EP1056468A1 - Verfahren zur förderung der proliferation von embryonalen stammzellen - Google Patents

Verfahren zur förderung der proliferation von embryonalen stammzellen

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Publication number
EP1056468A1
EP1056468A1 EP99907048A EP99907048A EP1056468A1 EP 1056468 A1 EP1056468 A1 EP 1056468A1 EP 99907048 A EP99907048 A EP 99907048A EP 99907048 A EP99907048 A EP 99907048A EP 1056468 A1 EP1056468 A1 EP 1056468A1
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European Patent Office
Prior art keywords
seq
group
ala
tyr
active agent
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English (en)
French (fr)
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Kathleen E. Rodgers
Gere Dizerega
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University of Southern California USC
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University of Southern California USC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/085—Angiotensins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/14—Angiotensins: Related peptides
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/32—Angiotensins [AT], angiotensinogen
    • 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

  • This present invention relates to methods for accelerating the proliferation of embryonic stem cells.
  • Embryonic stem (ES) cells may be maintained as totipotent cells in vitro for many generations. In the mouse, such cells have been demonstrated as continuously growing cell lines derived from the inner cell mass of 3.5 day blastocysts of the mouse (Wiles and Keller, Development 111:259-267 (1991)). Reintroduction of such cells into the mouse blastocysts illustrates the ability of ES cells to generate all lineages of mouse cells (Bradley, et al. Nature 309:255-256 (1984)).
  • ES cells when allowed to form three-dimensional structures known as embryoid bodies (EBs), ES cells will differentiate into many cell types including those of the hematopoietic system (Evans and Kaufman, Nature 292:154-156 (1981); Martin, Proc. Natl. Acad. Sci. 78:7634-7638 (1981); Doetschman et al. J. Embryol. Exp. Morphol. 87:27-45 (1985)).
  • ES cells provides a unique system with which to analyze both the cellular and molecular events involved in the first stages of lineage determination.
  • the present invention provides methods that increase proliferation of embryonic stem cells that are useful in rapidly providing a large population of such cells for use in replacement therapy and for making a large population of transfected embryonic stem cells for use in replacement therapy.
  • the present invention provides methods that promote embryonic stem cell proliferation by contacting the cells with angiotensinogen, angiotensin I ("Al”), Al analogues, Al fragments and analogues thereof, angiotensin II (“All"), All analogues, All fragments or analogues thereof or All AT 2 type 2
  • an improved cell culture medium for the proliferation of embryonic stem cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, Al, Al analogues, Al fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or All AT type 2 receptor agonists.
  • kits for the propagation of embryonic stem cells comprising an effective amount of angiotensinogen, Al, Al analogues, Al fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or All AT 2 type 2 receptor agonists, and instructions for culturing the cells.
  • kits for the propagation of embryonic stem cells comprise an effective amount of angiotensinogen, Al, Al analogues, Al fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or All AT 2 type 2 receptor agonists, and instructions for culturing the cells.
  • Preferred embodiments of the kit further comprise cell culture growth medium, a sterile container, other growth factors, and an antibiotic supplement.
  • stem cells refers to totipotent cells having the ability to differentiate into many different cell lineages.
  • One such cell type is the embryonic stem cell ("ES").
  • ES embryonic stem cell
  • proliferation encompasses both cellular self renewal and cellular proliferation with accompanying differentiation.
  • active agents refers to the group of compounds comprising angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof and All AT 2 type 2 receptor agonists.
  • angiotensin I (Al) which is converted to All by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from Al (Asp-Arg-Nal-Tyr-Ile-His-Pro-Phe-His-Leu [SEQ ID ⁇ O:37]). All is a known pressor agent and is commercially available. The use of All analogues and
  • a peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) is p-aminophenylalanine6-AII ["(p-NH 2 -Phe)6-AII)"], Asp-Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH 2 -Phe (Speth and Kim, BBRC 169:997-1006 (1990).
  • This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97 (1994); Bryson, et al, Eur. J. Pharmacol.
  • AII(l-7) All residues 1-7) or other fragments of All to evaluate their activity.
