EP1520009A2 - Verfahren zur gewinnung von tierischen oder menschlichen adulten stammzellen, und deren therapeutische verwendung - Google Patents

Verfahren zur gewinnung von tierischen oder menschlichen adulten stammzellen, und deren therapeutische verwendung

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Publication number
EP1520009A2
EP1520009A2 EP03761663A EP03761663A EP1520009A2 EP 1520009 A2 EP1520009 A2 EP 1520009A2 EP 03761663 A EP03761663 A EP 03761663A EP 03761663 A EP03761663 A EP 03761663A EP 1520009 A2 EP1520009 A2 EP 1520009A2
Authority
EP
European Patent Office
Prior art keywords
cells
cell
stem cells
tissue
animal
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP03761663A
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English (en)
French (fr)
Inventor
Didier Montarras
Nicolas Borenstein
Christian Pinset
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fondation de l'Avenir
Centre National de la Recherche Scientifique CNRS
Celogos SA
Institut Pasteur
Original Assignee
Fondation de l'Avenir
Centre National de la Recherche Scientifique CNRS
Celogos SA
Institut Pasteur
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 Fondation de l'Avenir, Centre National de la Recherche Scientifique CNRS, Celogos SA, Institut Pasteur filed Critical Fondation de l'Avenir
Publication of EP1520009A2 publication Critical patent/EP1520009A2/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • the present invention relates to the field of tissue regeneration and repair by cell transplantation. More particularly, the present invention relates to a process for the preparation of adult stem cells which preserves the diversity and plasticity of these cells. The present invention also relates to the use of these stem cells obtained by the extraction process proposed in therapeutic applications, including gene and cell therapy.
  • the present invention provides a method of cell preparation meeting the need mentioned above. More particularly, the present invention relates to a cell preparation process capable of preserving the diversity and plasticity of vertebrate stem cells preferably originating from a tissue biopsy for their later use in the context of tissue regeneration or repair by transplantation. in an animal, like a human being.
  • the invention further relates to a defined culture medium, particularly useful during the implementation of the cell preparation process.
  • the present invention also relates to the cell preparation or the stem cells obtained by the method of preparation of the invention.
  • the present invention also relates to the use of these stem cells obtained by the preparation process proposed in therapeutic applications, including gene or cell therapy.
  • the present invention also relates to a cellular composition comprising human or animal stem cells obtained according to the method of preparation of the invention.
  • the present invention also relates to a process for the preparation of stem cells capable of repairing or regenerating in vivo tissues or organs of a vertebrate.
  • the present invention also relates to a treatment method, characterized by the implantation of autologous or heterologous animal stem cells obtained according to the method of the invention in an animal.
  • the originality of the present invention relates to the fact that, unlike the cell preparation methods used in the field, the method of preparation of the present invention makes it possible: - to extract the cells under defined conditions without animal protein of origin extractive, therefore under sanitary conditions; to maintain cell viability through the use of antioxidant and anti-apoptotic molecules; - to preserve cellular diversity; to maintain the potential of cellular plasticity; maintain the potential for differentiation; improve implantation, colonization and differentiation of transplanted cells; and - to transplant the cells without having to go through an expansion step in in vitro culture.
  • Figures 1 to 5 are photomicrographs illustrating the detection of muscle cells grafted into the heart of sheep.
  • the cells were obtained by enzymatic dissociation of a skeletal muscle biopsy and then directly implanted, without expansion phase in in vitro culture, in the myocardium of the same sheep (autologous graft).
  • the presence of skeletal muscle cells is revealed using the antibody MY32 which specifically recognizes the heavy chain of skeletal myosin.
  • Figure 1 illustrates the detection of skeletal muscle cells grafted with the MY32 antibody by the presence, inside the wall of the myocardium, of massive grafting areas (2 to 9 mm in diameter) or of discrete foci (bar scale, 250 microns).
  • Figure 2 illustrates the immunodetection of skeletal muscle cells with the antibody MY32.
  • the fibers marked by the MY32 antibody are generally aligned with the network of native cardiomyocytes. Some fibers are intensely marked while others are only weakly marked; native cardiomyocytes are not marked (Scale bar, 500 microns).
