustment of buffer pH. SDS gel electrophoresis shows that two major proteins with apparent molecular weights of 1 1 5,000 and 1 80,000 Daltons are specific to the active fractions. Further purification of ESRF may be achieved by heparin agarose affinity chromatography.
  • ESRF electrospray re-semiconductor
  • the sample is loaded in 20 mM phosphate buffer, pH3.5, containing 0.1 % CHAPS. Under these conditions most material does not bind to the column and a significant purification of ESRF can be obtained.
  • ESRF is not eluted with 1 00mM salt, but elutes over a broad range of higher salt concentrations.
  • the heterogenous elution profile is suggestive of variable glycosylation. (Similarly heterogenous elution profiles are also obtained on anion exchange chromatography) . Bioactive fractions contain very low levels of protein (undetectable at 280nm).
  • Protein is also undetectable by either coomassie blue staining, conventional silver staining, or silver staining combined with periodic acid preincubation (for improved detection of glycoproteins) of SDS PAGE gels loaded with up to 10-fold higher amounts of material than required for full biological activity.
  • the low amount of protein present indicates that ESRF is fully active at nanomoiar concentrations.
  • the concentration of IL-6 in the conditioned medium was determined using the highly sensitive B9 cell proliferation assay.
  • Conditioned medium did induce proliferation of B9 cells but the mitogenic response 'equated to a concentration of less than 5 pg/ml IL-6. That this activity was due to IL-6 was confirmed by the use of a neutralizing IL-6 soluble receptor antibody, RS1 3.
  • the bioactivity was inhibited totally in the presence of RS1 3.
  • inhibition of ES cell differentiation by D7A3-PE cell conditioned medium was not modified by the addition of RS1 3, indicating that this low level of IL-6 is not responsible for the effect of this media.
  • This conclusion was further substantiated by the finding that the conditioned medium was not mitogenic for BAF-mgp 1 30 cells. The latter are responsive to IL-6 only in the presence of soluble IL-6 receptor, so the negative result in this assay indicates that the D7A3-PE ceil conditioned media does not contain any IL-6 soluble receptor.
  • CNTF The potential involvement of CNTF was examined both by specific immunodepletion of conditioned medium with the 4-68 anti-CNTF mAb, and by the use of a neutralizing anti-CNTF antiserum. Neither treatment affected the ability of D7A3-PE cell conditioned medium to inhibit ES cell differentiation. Furthermore, activity was retained on addition to RS1 3 anti-IL-6R antiserum to medium previously depleted with anti-CNTF.
  • the self-renewal factor is distinct from mouse oncostatin M and from cardiotrophin-1
  • 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, has an apparent molecular weight in excess of 100,000 Daltons. It is quantitatively retained by ultrafiltration membranes with a cut-off of 100,000 Daltons even in the presence of 4M urea and has a mobility on size exclusion chromatography corresponding to a molecular weight greater than 100,000 Daltons.
  • 1 .8 ESRF is active on LIF-receptor deficient ES cells
  • LIF and related cytokines act through a heterodimeric receptor consisting of the low affinity LIF receptor and gp 1 30.
  • ES cells lacking LIF receptor were generated by two rounds of gene targeting. These cells can be propagated using the combination of IL-6 and soluble IL-6 receptor which acts via gp1 30 homodimers without involvement of LIF-receptor.
  • LIF-receptor negative ES cells are pluripotent and contribute to multiple tissues in chimaeras. They show no self-renewal response to LIF, CNTF, oncostatin M or cardiotrophin-1 . As shown in Figure 1 , however, LIF-receptor deficient ES cells remain responsive to ESRF. This finding confirms that ESRF is distinct from the LIF group of cytokines. 1 .9
  • the self-renewal factor (ESRF) does not act through direct interaction with gp130
  • ESRF administration causes no apparent increase in STAT3 activity as determined by gel shift analysis and no induction of tis 1 1 transcripts by RNase protection assay. This indicates that ESRF action is mediated via a distinct intracellular signalling pathway(s) from gp 130 signal transduction.