  • AII(l-7) elicits some, but not the full range of effects elicited by AIL (Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and
  • a preferred class of AT2 agonists for use in accordance with the present invention comprises angiotensinogen, Al, Al analogues, Al fragments and analogues thereof, All, angiotensin II analogues, All fragments or analogues thereof or All AT 2 type 2 receptor agonists having p-NH-Phe in a position corresponding to a position 6 of AIL
  • various nonpeptidic agents e.g., peptidomimetics
  • having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.
  • AT 2 type 2 receptor agonists of particular interest in accordance with the present invention are characterized as comprising a sequence consisting of at least three contiguous amino acids of groups R"-R 8 in the sequence of general formula I
  • R A is suitably selected from Asp, Glu, Asn, Acpc (1- aminocyclopentane carboxylic acid), Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 ), Gly, Asp(NH 2 ) and Sue,
  • R B is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc, Lys and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer, Ala, and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;
  • R 6 is His, Arg or 6-NH 2 -Phe
  • R 7 is Pro or Ala
  • R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R 4 as a terminal Tyr group.
  • AT2 agonists useful in the practice of the invention include the AH analogues set forth above subject to the restriction that R 6 is p-NH 2 -Phe.
  • R A and R B are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys.
  • Particularly preferred embodiments of this class include the following: AH, AIII or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AH(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AH(l-7), Asp-Arg-Val-Tyr-Ile-His-Pro ⁇ SEQ ID NO:4]; AII(2-7).
  • a further class of particularly preferred compounds in accordance with the present invention consists of those with the following general structure:
  • RI is selected from the group consisting of H or Asp;
  • R2 is selected from the group consisting of He, Val, Leu, norLeu and Ala
  • R3 is selected from the group consisting of He, Val, Leu, norLeu and Ala
  • R4 is selected from the group consisting of His and aminoPhe.
  • R5 is either Phe or H.
  • Preferred embodiments of this class of the invention include SEQ ID NO:l,
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:34,
  • SEQ ID NO:38 Particularly preferred embodiments of this class include SEQ ID NO:38.
  • Particularly preferred embodiments of this class include SEQ ID NO:38.
  • R 2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;
  • R 6 is His, Arg or 6-NH 2 -Phe;
  • R 7 is Pro or Ala;
  • R is selected from the group consisting of Phe, Phe(Br), He and Tyr.
  • a particularly preferred subclass of the compounds of general formula II has the formula
  • R 2 , R 3 and R 5 are as previously defined.
  • Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2].
  • Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His- Pro-Phe [SEQ ID NO:17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18].
  • the fragment AII(4-8) was ineffective in repeated tests; this is believed to be due to the exposed tyrosine on the N-terminus.
  • Bet 1 carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt (betaine)
  • AH and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974).
  • neutral side chains in position R 3 , R 5 and R 7 may be involved in maintaining the appropriate distance between active groups in positions R , R and R 8 primarily responsible for binding to receptors and/or intrinsic activity.
  • Hydrophobic side chains in positions R 3 , R 5 and R 8 may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.
  • Arg and Lys are particularly preferred as R z
  • R 3 may be involved in the formation of linear or nonlinear hydrogen bonds with R 5 (in the gamma turn model) or R 6 (in the beta turn model). R 3 would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R 3 may suitably be selected from Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr. In another preferred embodiment, R 3 is Lys.
  • R 4 is preferably selected from Tyr, Thr, Tyr (PO 3 ) 2 , homoSer, Ser and azaTyr.
  • Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).
  • R 4 is Ala.
  • an amino acid with a ⁇ aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R 5 , it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, Gly and Val.
  • R 6 is His, Arg or 6-NH 2 -Phe.
  • the unique properties of the imidazole ring of histidine e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to
  • R 12 contribute to its particular utility as R 6 .
  • conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R 7 .
  • R 7 should be Pro in order to provide the most desirable orientation of R 8 .
  • both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.
  • Analogues of particular interest include the following:
  • polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide
  • these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein inco ⁇ orated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.
