[go: up one dir, main page]

WO1994007997A1 - Proliferation a long terme de cellules germinales primordiales - Google Patents

Proliferation a long terme de cellules germinales primordiales Download PDF

Info

Publication number
WO1994007997A1
WO1994007997A1 PCT/US1993/009536 US9309536W WO9407997A1 WO 1994007997 A1 WO1994007997 A1 WO 1994007997A1 US 9309536 W US9309536 W US 9309536W WO 9407997 A1 WO9407997 A1 WO 9407997A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
culture
animal
primordial germ
factor
Prior art date
Application number
PCT/US1993/009536
Other languages
English (en)
Inventor
Peter J. Donovan
James L. Resnick
Original Assignee
The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services
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
Application filed by The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services filed Critical The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services
Priority to AU61363/94A priority Critical patent/AU6136394A/en
Publication of WO1994007997A1 publication Critical patent/WO1994007997A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • C12N5/0608—Germ cells
    • C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the progenitors of the germ line arise extragonadally and move to the developing gonad early in embryonic development. Entry of the PGCs into the gonad results in several well characterized changes in the germ cell. In the male, PGCs enter mitotic arrest, whereas in the female they enter the first meiotic prophase. Loss of a number of antigenic determinants from the germ cell surface also occurs in both sexes. The decreasing ability of successively older embryonic mouse gonads to generate germ cell derived tumors (teratomas) when grafted into the testis capsule of syngeneic adult hosts is also suggestive of major changes in germ cell phenotype.
  • primordial germ cells can be identified histochemically, due to their high content of alkaline phosphatase, at 8.5 days post coitum (dpc) at the root of the allantois. At subsequent developmental stages they are found in the hindgut (9.5 dpc), the hindgut mesentery (9.5-1 1.5 dpc), and the dorsal body wall (from 10.5 dpc). By 12.5 dpc the PGCs are largely confined to the developing gonad. PGC ultrastructure, and the movements of cells identified as PGCs in squash preparations suggest that migration to the gonad involves active locomotion.
  • PGCs do adhere to outgrowths of embryonic somatic cells, and furthermore, isolated PGCs adhere to embryo-derived cell lines.
  • Murine primordial germ cells are first identifiable as a population of approximately eight alkaline phosphatase (APase) positive cells in the 7.0 days post coitum embryo. During the subsequent six days of development they proliferate to give rise to the approximately 25,000 cells that establish the meiotic population. Steel factor (SLF) is required for PGC survival both in vivo (Bennett, D., ).
  • APF alkaline phosphatase
  • One embodiment of the present invention is a method of culturing primordial germ cells from an animal in vitro for a period of time longer than seven days.
  • This method comprises growing the primordial germ cells in the presence of a fibroblast growth factor and leukemia inhibitory factor.
  • the fibroblast growth factor most preferably is basic fibroblasf growth factor (bFGF).
  • the primordial germ cells can advantageously be grown in the presence of steel factor, and most preferably, the primordial germ cells are grown in the presence of both soluble steel factor and membrane-bound steel factor.
  • a further preferred embodiment of the present invention includes growing the primordial germ cells on a layer of feeder cells, with the feeder cells advantageously being STO cells.
  • the feeder cells can provide the source of steel factor, and preferably provide a source of both soluble steel factor and membrane-bound steel factor.
  • the feeder cells are mitotically inactive fibroblasts.
  • these fibroblasts are murine STO cells or an analogue thereof from a non- murine animal.
  • the steel factor, fibroblast growth factor and leukemia inhibitory factor are from the same animal as the primordial germ cells.
  • these factors can most advantageously be derived from an animal more highly evolved than the animal from which the primordial germ cells are derived.
  • the primordial germ cells are isolated by dissection from embryos and the animal is selected from the group consisting of a chicken, a cow, a sheep and a pig.
  • the primordial germ cells can be derived from a mouse.
  • the primordial germ cells can also be advantageously grown on feeder cells to form a culture. Additionally the growth of these primordial germ cells can comprise splitting the culture and growing the primordial germ cells on fresh feeder cells.
  • the primordial germ cells are grown in the presence of cholera toxin.
  • a different aspect of the present invention includes a culture of primordial germ cells that will grow for a period longer than seven days. Most preferably the culture will grow for a period longer than fourteen days. Even more preferably the culture is a continuous culture that will grow for an indefinite period.