  • Figure 3 shows grafted cells differentiating into muscle fibers and forming organized sarcomeres (Scale bar, 25 microns).
  • FIG. 4 illustrates the immunohistochemical specificity of the antibody MY32.
  • FIG. 5 illustrates the co-detection of grafted skeletal muscle cells and cardiomyocytes with the antibody MY32 and an antibody directed against connexin-43, a protein specific to “gap junctions” or communicating junctions (red dots). This test did not demonstrate the presence of connexin-43 inside the grafted cells or between the grafted cells and their microenvironment.
  • the present invention therefore relates to the development of a process for the preparation of animal or human stem cells which preserves the diversity and plasticity of these cells preferably originating from a tissue biopsy for their subsequent use in as part of tissue regeneration or repair by direct transplantation of cells, without expansion in in vitro culture, thus prepared in an animal or in humans.
  • the initial preparation from a tissue biopsy is heterogeneous; for example, that from a skeletal muscle biospy may contain myoblasts, fibroblasts, adipocytes, peripheral nerve cells, endothelial cells and smooth muscle cells, in addition to pluripotent stem cells.
  • stem cell is meant cells which have both the ability to self-renew and the ability to differentiate into different types of cell precursors.
  • stem cell diversity is meant stem cells which are classified according to their stage of development and their tissue origin. More particularly, we mean a classification of embryonic or adult stem cells according to their tissue origin, for example, skin or neuronal or muscle stem cells.
  • tissue of stem cells is meant the capacity for these cells to cross line boundaries, for example the capacity of hematopoietic cells to differentiate into hepatocytes.
  • tissue regeneration is meant the capacity to reform tissue either by activation of the tissue progenitor cells (for example tissue of the skin, liver, heart, bone or nervous tissue), or by direct transplantation , without expansion in in vitro culture, of these progenitor cells. More particularly, it is then a new tissue formation.
  • tissue repair is meant an operation which makes it possible to overcome a deficit without having to resort to a regeneration process. Tissue repair results in an exogenous supply of cells which may be different from the cells of the recipient tissue.
  • cell transplantation is meant an operation which is characterized by the supply of isolated cells. This transplantation can be carried out, for example, by direct injection, without expansion in in vitro culture, in the tissue or in the afferent circulation.
  • animal is meant any living organism which may be subject to cell transplantation, and this includes vertebrate beings such as in particular human beings, domestic and wild animals, in particular birds. ii) Method for preparing stem cells
  • a first aspect of the present invention relates to a process for the preparation of human or animal stem cells.
  • This process essentially consists of the following steps: a) cell extraction; b) mechanical dissociation; c) enzymatic dissociation; and maintaining the cells in a specific medium making it possible to preserve the diversity and the plasticity, said method excluding the cultivation of the cells, that is to say an expansion phase in in vitro culture.
  • tissue extraction is obtained first of all following a biopsy to provide a tissue fragment, such as a muscle, liver or skin fragment.
  • a tissue fragment such as a muscle, liver or skin fragment.
  • a muscle biopsy is performed under local anesthesia following an incision or even with a needle.
  • the tissue fragment thus obtained is then moistened in a defined medium, such as DME / 202 in the presence of protective factors and factors inhibiting cell differentiation (Pinset and Montarras), (1994) Cell Biology: A laboratory Handbook. Press Academy).
  • the medium defined is a medium hereinafter called DPM (Diversity Protecting Medium) and comprises at least: a basic nutritive medium buffered with buffers dependent or independent of the C0 2 concentration.
  • DPM Diversity Protecting Medium
  • the media used are, in most cases, made up of a mixture in a 1: 1 ratio of DME type and F12 type media.
  • a protective factor such as:
  • antioxidants ascorbic acid, N-acetyl cysteine
  • anti-caspases Dose: about 0.1 mU to 10 mU
  • hormones such as glucocorticoids, insulin, retinoic acid, thyroid hormone, mineralocorticoids, IGF1 or IGF2 at physiological doses preferably ranging from 10 "6 to 10 " 9 M.
  • FGF Fibroblast Growth Factors
  • factors in the FGF family three factors are particularly important and play a role. similar role, FGF-2, FGF-6 and FGF-10.
  • concentrations preferably ranging from 0.1 to 100 ⁇ g / ml.
  • EGF Epidermal Growth Factor
  • LIF Leukemia Inhibitory Factor
  • This DPM medium thus defined is used during the cell preparation process.