  • a method of increasing in vitro and ex vivo embryonic stem cell proliferation by exposure to angiotensinogen, Al, Al analogues, Al fragments, All, All analogues, AH fragments or analogues thereof or AH AT 2 type 2 receptor agonists ("active agents") is disclosed.
  • Experimental conditions for the isolation, purification, ex vivo growth and in vivo mobilization of embryonic stem cells have been reported (Wulf, et al. 1993. EMBO, 12(13): 5065- 5074; Wiles, 1991. Development, 111:259-267; Guillemot, et al. 1996. Blood, 88(7): 2722-2731).
  • Embryonic stem cells may be obtained freshly from a host such as a murine animal, e.g. a mouse, rat, guinea pig, Chinese hamster or other small quantities of embryonic stem cells.
  • the cells may be grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • a G418-resistant subclone (CCEG2) of the 129/Sv-derived ES line CCE (Robertson et al., 1986. Nature, 323: 445-448), adapted to grow in the presence of LIF and without feeder cells, is used.
  • DME Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • LIF leukemia inhibitory factor
  • MTG monothioglycerol
  • Embryonic stem cells are suspended in culture medium and incubated in the presence of, preferably, between about 0.1 ng/ml and about 10 mg/ml of the active agents of the invention.
  • the cells are expanded for a period of between 8 and 21 days and cellular proliferation is assessed via any one of a variety of techniques well known in the art, including, but not limited to, bromodeoxyuridine inco ⁇ oration (Vicario-Abejon et al., 1995), 3 H-thymidine inco ⁇ oration (Fredericksen et al, 1988), or antibody labeling of a protein present in higher concentration in proliferating cells than in non-proliferating cells.
  • proliferation of embryonic stem cells is assessed by reactivity to an antibody
  • PCNA proliferating cell nuclear antigen
  • Viable cells may also be identified using a technique such as the trypan blue exclusion assay.
  • stem cells that have been cultured in the presence of the active agents are used, either alone or in combination with another compound of interest such as a cytokine, for studying embryonic stem cell proliferation in the research laboratory. Proliferation is then measured as described above.
  • embryonic stem cells may be exposed to the active agents and the level of gene expression, either specifically or generally, is determined. This may be accomplished by any of several techniques well known and widely available to one skilled in the art. Such techniques include nuclease protection assay (Multi-NPA, Ambion, Inc., Austin, TX), northern blot, RT-PCR, competitive PCR, relative PCR, RNase protection (RPA II, Ambion, Inc., Austin, TX).
  • the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10 6 and 50 x 10 6 cells per ml in an appropriate medium containing an effective amount (generally 0.1 ng/ml to 10 mg/ml) of the active agents. The cells are then incubated for an appropriate period of time and assayed for proliferation as described above.
  • RNA may be isolated and an assay performed to determine the level of expression of RNA encoding a protein expressed in proliferating cells such as PCNA.
  • assessment of the in vivo proliferative effect of the active agents on stem cells is done by histochemical evaluations of various tissues.
  • in vivo proliferation of ES cells is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating ES cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA, or cyclin; Zymed Laboratories).
  • PCNA proliferating cell nuclear antigen
  • Embryonic stem cell proliferation may also be assessed by examination of changes in gene expression, phenotype, mo ⁇ holgy, by genetic detection (for example, the donor cell is engineered to differ from the recipient by having a different allele of one gene), or any other method that distinguishes proliferating stem cells from non- proliferating stem cells.
  • the stem cell may be transfected with a marker gene that that allows for genetic detection of the transfected cell. Following administration of such a transfected stem cell after exposure of cells to the active agents, the stem cell is administered to a patient having a deficiency in one or more cells of the same or various lineages.
  • a sample which may comprise a bodily fluid such as blood or a tissue sample, is removed from the patient and the presence and quantity of stem cells in the patient may be measured by detection of the marker gene.
  • the amount of marker gene detected indicates the amount of stem cells present in the patient at a certain point in time.
  • the marker gene may be detected by any of several techniques well known and widely available
  • Such techniques may include Southern blot, Northern blot, PCR, RT-PCR, and RNase protection assay.