  • An even more preferable culture can advantageously comprise basic fibroblast growth factor, and even more preferably the culture additionally comprises steel factor.
  • the culture can most preferably comprise both soluble steel factor and membrane-bound steel factor, and even still more preferably can also comprise leukemia inhibitory factor.
  • the culture can advantageously include the primordial germ cells on a layer of feeder cells, while the feeder cells are most advantageously mitotically inactive fibroblasts.
  • Yet another embodiment of the present invention is a method of producing a chimeric animal, including growing primordial germ cells in the presence of a fibroblast growth factor and leukemia inhibitory factor to form a primordial germ cell culture, isolating one or more cells from the culture, inserting the one or more cells into a developing embryo, and growing ⁇ an animal from the embryo.
  • One preferable embodiment of this embodiment includes the animal being selected from the group consisting of a cow, a pig, a chicken, a mouse and a sheep.
  • Still another embodiment of the present invention is a method of producing a transgenic animal, including growing primordial germ cells having a given genotype in the presence of a fibroblast growth factor and leukemia inhibitory factor to form a primordial germ cell culture, isolating one or more cells from the culture, inserting the one or more cells into a developing embryo, growing an animal from the embryo, wherein the one Or more cells contribute to the germline of the animal, and allowing the animal to reproduce wherein the given genotype is present in the resulting offspring.
  • One preferred embodiment of this method includes the animal being selected from the group consisting of a cow, a pig, a mouse and a sheep.
  • a further preferred embodiment of the present invention includes inserting one or more cells into a sterile animal with no germ cells. In this preferred embodiment, the injection is advantageously into one or more cells of a post-implantation embryo.
  • An even more preferred embodiment includes injecting through the uterus of the animal in which the previous growing step occurs. Description of the Figures
  • FIG. 1 Analysis of PGC proliferation in culture. The effect of mLIF concentration on PGC proliferation in culture. The number of PGCs present after 1 and 3 days of culture on confluent STO cell feeder layers. Bars represent the mean plus/minus the standard deviation of five replicate cultures. Bars represent the mean plus/minus the standard deviation of five replicate cultures. Each experiment was done four times.
  • FIG. 1 The effect of bFGF concentration on PGC Proliferation in culture.
  • fibroblast growth factor and especially basic Fibroblast Growth Factor (bFGF) stimulates PGC proliferation in vitro.
  • bFGF in the presence of Steel Factor (SLF) and Leukemia Inhibitory Factor (LIF), stimulates long-term proliferation of PGCs leading to the derivation of large colonies of cells. These colonies of cells (we term embryonic germ (EG) cells) resemble embryonic stem (ES) cells.
  • ES cells are pluripotent cells derived from pre-implantation embryos or feeder-dependent embryonal carcinoma (EC) cells. These embryonal carcinoma cells are pluripotent stem cells from PGC-derived tumors (teratomas)
  • Our discovery provides the first system for long-term culture of PGCs or PGC-derived cell lines. We have previously shown that the transmembrane form of Steel Factor is required for
  • STO cells are mitotically inactive fibroblast cells; such cells, or analgous cells, can be isolated from a wide variety of animals. Studies by Hogan and her colleagues have shown that this factor is probably LIF. Nature, 353:750-752 (1991). LIF exists as both a diffusible form (LIF-D) and a matrix-associated form (LIF-M). Although STO cells produce LIF, we have found that addition of recombinant, murine LIF (mLIF) to the culture medium enhances murine PGC proliferation on STO cells in a dose-dependent manner, with a peak response around 1000 units/ml (see Figure 1). STO cells also produce steel factor in at least two forms: a soluble form and a membrane-bound form.
  • Example 1 Example 1
  • Embryos were derived from matings of the outbred mouse strain MF1 (OLAC), the finding of a vaginal plug was denoted day 0 of pregnancy.
  • Germ-cell-containing fragments of embryos were isolated by sterile dissection in Ca + + /Mg + + -free phosphate buffered saline (PBS), washed in several changes of PBS, and triturated repeatedly in a small volume of PBS to yield a single-cell suspension. Small aliquots of this germ-cell-containing suspension were added to preformed STO feeder layers at one embryo equivalent per feeder layer and allowed to attach for 16 hr.
  • PBS Ca + + /Mg + + + -free phosphate buffered saline