  • the samples from biopsies are kept either at room temperature, or stored, preferably, between 4 and 6 ° C in the DPM for a period preferably not exceeding 48 hours. It is clear that the precise composition of the DPM may vary depending on the type of tissue biopsy.
  • the tissue fragment maintained in the DPM medium is first minced until a cellular homogenate is obtained. This is preferably done using sterile surgical scissors but any other similar tool can be used. Thereafter, the homogeneous tissue extract or the tissue homogenate is preferably released from a portion of the erythrocytes and adipocytes by means of a washing and centrifugation step by gentle sedimentation of 1 to 10 g (Pinset and Montarras. 1994. Cell Biology: A Laboratory Handbook. Académie Press).
  • step c) of the process the tissue homogenate obtained in step b) is subjected to an enzymatic digestion to optimize cell dissociation.
  • proteolytic enzymes of bacterial origin such as collagenase and / or pronase, are preferably used.
  • trypsin produced by genetic engineering or any other acceptable enzyme.
  • These enzymes can be used alone or in combination at concentrations varying, preferably, from 0.1 to 0.5%.
  • step c) of the method the pellet of tissue fragments obtained in step b) is suspended in DPM medium supplemented with collagenase at a final concentration of 0.4 g per 100 ml.
  • the enzymatic digestion ranging from 5 to 20 minutes can be repeated several times.
  • the temperature of the enzymatic reaction is preferably between 20 ° C and 37 ° C with external agitation.
  • the enzymatic digestion preferably takes place in several stages, and this, up to five stages. At each stage, the fragments and the cells are separated by centrifugation at 10 g.
  • the fragment pellet is then resuspended in the DPM medium supplemented with proteolytic enzymes and then subjected to a new digestion step.
  • the cells present in the supernatant are harvested by centrifugation at 200 g and resuspended in DPM medium. This results in a cell suspension which adds the cells extracted from the different digestion stages.
  • the cell suspension is then filtered through a nylon filter, the pore diameter of which is preferably approximately 34 microns, to remove the tissue fragments.
  • a cell suspension resulting from a muscle biopsy which maintains its potential colonization and cellular plasticity.
  • a cell suspension resulting from a muscle biopsy which maintains its potential colonization and cellular plasticity.
  • 1 x 10 6 to 2 x 10 6 cells for one gram of tissue, it is possible to prepare from 1 x 10 6 to 2 x 10 6 cells.
  • the effectiveness of the methods for maintaining cell diversity and plasticity is evaluated by in vivo tests and ex vivo tests. In the former, the cell suspensions obtained are reintroduced into the animal after labeling.
  • a fluorescent molecule such as the Green Fluorescent Protein will be used.
  • Cell labeling makes it possible to follow the fate of cells injected into the body, to analyze their participation in the different tissue and the differentiation of the injected cells.
  • the injected cells participate in the neoformation of different tissues such as skeletal and cardiac muscle, vessels, tendons, cartilage, bone, hematopoietic tissue. This diversity and cellular plasticity is dependent on the injection site, emphasizing the importance of the environment in cell development.
  • Ex vivo tests also make it possible to measure cell diversity and cell plasticity. These are based on clonal analysis and on the response to different inducers of differentiation. Among these, mention may be made of: growth factors such as insulin and IGFs, TGFs, BMPs, VEGF; hormones such as retinoic acid derivatives, thyroid hormones, glucocorticoids; factors of the extracellular matrix; vitamins and agents for mineralization; components of the extracellular matrix.
  • growth factors such as insulin and IGFs, TGFs, BMPs, VEGF
  • hormones such as retinoic acid derivatives, thyroid hormones, glucocorticoids
  • factors of the extracellular matrix such as retinoic acid derivatives, thyroid hormones, glucocorticoids
  • factors of the extracellular matrix such as retinoic acid derivatives, thyroid hormones, glucocorticoids
  • factors of the extracellular matrix such as retinoic acid derivatives, thyroid hormones, glucocorticoids
  • Cell identification is done by immunoassays using specific staining and specific antibodies, which allow both identification and quantification, and by transcriptomic analysis.
  • Cells thus prepared without expansion in in vitro culture are a source of cells suitable for cell therapy and can either be reimplanted directly into an organism, or stored for later reimplantation.
  • the method of the present invention may include an additional step, namely a freezing step. This optional process step offers the following advantages: preservation of tissue samples; dissociation between biopsy preparation and cell reimplantation;