  • the cells can be transfected with a drug resistance marker gene, such as the neomycin resistance gene.
  • a drug such as neomycin, to which expression of the neo gene confers resistance. Only those cells that express the drug resistance gene will survive exposure to the drug, allowing for quantitation of the number of embryonic stem cells in the sample.
  • the active agents may be administered at a level of between about 0.1 ng/kg to about 10 mg/kg.
  • the active agents may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intra-arterial, intravenous, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.
  • the active agents may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions) and may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • active agents of the invention can be administered as the sole active agent, they can also be used in combination with one or more other compounds.
  • the active agents and other compounds can be formulated as separate compositions that are given at the same
  • the active agents and other compounds can be given as a single composition.
  • the active agents are administered in combination with leukemia inhibitory factor.
  • the active agents are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the active agents may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
  • liquid or semi-liquid preparations suitable for penetration through the skin e.g., liniments, lotions, ointments, creams, or pastes
  • drops suitable for administration to the eye, ear, or nose e.g., liniments, lotions, ointments, creams, or pastes
  • the dosage regimen for increasing in vivo proliferation of stem cells with the active agents is based on a variety of factors, including the type of injury or deficiency, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed.
  • the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Dosage levels of the order of between 0.1 ng/kg and 10 mg/kg of the active agents are useful for all methods of use disclosed herein.
  • the active agents are administered topically.
  • a suitable topical dose of active ingredient of the active agents is preferably between about 0.1 mg/kg and about 10 mg/kg administered twice daily.
  • the active ingredient may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1 % to 1 % of the formulation.
  • an improved cell culture medium for the proliferation of stem cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of the active agents of the invention, as described above.
  • Any cell culture media that can support the growth of stem cells can be used with the present invention.
  • Such cell culture media include, but are not limited to Basal Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-MEM® Reduced-Serum Medium, RPMI Medium, and Macrophage-SFM Medium or combinations thereof.
  • the improved cell culture medium can be supplied in either a concentrated
  • the cell culture may be either chemically defined, or may contain a serum supplement.
  • Culture media is commercially available from many sources, such as GIBCO BRL (Gaithersburg, MD) and Sigma (St. Louis, MO)
  • kits for the propagation of embryonic stem cells comprising an effective amount of angiotensinogen, Al, Al analogues, Al fragments and analogues thereof, AH, AH analogues, All fragments or analogues thereof or AH AT 2 type 2 receptor agonists, as described above.
  • the kit further comprises cell culture growth medium.
  • Any cell culture media that can support the growth of embryonic stem cells can be used with the present invention. Examples of such cell culture media are described above.
  • the improved cell culture medium can be supplied in either a concentrated
  • the cell culture may be either chemically defined, or may contain a serum supplement.
  • the kit further comprises a sterile container.
  • the sterile container can comprise either a sealed container, such as a cell culture flask, a roller bottle, or a centrifuge tube, or a non-sealed container, such as a cell culture plate or microtiter plate (Nunc; Naperville, IL).
  • the kit further comprises an amount of leukemia inhibitory factor (LIF) sufficient to prevent differentiation to cells beyond the ESC.
  • LIF leukemia inhibitory factor
  • the kit comprises between about 10 units/ml and about 1000 units/ml of LIF.
  • the kit further comprises an antibiotic supplement for inclusion in the reconstituted cell growth medium.
  • antibiotic supplements include, but are not limited to actimonycin D,
  • Fungizone® kanamycin, neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GIBCO).
  • EF cells are feeder cells used to produce proper culture conditions for the proliferation of the embryonal stem cells (ESC). EF cells were isolated from mouse embryos at day 14 of gestation (the kind gift of Dr. Peter Laird, Univ. of Southern California) by the following procedure:
  • Steps 5 and 6 were repeated four additional times with the tissue pieces
  • a tube of primary EF cells (described above) was thawed and expanded for several passages in HEPES-buffered (pH 7.3) DMEM, high glucose, supplemented with 10%) fetal calf serum (FCS) and antibiotics.