  • STO cells were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), glutamine (4 Mm), penicillin, and streptomycin (DME). The cells were harvested just prior to confluence by trypsinization (0.05% trypsin, 0.02% EDTA in PBS), washed, and irradiated (600 rads) in a cesium gamma source. Irradiated STO cells were adjusted to 5 x 10 4 cells per ml in DME and 200 ⁇ per well were added to 96 well plates (Costar) or 2 ml to 30 mm diameter tissue culture petri dishes.
  • FCS fetal calf serum
  • DME penicillin
  • DME streptomycin
  • Example 2 Because of its ability to stimulate both LIF-D and LIF-M production by feeder cells, we analyzed the effect of bFGF alone on PGC proliferation, as in Example 2.
  • Example 2 Because of its ability to stimulate both LIF-D and LIF-M production by feeder cells, we analyzed the effect of bFGF alone on PGC proliferation, as in Example 2.
  • both LIF and bFGF stimulated PGC proliferation but the number of viable PGCs continued to decline after 5 days of incubation on STO cells. This result was consistent with our previous observations which showed that PGCs could not be maintained in culture for longer than 7 days.
  • bFGF could increase the lifespan of PGCs in vitro
  • PGCs were identified by alkaline phosphatase histochemistry. Briefly, histochemical and immunocytochemical staining was carried out on tissue fixed for 1 hr, or cells fixed for 15 min, at room temperature in 4% para-formaldehyde in PBS. Alkaline phosphatase staining was performed at room temperature in 25 Mm Tris-maleate buffer (pH 9.0) containing 0.15 M NaCI.
  • Tissue blocks, frozen sections, or cell cultures were incubated for 15 min in a solution of 0.4 mg ml sodium alpha naphthyl phosphate, 1 mg/ml Fast Red TR salt, and 4 mM MgCI 2 .
  • Enzyme activity was localized by the appearance of a red reaction product which was inhibited, in control experiments, by the presence of 1 mM levamisole, a specific inhibitor of the enzyme.
  • PGCs identified by APase activity one day after isolation from 8.5 dpc embryos and plating onto STO cell feeder layers in bFGF and LIF. PGCs existed as single cells, with no evidence of clumps or cell colonies. b. Three days after isolation, PGCs were beginning to form small colonies, either by clonal division or aggregation. Some single PGCs were still identifiable. c. Five days after isolation, PGCs were found in increasingly larger colonies. Many small colonies were found clustered near each other. d. Small colony of EG cells growing as a cellular monolayer on top of the STO feeder layer. e. Two colonies of EG cells. The smaller colony was growing as a monolayer, while the large colony had become multilayered. f. Large, several hundred micrometer, multilayered EG cell colony identified 9 days after plating PGCs onto confluent STO cell feeder layers.
  • PGCs demonstrate APase activity from 7.0 dpc to 15.5 dpc.
  • Another classical marker of PGCs is the carbohydrate differentiation antigen SSEA-1 , which is expressed on the PGC surface from 8.5 dpc to 14.5 dpc (Fox N., et al., Dev. Biol., 83, 391-398 (1981 ))
  • Ascitic fluids of the two monoclonal antibodies were used at 1 :100 dilution and the rhodamine-labeled rabbit anti-mouse second antibody (Nordic) at 1 :50.
  • PGCs Cultures of PGCs were established as described above. For immunocytochemistry protocols, cultures were fixed with 4% para-formaldehyde in phosphate buffered saline (PBS), washed three times with PBS, and then incubated with primary (anti-SSEA-1) antibody (diluted in 10% heat inactivated normal goat serum) for 1 hour at room temperature. The cultures were then washed three times with PBS and incubated with the peroxidase-conjugated secondary (goat anti-mouse polyvalent immunoglobulin 1 :50 dilution, NORDIC) antibody f ⁇ r 30 minutes at room temperature.
  • PBS phosphate buffered saline
  • the first and second antibodies were applied for 1 hr after blocking nonspecific binding sites with 10% heat inactivated normal rabbit serum in 4% bovine serum albumin in PBS. Extensive washing with PBS followed each antibody application. Positive antibody staining was identified by peroxidase histochemistry using DAB, hydrogen peroxide, cobalt and nickel chloride. Positive cells were stained dark brown in color.
  • SF and bFGF relate to normal PGCs in vivo. While not wishing -to be bound by any particular explanation for this effect, three possibilities seem likely: (1) EG cells are simply proliferating
  • EG cells are PGCs that have become immortalized in culture; or (3) EG cells are a differentiated or de-differentiated derivative of PGCs. Regardless of whichever of these possibilities proves to be correct these cells represent an invaluable reagent for the analysis of
  • PGC development and can be used to study such key issues as germ line imprinting, recombination, sexual differentiation and pluripotency.
  • ES and EC cells Two other cell types that have APase activity and express the SSEA-1 antigen are pluripotent ES and EC cells. Interestingly, as discussed above, large colonies of EG cells closely resemble feeder-dependent EC cells or ES cells grown on STO feeder layers. How EG cells are related to EC cells or ES cells remains to be determined.