  • 10 6 to 2 x 10 6 cells from 1 gram of tissue are placed in the presence of DPM medium supplemented with cryopreservative agents, such as DMSO.
  • cryopreservative agents such as DMSO.
  • concentration used is preferably 10%.
  • the cells are maintained at 20 ° C. for a period of
  • P-Cam as an endothelial cell marker
  • N-Cam as a marker for neuronal and muscle cells
  • GFAP as a marker for glial cells
  • Myf5, Pax3, Pax7, C-met and M-cadherin as markers of muscle cells
  • This characterization can also be undertaken at the level of transcription by the use of biochips containing oligo- nucleotides encoding cellular genes (eg, specific transcription factors and factors in the cell cycle machinery) used to identify cells in the cell suspension.
  • biochips containing oligo- nucleotides encoding cellular genes eg, specific transcription factors and factors in the cell cycle machinery
  • said stem cells are animal or human stem cells chosen from the group consisting of progenitor stem cells from a tissue.
  • the tissue is chosen from the group consisting of the skin, the liver, the heart, the bone and the nervous tissues.
  • Therapeutic uses A second aspect of the present invention relates to the use of a cell suspension or of stem cells obtained by the process of the invention in therapeutic applications, including gene therapy, following a transplantation of these cells in a human being, for example. More particularly, the present invention proposes to use the cells prepared by the method of the invention in the context of tissue repair and / or regeneration by cell transplantation without expansion in in vitro culture. The cells thus prepared will also be used as vectors in gene therapy.
  • the adult stem cells obtained by the preparation process according to the invention can be used in the context of a repair of bone tissue.
  • it is a question of transplanting the cells directly, without expansion in in vitro culture, to a site of bone repair.
  • cells or some of them can adopt the bone phenotype and thus participate in the repair of damaged tissue.
  • the method of cell preparation of the present invention can also provide a suspension of adult mammalian stem cells which can be used in restoring the hematopoietic potential.
  • the capacity of muscle biopsy stem cells to colonize the bone marrow of irradiated mice and to contribute to all hematopoietic lines obliges to consider the repairing potential of muscle biopsy cells in the case, for example, of leukemias.
  • transplants of cultivated muscle cells in the muscle are ineffective and characterized by massive death of the reimplanted cells within hours of their injection. It is therefore proposed that the process for preparing stem cells according to the invention which does not include an expansion phase in in vitro culture can contribute to the repair of muscle tissue.
  • the cells contained in the suspension will be able to respond to micro-environments and thus restore functions or repair muscle tissue.
  • the subject of the invention is also the use of human or animal stem cells obtained according to the method according to the invention, for the preparation of a medicament intended for the treatment of diseases by cell therapy or gene therapy.
  • the present invention further relates to a stem cell obtained according to the method according to the invention.
  • the present invention also relates to a treatment method comprising a step of implanting autologous or heterologous animal stem cells obtained according to the method according to the invention in an animal or in humans.
  • the present invention also relates to a cellular composition comprising human or animal stem cells obtained according to the method according to the invention.
  • the stem cells have a colonization capacity and an capacity to allow functional recovery.
  • cardiac tissue does not have stem cells capable of repairing this tissue in the adult state after injury.
  • ischemia of the heart is always accompanied by insufficient contraction, which, if significant, leads to heart failure.
  • the objective of cell therapy in this indication is to restore the contraction function.
  • muscle cells amplified in culture This first step shows the feasibility of the approach, but also, certain limitations of the strategy adopted.
  • the amplification of a large number of muscle cells in environments comprising animal fluids such as fetal calf serum, and their transplantation into heterotypic tissue are operations that are both cumbersome and not without potential health danger (viral contaminants, prions , etc.).
  • the essential limitation of this process lies in the fact that the cells thus injected have a restricted plasticity which only give skeletal muscle cells.
  • the process for the preparation of stem cells of the present invention without an expansion phase in in vitro culture avoids the impoverishment of cell diversity by the culture.