  • the biological response of ES cells to ESRF is also distinguishable from their response to LIF or related cytokines.
  • the ES cells are characteristically more compact in the presence of ESRF and form very tight, rounded, colonies which have a tendency to detach from the culture surface. On transfer to LIF-containing medium these colonies adopt a slightly more flattened and spread appearance, typical of ES cells cultured in LIF. Undifferentiated colonies can be maintained for at least 1 1 days using ESRF alone. However, the colony size does not appear to increase after the first 4 days, in contrast to the effect of LIF which promotes continuous stem cell expansion. Additive or synergistic effects on stem cell propagation are apparent on combination of sub-optimal amounts of LIF and ESRF. These data are consistent with distinctive effects on the self-renewal process.
  • ESRF sustains the undifferentiated phenotype of ES cells in vitro. This effect persists for at least 1 1 days. However, proliferation is limited and cultures cease to expand after about four days. This contrasts with cultures propagated in the presence of LIF which undergo a continuous increase in stem cell numbers.
  • ES cells maintained in ESRF were cultured at clonal density in the presence of ESRF for four days. They were then transferred to LIF-containing medium to facilitate expansion of the stem cells prior to blastocyst injection. This protocol was applied to the ES cell line Zin40 which carries a constitutively expressed nuclear localised ⁇ - galactosidase marker.
  • Zin40 cells plated in control medium underwent complete differentiation and did not give rise to any ES cell colonies upon replating in the presence of LIF.
  • Cells plated in ESRF remained undifferentiated as described above and engendered many alkaline phosphatase positive stem cell colonies on replating.
  • Cells from replated and expanded cultures contributed extensively to chimaeras as determined by the widespread presence of ⁇ -galactosidase positive cells in mid- gestation embryos. These chimaeric embryos were morphologically normal. The levels of Zin40 contribution were comparable to those obtained from parallel injections of cells maintained in LIF only.
  • DIA/LIF-deficient ES cells allowed us to study the role of this cytokine in stem cell renewal and differentiation and to characterize other factors involved in the regulation of this process.
  • the results establish that DIA/LIF-deficient differentiating cultures retain some capacity for maintaining a population of undifferentiated cells.
  • the findings indicate that factors and signalling pathways other than those characterised to date have the capacity to maintain the self-renewal of piuripotential ES cells. This may prove of fundamental importance for the isolation and propagation of stem cells from other species.
  • ES murine piuripotential embryonic stem
  • cytokines The proliferation of ES cells in vitro is sustained through the activation of intracellular processes associated with the signal transducer gp1 30.
  • DIA/LIF cytokine Differentiation Inhibiting Activity
  • CNTF ciliary neurotrophic factor
  • IL-6/slL-6R soluble interleukin-6 receptor
  • cardiotrophin-1 and oncostatin M can each activate gp 1 30 and support ES cell propagation.
  • the involvement of either CNTF or IL-6/sIL-6R in our system has been precluded through the use of neutralising antisera against these factors.
  • the cell line designated D7A3-PE has been deposited at the European Collection of Animal Cell Cultures (ECCAC) on 1 8th November 1 994 under Accession No 941 1 1 845.
  • Rat blastocysts were placed in culture on irradiated feeder layers of C3H10T1 /2 derived feeder cells in standard ES cell culture medium containing LIF plus ESRF. Inner cell mass clumps were picked, dissociated in trypsin and replated in identical conditions. Colonies of small, morphologically undifferentiated cells which arose were picked individually, dissociated and replaced. In this way stem cell cultures have reproducibiy been initiated from approximately 50% of rat blastocysts of Fischer and Sprague Dawley, and at a lower percentage of BDIX and DA strains.
  • the undifferentiated cells can be maintained for prolonged periods and expanded extensively, by regular passaging. They can be frozen by conventional procedures and recovered from storage in liquid nitrogen. In more detail, the procedure was as follows:
  • Feeder layers are prepared from DIA-M fibroblasts.
  • DIA-M cells are a clonal derivative of C3H 10T112 mouse embryo fibroblasts stably transfected with an expression vector containing a cDNA for the matrix-associated form of mouse LIF (Rathjen et al., 1 990) and an IRES-linked neo selectable marker. These cells express high levels of recombinant matrix LIF.
  • Feeder layers are made by dispensing approximately 75,000 gamma-irradiated DIA-M cells to a 1 5 mm diameter well of a 4-well tissue-culture plate (from Nunclon) . 0.5 ml medium 2 (Appendix 1 ) is added to each well.
  • the zona-free embryos are cultured in these wells for 5 days. After the embryos attach to the feeders (after 2-3 days) the medium (medium 2) is changed daily.
  • Dishes are incubated at 37°C, 7%CO 2 in air, and the medium is changed daily. These culture conditions are used in all further steps.
  • Colonies to be trypsinised are rinsed briefly in 3 changes of phosphate-buffered saline and incubated in .05% trypsin with 0.5 mM EDTA and 0.1 % chicken serum for 45-60 seconds. They are then transferred to a drop of medium 3, disaggregated into single cells and small clumps by trituration, and added to a new well (as described in step 3 above) .
  • colonies may be physically broken up into clumps without trypsin treatment by cutting them apart with glass needles and triturating the fragments with a finely drawn pipette.
  • a) Host embryos at the early blastocyst stage are flushed from uteri of female rats in the morning of the 5th day of pregnancy (early blastocyst stage) using medium 1 . They are cultured for 24 hours in medium 4, and the next morning disaggregated cells are injected into the blastocyst cavity. The embryos are allowed to recover in the incubator for two hours, and are then transferred to the uteri of rats in the 5th day of pseudopregnancy.
  • b) Host embryos at the early blastocyst stage are collected as in 2.2.1 1 .a. They are injected immediately with disaggregated cells and transferred immediately to the uteri of rats in the 4th day of pseudopregnancy.
  • Cells and host blastocysts are derived from strains with different coat colours, and chimaeric animals can be identified by colour about a week after birth.
  • micro satellite markers can be used to distinguish donor cells and host derived populations.
  • Cells may be transfected by the procedure described in Appendix 7.
  • Cells may be frozen by the procedure described in Appendix 8.
  • the ES cell status of the undifferentiated cells after extended culture periods is evidenced by one or more of: (i) maintenance of overt ES cell morphology; (ii) induction of overt differentiation by withdrawal of cytokines, (iii) expression of the early embryo and ES cell specific antigen SSEA-1 , (iv) expression of the pluripotent cell specific transcription factor Oct-3/4, (v) expression of alkaline phosphatase; and (vi) non-expression of H1 9.
  • rat ES cell cultures The characteristic stem cell morphology of rat ES cell cultures is shown in figure 2. These cells are morphologically indistinguishable from mouse ES cells cultured under the same conditions.
  • the rat chromosome complement of the cultures is shown in Figure 3. Expression of markers is shown in Figure 4; upper panel alkaline phosphatase, lower panel Oct-4 immunostaining.
  • Figure 5 shows examples of rat ES cell differentiation; a. trophoblast, b. parietal endoderm, c. unidentified bipolar cells.
  • the rat cells express the same markers. Alkaline phosphatase is expressed by primordial germ cells in the rat and mouse, and by mouse ES cells.
  • the rat cell lines we have derived are also strongly positive for alkaline phosphatase, but cease to express this enzyme when the cells differentiate.
  • the rat cells stain positively with an antibody to SSEA-1 (stage-specific embryonic antigen 1 ) which is also expressed by primordial germ cells and mouse ES cells.
  • SSEA-1 stage-specific embryonic antigen 1
  • colonies may be stored in liquid nitrogen and subsequently thawed and maintained in culture with no increase in incidence of differentiation, or alteration of ploidy.
  • the maintenance of an undifferentiated phenotype in these lines is dependent upon 3 factors, each of which should be present in the system if cell lines are to remain viable and undifferentiated for more than a few passages.
  • the medium must contain soluble DIA/LIF and ESRF (see sections 2.1 ), and the cells are preferably grown upon feeder layers of mitotically inactivated DIA-M cells (section 2.2.2). If either LIF or ESRF is removed from the systems, the cells will all eventually differentiate (mostly as trophoblast-like cells) or die (Table 1 , below) . Once established, rat cells can be propogated on alternative feeder layers or even on gelatin-coated plastic. Later passage cells may also be grown in the absence of ESRF. maximum passage number with DIA/LIF, ESRF, and DIA-M feeders > 25
  • Chimaeras may be produced from these cells by introduction into host embryos using the procedures given in section 2.2.1 1 .
  • Genetically modified rats may be produced by transgene integration or by gene targetting in rat ES cells followed by Chimaera production and germline transmission.
  • the effects of conditioned medium and ammonium sulphate fractionated ESRF on the multipotent human teratocarcinoma cell line GCT 27X1 were examined.
  • the assay involves growth of cell colonies from single cells in the absence of a feeder layer in serum-containing medium over a 10-14 day period. Cells are observed on a daily basis using phase contrast microscopy. Under the conditions of the assay, cell survival and colony formation in control medium is low.
  • ESRF preparations consistently demonstrated a positive effect on stem cell survival in these assays. Cell viability was enhanced in the early part of the assay and final colony number was increased substantially. Thus ESRF or an activity associated with it is active in promoting in vitro survival of human pluripotent teratocarcinoma stem cells.
  • ESRF medium 80% GMEM, 20% fetal calf serum, 2000 units human DIA/ml, and either 50 units/ml penicillin, 50 mg/ml streptomycin, or 50 mg/ml gentamycin.
  • Semi-purified ESRF is added at a concentration determined by bioassay of mouse ES cells (see Appendix 9) .
  • Colonies of cells to be karyotyped are picked from their dishes and cultured in Colcemid at 0.8 mg/ml for 2 hours in medium 3, in dishes containing feeder layers of DIA-M cells.
  • the dish is then filled with freshly made up 3: 1 fixative (3 parts absolute ethanol to 1 part glacial acetic acid) and left for 1 hour.
  • Primary antibody affinity-purified anti-oct-4 antiserum from rabbits Palmieri et al., 1 995) is diluted 1 :5000 in PBS and added to the cells for 30 minutes.
  • biotinylated secondary antibody (goat anti-rabbit) is prepared according to the protocol of the Vectastain kit (Vector Laboratories) and added to the cells for 30 minutes.
  • the "ABC reagent” is prepared according to the protocol of the Vectastain kit (Vector Laboratories), allowed to stand for 30 minutes, and added to the cells for 30 minutes.
  • Peroxidase substrate is prepared from the "VIP kit” supplied by Vector Laboratories according to instructions.
  • Peroxidase substrate is added to the cells fro 2-1 0 minutes, until a suitable intensity of color develops.
  • the probe is synthesized from a Stu 1 fragment of a Bluescript plasmid carrying the POU homeodomain of the oct-4 gene, with 1 .75 M digoxigenin-1 1 -UTP in the synthesis mixture .
  • Cells are rehydrated through the methanol series, rinsed with PBT, and washed 3 times in RIPA ( 1 % NP-40, 0.5% NaDOC, 0.1 % SDS, 1 mM EDTA and 50 mM Tris in 1 50 mM NaCl) .
  • hybridisation buffe ⁇ PBT hybridisation buffer:50% formamide in 10xSSC pH 4.5, with 0.05% of a stock solution of 1 00 mg/ml heparin, and 0.1 % Tween 20), and then with 1 00% hybridisation buffer.
  • Hybridisation buffer with herring sperm DNA ( 1 00 ⁇ g/ml) and yeast transfer RNA ( 1 0 mg/ml) is added to the cells, and the dishes are incubated at 70°C overnight.
  • the probe is denatured, added to the wells at a dilution of 1 : 1 00 - 1 :200, and the dishes incubated at 70°C overnight.
  • Sheep serum is inactivated by heating to 30 minutes at 70°C with regular shaking. 10% inactivated sheep serum in TBST is added to the cells and the plates are left at room temperature for 1 hour.
  • the wash is removed and cells stained in the dark in a solution of 4.5 ul NBT and 3.5 ul BCIP (x-phosphate) per ml alkaline phosphatase buffer.
  • APPENDIX 5 Alkaline phosphatase staining of cultured cells
  • Cells are washed in distilled water and observed immediately. If necessary, cells can be stored in 50% glycerine in water.
  • Primary antibody (monoclonal anti-SSEA-1 , obtained from J. Ansell) is diluted 1 : 1000 in serum-free medium with 0.1 5% BSA and buffered with Hepes. This is added to the cells and the dishes incubated at 4°C for 45 minutes.
  • the secondary antibody (anti-mouse IgM, FITC labelled) is diluted 1 : 10 with medium, added to the cells for 30 minutes at 4°C.
  • Cells are trypsinised and transferred to fresh wells containing feeder layers and medium as usual.
  • Transfection mix (to a final concentration of 50 ul/ml BES, 0.01 2 M CaCI2 and 1 ug/ml DNA) is added to wells and the cells incubated overnight.
  • Transfected cells are selected and/or identified according to the method determined by the incorporated DNA.
  • APPENDIX 8 Freezing and thawing of cells
  • ESRF-producing adherent cell fine (D7A3-PE) is grown to near-confluency in GMEM/10% FCS. Currently, approximately 30-40 large ( 1 75 sq.cm.) flasks are used.
  • Conditioned medium is harvested, centrifuged to remove detached cells, and filter-sterilised. Approximately 1000 ml of medium is collected before proceeding to step 5.
  • Conditioned medium is brought to 35% saturation with ammonium sulphate. Saturated ammonium sulphate is added at 5 ml/min with stirring n the cold room and left for a further 60 min. The precipitated protein is pelleted by centrifugation and constitutes approximately 20% of the total protein in the conditioned medium (Bradford assay).
  • the precipitate is solubilised in 2M urea.
  • the centrifugation (step 5) is done in polypropylene bottles and the precipitate ends up as a viscous smear down the length of the bottle.
  • the solubilisation is done on a rocking roller apparatus for 1 h at room temperature. 1 5-20 ml in 6 bottles.
  • the solubilised material is finally dialysed overnight against tissue culture- grade PBS (in cold room) then frozen in aliquots.
  • the basic protocol is the same in each case.
  • ES cells are seeded on to gelatinised 24-well plates at a density of 5,000 cells per well in 0.5 ml GMEM/1 0% FCS. Antibiotic is used at an appropriate concentration (unless the ESRF sample is filter sterilised) .
  • the cells are stained for alkaline phosphatase activity (Sigma Alkaline Phosphatase Leukocyte kit, cat. no. 86-R) or ⁇ -galactosidase activity (X-gal stain) .
  • 1 unit of ES cell activity is defined as the minimal amount which produces typical ES-cell phenotype in 0.5 ml assay at the 4d point as described above (n.b. at this concentration the majority of cell will not have ES-cell phenotype).
  • S1 , S2, P1 and P2 were desalted/exchanged into PBS on Pharmacia PD 1 0 (S) or BioGel P6-DG (P) columns.
  • APPENDIX 1 1 Lectin-affinity chromatography of ESRF
  • solubilised material was exchanged into lectin buffer* on a Pharmacia PD-1 0 column.
  • wash-through and sugar-eluted material was exchanged into 20mM sodium phosphate pH 7.3 and assayed for ESRF activity.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une nouvelle cytokine ayant la capacité d'inhiber la différenciation des cellules souches (ES). La cytokine est appelée ESRF et se caractérise par sa capacité à inhiber la différenciation des cellules ES (i) en l'absence de DIA (activité inhibant la différenciation)/LIF(facteur inhibiteur de la leucémie myéloïde), (ii) en l'absence de cytokines qui agissent par l'intermédiaire de la gp130, (iii) en l'absence de l'interaction avec la gp130, et (iv) en l'absence de l'interaction avec le récepteur de LIF. L'invention concerne également des procédés de dérivation et de propagation des cellules ES, des cellules ES elles-mêmes et des lignées cellulaires utilisées dans la dérivation et le dosage de nouveaux facteurs.
PCT/GB1997/000426 1996-02-16 1997-02-14 Cytokine exprimee par des cellules souches embryonnaires a deficience dia/lif pour l'inhibition de la differenciation WO1997030151A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU18027/97A AU1802797A (en) 1996-02-16 1997-02-14 Cytokine expressed by dia/lif-deficient embryonic stem cells for the inhibition of differentiation
JP9529116A JP2000505294A (ja) 1996-02-16 1997-02-14 分化の阻害のためのｄｉａ／ｌｉｆ―欠損胚幹細胞により発現されるサイトカイン
EP97903477A EP0880584A1 (fr) 1996-02-16 1997-02-14 Cytokine exprimee par des cellules souches embryonnaires a deficience dia/lif pour l'inhibition de la differenciation

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9603244.6 1996-02-16
GBGB9603244.6A GB9603244D0 (en) 1996-02-16 1996-02-16 Novel cytokine
GBGB9611319.6A GB9611319D0 (en) 1996-05-31 1996-05-31 Novel cytokine
GB9611319.6 1996-05-31

Publications (1)

Publication Number Publication Date
WO1997030151A1 true WO1997030151A1 (fr) 1997-08-21

Family

ID=26308723

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/000426 WO1997030151A1 (fr) 1996-02-16 1997-02-14 Cytokine exprimee par des cellules souches embryonnaires a deficience dia/lif pour l'inhibition de la differenciation

Country Status (5)

Country Link
EP (1) EP0880584A1 (fr)
JP (1) JP2000505294A (fr)
AU (1) AU1802797A (fr)
CA (1) CA2246712A1 (fr)
WO (1) WO1997030151A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999027076A1 (fr) * 1997-11-25 1999-06-03 Arc Genomic Research Cellules souches embryonnaires multipotentes et procedes permettant de les obtenir
WO2000015764A2 (fr) * 1998-09-11 2000-03-23 University Of Edinburgh Proliferation et/ou obtention de cellules souches embryonnaires
US6642048B2 (en) 2000-01-11 2003-11-04 Geron Corporation Conditioned media for propagating human pluripotent stem cells
US6800480B1 (en) 1997-10-23 2004-10-05 Geron Corporation Methods and materials for the growth of primate-derived primordial stem cells in feeder-free culture
US7297539B2 (en) 2000-01-11 2007-11-20 Geron Corporation Medium for growing human embryonic stem cells
US7455983B2 (en) 2000-01-11 2008-11-25 Geron Corporation Medium for growing human embryonic stem cells
US7687266B2 (en) 2002-01-30 2010-03-30 University Of Edinburgh Pluripotency determining factors and uses thereof
WO2014130706A1 (fr) * 2013-02-20 2014-08-28 Regeneron Pharmaceuticals, Inc. Modification génétique de rats
US9546384B2 (en) 2013-12-11 2017-01-17 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a mouse genome
US10385359B2 (en) 2013-04-16 2019-08-20 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
RU2774076C2 (ru) * 2013-02-20 2022-06-15 Регенерон Фармасютикалс, Инк. Генетическая модификация крыс

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990002183A1 (fr) * 1988-08-18 1990-03-08 Genetics Institute, Inc. Production d'une lymphokine nouvelle presentant une activite inhibitrice de la differenciation
JPH05304951A (ja) * 1992-04-06 1993-11-19 N T Sci:Kk 初期胚及び胚性幹細胞の培養液
JPH0751060A (ja) * 1993-08-11 1995-02-28 Tosoh Corp 胚性幹細胞の培養液
JPH08154681A (ja) * 1994-12-01 1996-06-18 Ito Ham Kk ウシｌｉｆ 遺伝子

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990002183A1 (fr) * 1988-08-18 1990-03-08 Genetics Institute, Inc. Production d'une lymphokine nouvelle presentant une activite inhibitrice de la differenciation
JPH05304951A (ja) * 1992-04-06 1993-11-19 N T Sci:Kk 初期胚及び胚性幹細胞の培養液
JPH0751060A (ja) * 1993-08-11 1995-02-28 Tosoh Corp 胚性幹細胞の培養液
JPH08154681A (ja) * 1994-12-01 1996-06-18 Ito Ham Kk ウシｌｉｆ 遺伝子