  • the animal On the fourth day, the animal was given prostaglandin F2 ⁇ as Estrumate (Cloprostenol); 4.0 ml in the morning and 2.0 ml in the afternoon.
  • the animal On the fifth day, the animal was in heat and was mated with two Simmental bulls. Thirty-five days later the animal was examined by ultrasound to confirm the pregnancy, to count the number of embryos and to determine their viability.
  • the paralumbar fossa of the animal was clipped and surgically prepped. Local anaesthetic was Lidocaine. The abdominal cavity was entered surgically and the uterine horn was exteriorized. Each embryo was located and isolated surgically.
  • the embryos were placed into cooled phosphate buffered saline (PBS) and the genital ridges were identified and dissected out.
  • the genital ridges were dissociated in trypsin/EDTA and the resultant cell suspension was plated onto confluent monolayers of irradiated STO cells.
  • Cultures were fed in DMEM plus 15% fetal bovine serum, Na pyruvate, L-glutamine, penicillin and streptomycin with added bFGF and LIF. To some of these cultures, cholera toxin was also added. Separate cultures were established containing either mouse or human recombinant LIF.
  • PGCs primordial germ cells
  • cholera toxin was a potent germ cell mitogen.
  • both the recombinant human and murine LIF functioned effectively.
  • native human LIF functions effectively in bovine cells while native murine LIF will function much less effectively.
  • STO cells from the same animal as the primordial germ cells will serve as effective feeder cells, while STO cells or their analogues from an animal less highly evolved will not function as well.
  • STO cells from a more highly evolved animal will provide effective continuous culture.
  • STO cells or quite similar analogs can be derived from a very large number of animals and can function as effective feeder cells for cultures of primordial germ cells from many such animals.
  • bFGF or LIF are expressed in the developing gonad or whether PGCs express an FGF-R or a LIF receptor remain to be determined. Nevertheless, the ability of bFGF and LIF to stimulate long-term PGC proliferation provides a unique tool for the analysis of PGC development and for the creation of chimeric an transgenic animals.
  • Chimeric and transgenic animals of many sorts can be made from these cell lines.
  • primordial germ cells are grown in the presence of a fibroblast growth factor and leukemia inhibitory factor as described above to form a primordial germ cell culture.
  • a fibroblast growth factor and leukemia inhibitory factor as described above to form a primordial germ cell culture.
  • these factors are present throughout a wide range of animals, including the pigs, chickens, cows, sheep and mice discussed above. We have found that these factors work best if they are from the same animal as the animal from which the cells are derived or if the factors are from an animal more highly evolved than the animal from which the cells are derived.
  • the human factors will function effectively with cells from most animals.
  • the murine factors will function effectively only with cells derived from mice.
  • One or more cells from the culture are then isolated and inserted into a developing embryo of the same animal as the cells from which the cells are derived.
  • the blastocysts or other suitable stage embryos can be used.
  • a whole chimeric animal can then be derived from this embryo by growing the embryo in utero.
  • Transgenic animals can also be created by following the procedure described above for creating a chimeric animal.
  • the one or more cells from the culture should contribute to the germline of the resulting animal.
  • the genotype of the primordial germ cells is present in the resulting offspring.
  • the cells can be inserted, such as through sterile injection, into a sterile embryo lacking germ cells.
  • the cells from the culture can also be inserted into a post-implantation embryo, such as through the uterus of the animal in which the embryo is growing.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Husbandry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de culture in vitro de cellules germinales primordiales animales sur une période de plus de sept jours. Ce procédé consiste à mettre en culture des cellules germinales primordiales en présence d'un facteur de croissance fibroblastique et d'un facteur inhibiteur de la leucémie. L'invention porte également sur des cultures cellulaires obtenues selon ledit procédé et des animaux chimères produits à l'aide de ces cultures.
PCT/US1993/009536 1992-10-06 1993-10-06 Proliferation a long terme de cellules germinales primordiales WO1994007997A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU61363/94A AU6136394A (en) 1992-10-06 1993-10-06 Long-term proliferation of primordial germ cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US95800992A 1992-10-06 1992-10-06
US07/958,009 1992-10-06