  • Anesthetic protocol premedication with midazolam, induction using etomidate, tracheal intubation and positive pressure ventilation with isoflurane in 100% oxygen. Surveillance is ensured by electrocardioscopy, invasive blood pressure, capnography. Postoperative analgesia with bupivacaine (intercostal block during thoracotomy), morphine and flunixin meglumine.
  • Skeletal muscle biopsy A skeletal muscle biopsy (approximately 10 g) is explanted sterile from the left femoral biceps of each sheep. The tissue thus removed is kept in DMEM (SIGMA) or DPM, as defined above, at room temperature until mechanical or enzymatic digestion. The plague of the animal is closed in the usual way. The animals recover for about three hours, then are re-anesthetized when the cells are ready to be reimplanted.
  • SIGMA DMEM
  • DPM DPM
  • the supernatant containing the isolated cells is stored in 20% DMEM (V / V) of fetal calf serum or DPM.
  • the pellet undergoes up to four additional digestions (Pinset, C. and Montarras, D. Cell Systems for Ex-vivo Studies of Myogenesis: A Protocol for the Isolation of Stable muscle cell populations from newborn to adult mice in Cell Biology: A Laboratory Handbook; second edition e. JF Celis, Académie Press; 1998).
  • the extracted cells are then filtered on a nylon filter with pores of 250 ⁇ m in diameter (Polylabo SA).
  • the cells thus prepared do not undergo an expansion phase in in vitro culture.
  • the cells are resuspended in 10 mL of DMEM without serum containing 25 ⁇ g / mL of 4'-6-diamino-2-phenylindole (DAPI, SIGMA) for 10 minutes or of DPM in the same conditions.
  • the cells are rinsed four times in DMEM or DPM in order to remove the DAPI.
  • Mononucleated cells are counted with a counting cell under fluorescence microscopy.
  • the cell preparation is then resuspended in 1.2 ml of DMEM or DPM without serum and stored at 4 ° C. until implantation without undergoing an expansion phase in in vitro culture.
  • Cell transplant The sheep previously anesthetized according to the method described above are placed in the right lateral decubitus position for a left thoracotomy on the 5th intercostal space. After pericardiotomy and suspension of the pericardium, the cells in DMEM or DPM are injected (0.1 ml per site) using an insulin syringe connected to a 27 G epijet, on 10 zones of the left ventricle . Coronary vessel mapping is established to identify injection sites in the myocardium. Epicardial stitches of location are made for at least two injections. The chest is closed in the usual way and the animals recover under an analgesic regime (morphine at 0.5 mg / kg IM BID, flunixin 1 mg / kg IM once) overnight inclusive.
  • an analgesic regime morphine at 0.5 mg / kg IM BID, flunixin 1 mg / kg IM once
  • Control cell cultures In order to confirm the presence of muscle precursor cells in the cell preparation, a 100 ⁇ L sample of the cell suspension is seeded in a culture dish containing DMEM or DPM with fetal calf serum. The cells are maintained as a control in an atmosphere containing 5% of CO 2 . Three to seven days after seeding, the cells are used for immunodetection of the specific regulatory factor MyoD (DAKO) as described by Montarras, D. et al. (Cultured Myf5 Null and MyoD Null Muscle Precursor Cells Display Distinct Growth Defects. Biol Cell 2000; 92: 565-72). Results
  • the cell injection procedure has no serious effect on animals.
  • a ventricular arrhythmia occurs when the needle is inserted and when the cells are injected, but resolves when the needle is withdrawn.
  • the cell count makes it possible to determine the injection of 1.7 +/- 0.3 x 10 7 mononuclear cells labeled with DAPI.
  • Skeletal myosin heavy chain expression (MY32) is detected at 3 weeks post-implantation in 8 animals out of 8, which confirms cell survival and myogenic expression of the implanted cells.
  • Large graft surfaces (2 to 9 mm in diameter) or discrete foci are observed inside the wall of the myocardium ( Figure 1). Isolated cells are also found in the adipose tissue of the pericardium.
  • fibers that are positive for expression of MY32 are usually aligned with native cardiomyocytes. Some of these fibers are strongly labeled with the antibody directed against MY32, while others are only weakly labeled ( Figure 2). These fibers are not electro-mechanically coupled to each other or to native cardiomyocytes as demonstrated by Connexin-43 negative immunohistochemistry.
  • the implanted cells develop organized sarcomeres and have either an elongated morphology characteristic of fused multinucleic myotubes, or a morphology which remains mononuclear ( Figure 3). These skeletal muscle cells have nuclei observed at the periphery or in the center. Replacement fibrosis and regions with mononuclear cells of the inflammatory response are also observed. No DAPI labeling is observed in the explanting hearts. This may be due to active cell division resulting in a dilution of the DAPI. Indeed, in cell cultures serving as controls, the
  • DAPI becomes undetectable after 6 to 8 doubling of the cell population.

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EP03761663A 2002-06-28 2003-06-27 Verfahren zur gewinnung von tierischen oder menschlichen adulten stammzellen, und deren therapeutische verwendung Withdrawn EP1520009A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CA002391638A CA2391638A1 (fr) 2002-06-28 2002-06-28 Procede pour maintenir la diversite et la plasticite cellulaire de cellules souches adultes de mammifere issues de biopsie tissulaire pour la therapie cellulaire
CA2391638 2002-06-28
PCT/FR2003/002010 WO2004003181A2 (fr) 2002-06-28 2003-06-27 Procede de preparation de cellules souches adultes animales ou humaines et utilisation desdites cellules souches en therapie.

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EP1520009A2 true EP1520009A2 (de) 2005-04-06

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EP03761663A Withdrawn EP1520009A2 (de) 2002-06-28 2003-06-27 Verfahren zur gewinnung von tierischen oder menschlichen adulten stammzellen, und deren therapeutische verwendung

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US (1) US20060121001A1 (de)
EP (1) EP1520009A2 (de)
AU (1) AU2003260637A1 (de)
CA (1) CA2391638A1 (de)
WO (1) WO2004003181A2 (de)

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Publication number Priority date Publication date Assignee Title
US6143508A (en) * 1989-06-29 2000-11-07 Rhone-Poulenc Rorer Pharmaceuticals Inc. Device and process for cell capture and recovery
US6069005A (en) * 1991-08-07 2000-05-30 Albert Einstein College Of Medicine Of Yeshwa University Hapatoblasts and method of isolating same
EP1025204A4 (de) * 1997-10-23 2001-02-28 Geron Corp Methoden und materialien zur kultivierung von von primaten abgeleiteten primordialen stammzellen
AU1603701A (en) * 1999-11-19 2001-05-30 Cold Spring Harbor Laboratory Transgenic mice expressing fluorescent protein under the control of the nestin promoter

Non-Patent Citations (1)

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Title
See references of WO2004003181A2 *

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AU2003260637A1 (en) 2004-01-19
US20060121001A1 (en) 2006-06-08
CA2391638A1 (fr) 2003-12-28
AU2003260637A8 (en) 2004-01-19
WO2004003181A3 (fr) 2004-04-15
WO2004003181A2 (fr) 2004-01-08

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