  • HEPES-buffered (pH 7.3) DMEM high glucose
  • FCS fetal calf serum
  • antibiotics antibiotics.
  • the feeder cells were thawed and placed in the flasks (one vial of irradiated feeders was sufficient for 8 25 cm 2 flasks). After preparation of the feeder layer for 24 hours, the mouse embryonal cells, isolated via standard procedures (Martin, Proc. Natl. Acad. Sci. 78:7634-7638 (1981); Evans and Kaufman, Nature 292:154-156 (1981), were thawed and added to the feeder layer.
  • ESC After growing to healthy colonies, but prior to differentiation (approximately 3 days after thawing), cells were harvested for assessment of the effect of AH on the proliferation of ESC.
  • the ESC were trypsinized from the tissue culture flask together with the feeder cells for replating under the appropriate conditions. Flasks containing ESC to be passaged were washed with 3-4 ml of HEPES-buffered saline, and 0.5 ml of 0.25% trypsin 1 mM EDTA was added. The cells were incubated at 37°C for 2-3 minutes. The flask was then shaken to detach the cells and 2 ml of ES medium was added. This mixture was pipetted up and down to achieve a single cell suspension.
  • the ESC were trypsinized from the tissue culture flask together with the feeder cells for replating under appropriate conditions.
  • the present invention by providing a method for enhanced proliferation of embryonic stem cells, will greatly increase the clinical benefits of embryonic stem transplantation. This is true both for increased “self-renewal", which will provide a larger supply of embryonic stem cells capable of generating a lineage of cells that are deficient in the patient, and for proliferation with differentiation which will provide a larger supply of the progenitor cells of different cell lineages. Similarly,
  • the number of cells per well was evaluated in the presence and absence of LIF.
  • the data were gathered 4 days after initiation of the cultures,
  • Ala3aminoPhe6 AH SEQ IDNO:38
  • the method of the present invention also increases the potential utility of embryonic stem cells as vehicles for gene therapy in certain disorders by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected embryonic stem cells.

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EP99907048A 1998-02-19 1999-02-16 Verfahren zur förderung der proliferation von embryonalen stammzellen Withdrawn EP1056468A1 (de)

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PCT/US1999/003243 WO1999042122A1 (en) 1998-02-19 1999-02-16 Method of promoting embryonic stem cell proliferation

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JP3880795B2 (ja) 1997-10-23 2007-02-14 ジェロン・コーポレーション フィーダー細胞を含まない培養物中で、霊長類由来始原幹細胞を増殖させるための方法
US7410798B2 (en) 2001-01-10 2008-08-12 Geron Corporation Culture system for rapid expansion of 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
US6730775B1 (en) 1999-03-23 2004-05-04 University Of Southern California Methods for limiting scar and adhesion formation
US7455983B2 (en) 2000-01-11 2008-11-25 Geron Corporation Medium for growing human embryonic stem cells
US6747008B1 (en) 2000-06-19 2004-06-08 University Of Southern California Methods for treating and preventing alopecia
DE60222409T2 (de) 2001-05-31 2008-09-25 Vicore Pharma Ab Trizyklische verbindungen, nützlich als angiotensin ii agonisten
JP5560391B2 (ja) 2005-06-22 2014-07-23 アステリアス バイオセラピューティクス インコーポレイテッド ヒト胚性幹細胞の懸濁培養法
EP2455388A1 (de) 2010-11-23 2012-05-23 LanthioPep B.V. Neuartige Rezeptoragonisten von Angiotensin des Typs 2 (AT2) und Verwendungen dafür
WO2021023698A1 (en) 2019-08-02 2021-02-11 Lanthiopep B.V Angiotensin type 2 (at2) receptor agonists for use in the treatment of cancer

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US5418159A (en) * 1989-01-10 1995-05-23 Amrad Corporation Limited Leukaemia inhibitory factor from livestock species and use thereof to enhance implantation and development of embryonic cells
US5015629A (en) * 1989-06-26 1991-05-14 University Of Southern California Tissue repair
US6335195B1 (en) * 1997-01-28 2002-01-01 Maret Corporation Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation

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