Publications (1)

Publication Number Publication Date
WO1994007997A1 true WO1994007997A1 (fr) 1994-04-14

Family

ID=25500481

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/009536 WO1994007997A1 (fr) 1992-10-06 1993-10-06 Proliferation a long terme de cellules germinales primordiales

Country Status (2)

Country Link
AU (1) AU6136394A (fr)
WO (1) WO1994007997A1 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995010599A1 (fr) * 1993-10-15 1995-04-20 The University Of Melbourne Cellules analogues a des cellules souches embryonnaires
WO1997025412A1 (fr) * 1996-01-09 1997-07-17 The Regents Of The University Of California Cellules embryonnaires d'ongules, du type cellules souches, procedes de production et d'utilisation de ces cellules aux fins de la fabrication d'ongules transgeniques
WO1998016630A1 (fr) * 1996-10-11 1998-04-23 The Texas A & M University System Techniques permettant la production de cellules sexuelles primordiales et d'especes animales transgeniques
WO1998039416A1 (fr) * 1997-03-06 1998-09-11 Infigen, Inc. Methode de clonage d'animaux
US6194635B1 (en) 1996-01-09 2001-02-27 The Regents Of The University Of California Embryonic germ cells, method for making same, and using the cells to produce a chimeric porcine
US6245566B1 (en) 1997-03-31 2001-06-12 The Johns Hopkins University School Of Medicine Human embryonic germ cell line and methods of use
EP0855441A3 (fr) * 1996-12-27 2002-04-17 National Agricultural Research Organization Isolement et purification de cellules souches germinales au moyen de particules magnétiques fonctionnant comme des anticorps et méthode pour l'établissement de lignées de cellules souches
US6395958B1 (en) 1997-03-06 2002-05-28 Infigen, Inc. Method of producing a polypeptide in an ungulate
US6635802B1 (en) 2000-01-10 2003-10-21 The Texas A&M University System Nuclear transfer using cells cultured in serum starvation media containing apoptosis inhibitors
US6667176B1 (en) 2000-01-11 2003-12-23 Geron Corporation cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells
WO2004094627A1 (fr) * 2003-04-21 2004-11-04 Seoul National University Industry Foundation Procede d'amelioration de l'efficacite de transmission d'une lignee germinale de cellules germinales primordiales aviaires
US7129084B2 (en) 2000-08-03 2006-10-31 Therapeutic Human Polyclonals, Inc. Production of humanized antibodies in transgenic animals
WO2006135824A1 (fr) * 2005-06-10 2006-12-21 Irm Llc Composes maintenant le caractere pluripotent de cellules souches embryonnaires
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
US9074181B2 (en) 2005-06-22 2015-07-07 Asterias Biotherapeutics, Inc. Suspension culture of human embryonic stem cells
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals
US12441983B2 (en) 2024-01-24 2025-10-14 Asterias Biotherapeutics, Inc. Suspension culture of human embryonic stem cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7958009B1 (en) * 2000-03-20 2011-06-07 Amazon.Com, Inc. Method and system for bidding on multiple auctions