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 9351, Derwent World Patents Index; Class B04, AN 93-408317, XP002031783 *
DATABASE WPI Section Ch Week 9517, Derwent World Patents Index; Class B04, AN 95-127351, XP002031782 *
DATABASE WPI Section Ch Week 9634, Derwent World Patents Index; Class B04, AN 96-336571, XP002031784 *
S. MACLEAN-HUNTER ET AL.: "Expression of a RLF/L-MYC minigene inhibits differentiation of embryonic stem cells and embroid body formation", ONCOGENE, vol. 9, no. 12, December 1994 (1994-12-01), pages 3509 - 3517, XP000673923 *
THOMSON J A ET AL: "ISOLATION OF A PRIMATE EMBRYONIC STEM CELL LINE", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 92, no. 17, August 1995 (1995-08-01), pages 7844 - 7848, XP000604992 *

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6800480B1 (en) 1997-10-23 2004-10-05 Geron Corporation Methods and materials for the growth of primate-derived primordial stem cells in feeder-free culture
US7413902B2 (en) 1997-10-23 2008-08-19 Geron Corporation Feeder-free culture method for embryonic stem cells or primate primordial stem cells
WO1999027076A1 (fr) * 1997-11-25 1999-06-03 Arc Genomic Research Cellules souches embryonnaires multipotentes et procedes permettant de les obtenir
US7371573B2 (en) 1998-09-11 2008-05-13 University Of Edinburgh Propagation and/or derivation of embryonic stem cells
WO2000015764A2 (fr) * 1998-09-11 2000-03-23 University Of Edinburgh Proliferation et/ou obtention de cellules souches embryonnaires
WO2000015764A3 (fr) * 1998-09-11 2000-06-29 Univ Edinburgh Proliferation et/ou obtention de cellules souches embryonnaires
US7595193B2 (en) 1998-09-11 2009-09-29 University Of Edinburgh Propagation and/or derivation of embryonic stem cells
US6875608B1 (en) 1998-09-11 2005-04-05 University Of Edinburgh Propagation and/or derivation of embryonic stem cells
US8637311B2 (en) 1998-10-23 2014-01-28 Asterias Biotherapeutics, Inc. Human embryonic stem cells genetically modified to contain a nucleic acid and cultured with fibroblast growth factor
US8951800B2 (en) 1998-10-23 2015-02-10 Asterias Biotherapeutics, Inc. Primate pluripotent stem cell expansion without feeder cells and in the presence of FGF and matrigel or Engelbreth-Holm-Swarm tumor cell preparation
US10059939B2 (en) 1998-10-23 2018-08-28 Asterias Biotherapeutics, Inc. Screening methods for human embryonic stem cells
US8097458B2 (en) 1998-10-23 2012-01-17 Geron Corporation Micro-carrier culture system for rapid expansion of human embryonic stem cells
US20120149025A1 (en) * 1998-10-23 2012-06-14 Geron Corporation Screening Methods for Human Embryonic Stem Cells
US7041438B2 (en) 2000-01-11 2006-05-09 Geron Corporation Use of human embryonic stem cells for drug screening and toxicity testing
US7455983B2 (en) 2000-01-11 2008-11-25 Geron Corporation Medium for growing human embryonic stem cells
US6667176B1 (en) 2000-01-11 2003-12-23 Geron Corporation cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells
US7297539B2 (en) 2000-01-11 2007-11-20 Geron Corporation Medium for growing human embryonic stem cells
US6642048B2 (en) 2000-01-11 2003-11-04 Geron Corporation Conditioned media for propagating human pluripotent stem cells
US7410798B2 (en) 2001-01-10 2008-08-12 Geron Corporation Culture system for rapid expansion of human embryonic stem cells
US7687266B2 (en) 2002-01-30 2010-03-30 University Of Edinburgh Pluripotency determining factors and uses thereof
US20100144039A1 (en) * 2002-01-30 2010-06-10 The University Of Edinburgh Pluripotency determining factors and uses thereof
WO2014130706A1 (fr) * 2013-02-20 2014-08-28 Regeneron Pharmaceuticals, Inc. Modification génétique de rats
KR20150119384A (ko) * 2013-02-20 2015-10-23 리제너론 파마슈티칼스 인코포레이티드 랫트의 유전자 변형
CN105121631A (zh) * 2013-02-20 2015-12-02 瑞泽恩制药公司 大鼠的遗传修饰
CN109913495B (zh) * 2013-02-20 2022-11-25 瑞泽恩制药公司 大鼠的遗传修饰
RU2774076C2 (ru) * 2013-02-20 2022-06-15 Регенерон Фармасютикалс, Инк. Генетическая модификация крыс
CN105121631B (zh) * 2013-02-20 2019-04-19 瑞泽恩制药公司 大鼠的遗传修饰
RU2690352C2 (ru) * 2013-02-20 2019-05-31 Регенерон Фармасютикалс, Инк. Генетическая модификация крыс
CN109913495A (zh) * 2013-02-20 2019-06-21 瑞泽恩制药公司 大鼠的遗传修饰
US10329582B2 (en) 2013-02-20 2019-06-25 Regeneron Pharmaceuticals, Inc. Genetic modification of rats
US10894965B2 (en) 2013-02-20 2021-01-19 Regeneron Pharmaceuticals, Inc. Genetic modification of rats
KR102209979B1 (ko) 2013-02-20 2021-02-01 리제너론 파마슈티칼스 인코포레이티드 랫트의 유전자 변형
US10385359B2 (en) 2013-04-16 2019-08-20 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
US10975390B2 (en) 2013-04-16 2021-04-13 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
US10711280B2 (en) 2013-12-11 2020-07-14 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a mouse ES cell genome
US10208317B2 (en) 2013-12-11 2019-02-19 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a mouse embryonic stem cell genome
US9546384B2 (en) 2013-12-11 2017-01-17 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a mouse genome
US11820997B2 (en) 2013-12-11 2023-11-21 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a genome

Also Published As

Publication number Publication date
JP2000505294A (ja) 2000-05-09
AU1802797A (en) 1997-09-02
EP0880584A1 (fr) 1998-12-02
CA2246712A1 (fr) 1997-08-21

Similar Documents

Publication Publication Date Title
AU717529B2 (en) Cultured inner cell mass cell lines derived from ungulate embryos
Pease et al. Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)
Zhang et al. CRISPR/Cas9‐mediated sheep MSTN gene knockout and promote sSMSCs differentiation
Shen et al. Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro.
EP0695351B1 (fr) Isolation, selection et propagation de cellules souches d'animaux transgeniques
US6603059B1 (en) Method of cloning animals
JP2023026679A (ja) ヒストンｈ３－リジントリメチル化を除去することによって体細胞核移入（ｓｃｎｔ）効率を増加させるための方法および組成物
JP2002511732A (ja) トランスジェニック動物を発生させるための組成物および方法
AU2002330465B2 (en) Screening method for a reprogramming agent, reprogramming agent screened by the method, method for using the reprogramming agent, method for differentiating an undifferentiated fusion cell, and production method of cells, tissues and organs.
Wiley et al. Epidermal growth factor receptor function in early mammalian development
JP2003503071A (ja) 脱分化レシピエント細胞への細胞質移入
WO1998039416A1 (fr) Methode de clonage d'animaux
US9012223B2 (en) Methods for enhancing genome stability and telomere elongation in embryonic stem cells
Mountford et al. Maintenance of pluripotential embryonic stem cells by stem cell selection
WO2003078967A2 (fr) Selection et differentiation de cellules souches
WO1997030151A1 (fr) Cytokine exprimee par des cellules souches embryonnaires a deficience dia/lif pour l'inhibition de la differenciation
JP2004516834A (ja) 胸腺上皮前駆細胞およびその使用
US20030032177A1 (en) Cell maintenance and proliferation
WO2008027241A2 (fr) Lignées de cellules souches embryonnaires non humaines et animaux transgéniques dérivés de celles-ci
JP2001224367A (ja) 不死化血管周皮細胞株
Newman-Smith Peri-implantation mouse development
Edwards Carnegie Stage Comment 1 Maternal mRNA transcribed 2 Embryonic mRNA transcribed Transcripts of HCGB
AU2002301559A1 (en) Cell maintenance and proliferation
MXPA98008103A (en) Cellular lines of internal cellular mass cultivated derivatives of embryos ungula
MXPA99008933A (es) Linea de celulas germinales embrionicas, humanas y metodos de uso

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 331127

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 1997903477

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2246712

Country of ref document: CA

Ref country code: CA

Ref document number: 2246712

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1997903477

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1997903477

Country of ref document: EP