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7958009B1 (en) * 2000-03-20 2011-06-07 Amazon.Com, Inc. Method and system for bidding on multiple auctions

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 9319, Derwent World Patents Index; Class B04, AN 93-159486, DONOVAN ET AL: "LONG TERM CULTURE OF PRIMORDIAL GERM CELLS-IN PRESENCE OF FIBROBLAST GROWTH FACTOR AND LEUKAEMIA INHIBITORY FACTOR,USEFUL IN RESEARCH AND PRODN.OF CHIMERIC AND TRANSGENIC ANIMALS" *
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.79173363 & J CELL PHYSIOL, (1979 MAR) 98 (3) 469-74 *
FILE SERVER STN KARLSRUHE,FILE MEDLINE ABSTRACT NO.85203765 & EXP LUNG RES, (1985) 8 (1) 53-66 *
MATSUI ET AL: "DERIVATION OF PLURIPOTENTIAL EMBRYONIC STEM CELLS FROM MURINE PRIMORDIAL GERM CELLS IN CULTURE", CELL, vol. 70, 4 September 1992 (1992-09-04), CAMBRIDGE,MASS.,USA, pages 841 - 847 *
RESNICK ET AL: "LONG-TERM PROLIFERATION OF MOUSE PRIMORDIAL GERM CELLS IN CULTURE", NATURE, vol. 359, 8 October 1992 (1992-10-08), LONDON,GB, pages 550 - 551 *

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995010599A1 (fr) * 1993-10-15 1995-04-20 The University Of Melbourne Cellules analogues a des cellules souches embryonnaires
US6194635B1 (en) 1996-01-09 2001-02-27 The Regents Of The University Of California Embryonic germ cells, method for making same, and using the cells to produce a chimeric porcine
WO1997025412A1 (fr) * 1996-01-09 1997-07-17 The Regents Of The University Of California Cellules embryonnaires d'ongules, du type cellules souches, procedes de production et d'utilisation de ces cellules aux fins de la fabrication d'ongules transgeniques
US6369294B1 (en) 1996-10-11 2002-04-09 Texas A&M University System Methods comprising apoptosis inhibitors for the generation of transgenic pigs
WO1998016630A1 (fr) * 1996-10-11 1998-04-23 The Texas A & M University System Techniques permettant la production de cellules sexuelles primordiales et d'especes animales transgeniques
US6271436B1 (en) 1996-10-11 2001-08-07 The Texas A & M University System Cells and methods for the generation of transgenic pigs
EP0855441A3 (fr) * 1996-12-27 2002-04-17 National Agricultural Research Organization Isolement et purification de cellules souches germinales au moyen de particules magnétiques fonctionnant comme des anticorps et méthode pour l'établissement de lignées de cellules souches
US6395958B1 (en) 1997-03-06 2002-05-28 Infigen, Inc. Method of producing a polypeptide in an ungulate
WO1998039416A1 (fr) * 1997-03-06 1998-09-11 Infigen, Inc. Methode de clonage d'animaux
US6603059B1 (en) 1997-03-06 2003-08-05 Infigen, Inc. Method of cloning animals
US6331406B1 (en) 1997-03-31 2001-12-18 The John Hopkins University School Of Medicine Human enbryonic germ cell and methods of use
US6562619B1 (en) 1997-03-31 2003-05-13 The Johns Hopkins University School Of Medicine Differentiation of human embryonic germ cells
US6245566B1 (en) 1997-03-31 2001-06-12 The Johns Hopkins University School Of Medicine Human embryonic germ cell line and methods of use
US8097458B2 (en) 1998-10-23 2012-01-17 Geron Corporation Micro-carrier culture system for rapid expansion of human embryonic stem cells
US10059939B2 (en) 1998-10-23 2018-08-28 Asterias Biotherapeutics, Inc. Screening methods for human embryonic stem cells
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
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
US6635802B1 (en) 2000-01-10 2003-10-21 The Texas A&M University System Nuclear transfer using cells cultured in serum starvation media containing apoptosis inhibitors
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
US7455983B2 (en) 2000-01-11 2008-11-25 Geron Corporation Medium for growing 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
US7129084B2 (en) 2000-08-03 2006-10-31 Therapeutic Human Polyclonals, Inc. Production of humanized antibodies in transgenic animals
US7476776B2 (en) 2003-04-21 2009-01-13 Seoul National University Industry Foundation Method for improving germline transmission efficiency of avian primordial germ cells
WO2004094627A1 (fr) * 2003-04-21 2004-11-04 Seoul National University Industry Foundation Procede d'amelioration de l'efficacite de transmission d'une lignee germinale de cellules germinales primordiales aviaires
WO2006135824A1 (fr) * 2005-06-10 2006-12-21 Irm Llc Composes maintenant le caractere pluripotent de cellules souches embryonnaires
US9074181B2 (en) 2005-06-22 2015-07-07 Asterias Biotherapeutics, Inc. Suspension culture of human embryonic stem cells
US10676714B2 (en) 2005-06-22 2020-06-09 Asterias Biotherapeutics, Inc. Suspension culture of human embryonic stem cells
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals
US12441983B2 (en) 2024-01-24 2025-10-14 Asterias Biotherapeutics, Inc. Suspension culture of human embryonic stem cells

Also Published As

Publication number Publication date
AU6136394A (en) 1994-04-26

Similar Documents

Publication Publication Date Title
CA1341469C (fr) Culture in vitro de cellules souches embryonnaires
WO1994007997A1 (fr) Proliferation a long terme de cellules germinales primordiales
Eistetter Pluripotent Embryonal Stem Cell Lines Can Be Established from Disaggregated Mouse Morulae: (mouse embryonal stem (ES) cells/disaggregated morulae/in vitro development/teratoma)
US7186883B2 (en) In vitro propagation of embryonic stem cells
Marques-Mari et al. Differentiation of germ cells and gametes from stem cells
US9434922B2 (en) Feeder-free method for culture of bovine and porcine spermatogonial stem cells
Park et al. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos
US6190910B1 (en) Mouse embryonic stem cell lines
US20050193435A1 (en) Prolonged culturing of avian (chicken) primordial germ cells (PGCs) using specific growth factors, use thereof to produce chimeric avians (chickens)
CA2250834A1 (fr) Lignees de cellules a bouton embryonnaire cultive, derivees d'embryons d'ongules
IL134366A (en) PRODUCTION OF AVIAN EMBRYONIC GERM (EG) CELL LINES BY PROLONGED CULTURING OF PGCs AND USE THEREOF FOR CLONING AND CHIMERIZATION
WO1995016770A1 (fr) Cellules souches embryonnaires derivees de cellules du stade preblastocyste d'ongule et leur utilisation pour produire des ongules chimeres et transgeniques clones
US20050132426A1 (en) Long-term cell culture compositions and genetically modified animals derived therefrom
CA2192982A1 (fr) Cellule eg d'ongule
US20050244958A1 (en) Production of avian embryonic germ (EG) cell lines by prolonged culturing of PGCs, use thereof for cloning and chimerization
Kawase et al. Effects of feeder cells on the establishment of ES cells from mice and rats: The difference between allogenetic and xenogenetic feeders
US20070117206A1 (en) Methods for clonal derivation of human blastocyst-derived stem cell lines
KR100479638B1 (ko) 전기자극 및 막항원 표시자를 이용한 핵이식용 공여체 준비방법 및 이를 이용한 복제동물의 생산방법
Torkzaban et al. Isolation and maintenance of calves type A spermatogonia in vitro: in influence of epidermal growth factor on number of cells, colony size and spermetogoia viability
KR20060089774A (ko) 서로 다른 개체로부터 유래된 체세포와 난자로부터 유래된 인간 배아 줄기세포 및 그 제조방법
AU779273B2 (en) Long-term cell culture compositions and genetically modified animals derived therefrom
Piedrahita Studies on the isolation of embryonic stem (ES) cells: Comparative behavior of murine, porcine, and ovine species
HK1003208B (en) Use of leukaemia inhibitory factor (lif) for the (in vitro) propagation of embryonic stem cells
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): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

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
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA