WO2004018652A2 - Procede permettant d'obtenir des cellules souches diploides pluripotentes et utilisation desdites cellules - Google Patents

Procede permettant d'obtenir des cellules souches diploides pluripotentes et utilisation desdites cellules Download PDF

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Publication number
WO2004018652A2
WO2004018652A2 PCT/DE2003/002734 DE0302734W WO2004018652A2 WO 2004018652 A2 WO2004018652 A2 WO 2004018652A2 DE 0302734 W DE0302734 W DE 0302734W WO 2004018652 A2 WO2004018652 A2 WO 2004018652A2
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WO
WIPO (PCT)
Prior art keywords
cells
tissue
stem cells
cell
tumor
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PCT/DE2003/002734
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German (de)
English (en)
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WO2004018652A3 (fr
Inventor
Ulrich Drews
Paul Schweizer
Christian Busch
Original Assignee
Ulrich Drews
Paul Schweizer
Christian Busch
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 Ulrich Drews, Paul Schweizer, Christian Busch filed Critical Ulrich Drews
Priority to AU2003266125A priority Critical patent/AU2003266125A1/en
Priority to DE10393633T priority patent/DE10393633D2/de
Publication of WO2004018652A2 publication Critical patent/WO2004018652A2/fr
Publication of WO2004018652A3 publication Critical patent/WO2004018652A3/fr

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    • 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
    • C12N5/0693—Tumour cells; Cancer cells
    • 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
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells

Definitions

  • the invention relates to a method for obtaining diploid, pluripotent stem cells from a tumor which is not a germline tumor, and the use of the stem cells thus contained for the therapeutic treatment of diseases, for the production of replacement tissue and replacement organs, and for the determination of the mode of action and side effect of medicaments in pharmaceutical development and environmental influences.
  • embryoblast cells are called pluripotent embryonic stem cells and are able to differentiate into each of the different body cell types. Since germ cells can also develop from embryonic stem cells, they belong to the germ line.
  • the stem cells are differentiated according to their properties and their origin as follows.
  • Embryonic stem cells (ES) have their origin in the inner cell mass of the embryo.
  • the area for the primordial germ cells is not yet determined within this cell mass, which is why the embryoblast is by definition part of the germ line. If such primordial germ cells are transplanted into adult animals, this leads to the development of typical teratoms, which is why they are also referred to as germ cell teratoms.
  • Stem cell lines that have emerged from germ cells GS, premordial germ cell lines
  • GS premordial germ cell lines
  • Mitotic activity is interrupted at a later point in time by gender-specific inhibitory factors from the follicular epithelial cells of the ovary or the supporting cells of the testes.
  • primordial germ cells can continue to share mitotically on the way to the development of dormant orgates and spermatogonia, whereby benign teratos or malignant teratocarcinomas are formed by differentiating these cell clusters, from which pluripotent stem cell lines can be isolated.
  • the human stem cell line Terra II was isolated from the lung metastasis of a human seminoma (Andrews in APMIS 106, 158-168, 1998).
  • stem cell lines can also be obtained from experimentally induced tere- toms.
  • the teratomas are created by injecting embryonic stem cells from the inner cell mass of the blastocyst into a recipient or experimental animal. Teratoma cells of this type also emerge from the germline and therefore belong to the germline tumors. In individual cases, however, they can also be isolated from a testicular teratocarcinoma that has developed from spermatogonia.
  • adult stem cells e.g. B. from the bone marrow as mesenchymal stem cells (MAPC, multipotent adult progenitor cells), with a cell suspension of the bone marrow of an adult donor using so-called FACS (fluorescence-activated cell sorter) is isolated.
  • FACS fluorescence-activated cell sorter
  • NAS neural adult stem cells
  • stem cells described above have so far been obtained essentially in animal experiments. However, transmission of animal cells to humans is currently problematic and human cell lines can currently only be obtained from ES blastocysts which are problematic according to the Embryo Protection Act.
  • Teratome contains tissue from the three cotyledons Ectoderm, Mesoderm and Endoderm.
  • Germ cell teratomas of this type are tumors that consist of female (ovarian teratoms) or male germ cells (testicular atoms) or in another place from primordial germ cells that are not in the right place during embryonic development.
  • Teratomas normally contain tissue from the three cotyledons that is already more or less differentiated. For example, the development of teeth and bones that are already visible in the X-ray image is described. Teratoms also show a tendency to degenerate, that is, to transition to malignant growth. Such a degenerate tumor is called teratocarcinoma.
  • teratomas do not only develop in the ovaries or testicles, but also on the tailbone. Because of its characteristic localization as an attachment to the coccyx, this species is called the coccyx teratom.
  • APNIS 106 158-168 (1998) the occurrence of pluripotent human embryonic stem cell lines in teratocarcinoma. However, as is expressly stated, these are also tumors from germline cells (GCT Germ Cell Tumors).
  • WO 01/59076 describes the pre- and post-meiotic origin of teratoms.
  • the teratoms are also described as chromosomally polymorphic.
  • the fusion of two mature oocytes acts as a trigger for the development of the atoms or spermatocytes or the prevention of the second meiotic division as the cause.
  • the aim of the invention is therefore to provide a source for cells, in particular stem cells, which have not yet completely lost their ability to differentiate into certain organs, i. H. are pluripotent and do not have the aforementioned problems, especially ethical and legal problems, and do not derive from the germline.
  • the coccyx is not a teratoma that develops from parts of the germline.
  • the coccyx teratom is very strongly differentiated and essentially contains all different types of tissue - in addition to already differentiated tissue and malignant structures, it also contains a non-malignant tissue in one area of undifferentiated embryonic blastem areas, in which one A large number of pluripotent stem cells are present which can be isolated in a manner known per se.
  • Options for this include FACS with magnetic beads; Cell culture and selective cultivation of stem cells by certain cell culture media (as with adult neuronal stem cells from the SVZ); the shredding of the tissue with a tissue chopper and the subsequent marking staining and staining of stem cells with selective (vital) dyes.
  • tumor tissue close to the spine is preferably used to isolate the desired stem cells. It would also be conceivable to use blastema cells from the center of the tumor that are not necessarily in the immediate vicinity of the coccyx.
  • the tissue which encloses the coccyx, in particular also contains the leg itself, is particularly preferably used. Such fabrics are about the size of a tangerine.
  • the tissues can be stored at 4 ° C with cooling for up to 6 hours.
  • the tissue is preferably stored using a conventional nutrient solution. Sterile HBSS is particularly suitable for this, which is also used for the extraction of adult SVZ cells.
  • tissue areas are preferably visually separated from one another under a microscope according to their differentiation.
  • a sharp knife such as a scalpel
  • tissue areas are preferably visually separated from one another under a microscope according to their differentiation.
  • the target tissue has a typical morphology that corresponds to that of an embryonic blastema tissue.
  • those whose structures are still largely undifferentiated are preferably processed further.
  • the cells contained therein are then released from their cell structure using methods known per se. Such disintegration of cell tissue is known to the person skilled in the art and can be carried out, for example, using one or more enzymes.
  • a cell suspension is preferably produced from the cells thus obtained.
  • the cells in suspension can then be selectively separated using a cell sorter, such as FACS, or magnetic beads using their characteristic surface markers.
  • the surface markers are known for the individual stem cells and are, for example, CEP-68 (chondrocyte expressed protein 68) for cartilage stem cells (E. Steck et al.; Biochemical Journal, Jan.
  • precursor or progenitor cells of the liver, pancreas and Langerhan islet cells, of neuronal tissue, of serous and mucous glands can be made from the coccyx teratoms, for example for the extraction of centroazinous cells of the exocrine pancreas or also sebaceous glands with hair attachments of the most varied types of epithelium, such as cubic epithelium, transitional or transitional epithelium, from which derivable urinary tract can be obtained, or also multi-layered, cornified and unhorned squamous epithelium, which is used for the extraction of vaginal mucosa, but also secretory epithelium, ciliated epithelium, multi-row ciliated epithelium for the production of respiratory and bone marrow epithelium as well as skeletal and tracheal cartilage cells and progenitor cells of smooth and striated muscles, high prismatic mucous epithelium for production isolate the colon
  • the precursor cells of dopaminergic neurons are to be isolated from the stem cells isolated according to the invention or can be further differentiated from these.
  • the cells obtained in this way can be cultivated, for example, on corresponding feeder cells. Cultivation methods of this type are diverse and are known to the person skilled in the art. In principle, the cells obtained according to the invention can also be cultivated without feeder cells.
  • the pluripotent stem cells obtained with the method according to the invention are particularly suitable for the production of replacement tissues and replacement organs.
  • stem cells and thus skeletal and tracheal cartilage cells and tissue can be produced from cells with the marker CEP 68;
  • Nestin-positive cells can be used to produce neural stem cells and all kinds of nerve tissue, in particular dopaminergic nerve cells as well as other nerve tissue in the brain and motor and sensory nerves.
  • Cells with the surface markers CD 34, Thy-1, TIE, TEK can be used to produce hematopoietic stem cells and thus cells of the hematopoietic system.
  • Cells with the AA4 marker can be used in particular to make endothelial cells, aortic-associated hematopoietic blastocells, hematopoietic primitive fetal liver progenitor cells and the tissue formed therefrom can be produced, bile duct and liver cells can be produced from OC.3 marker-positive stem cells and skin stem cells and corresponding skin tissue can be obtained from -2 -labeled cells.
  • PSCA and CK ⁇ mRNA positive cells are progenitor cells of the prostate.
  • Other general superordinate markers for stem cells are AP and FGF-4 and, for pluripotent stem cells, the corresponding stage-specific embryonic antigens 1, 3 and 4 (SSEA-1, SSEA-3 and SSEA-4).
  • the invention therefore also relates to the use of the cells obtained by the method according to the invention for the production of organs and parts of the body, such as blood cells, in particular erythrocytes, in particular leukocytes, lymphocytes, thrombocytes, etc., blood and lymphatic vessels, such as, for example, arteries and veins, liver and extracorporeal liver replacement devices, kidneys and artificial extracorporeal kidney cell-containing dialysis devices, muscle tissue, in particular cardiac muscle and motor muscles, hair-forming tissue, dopaminergic neurons, in particular for the therapy of Parkinson's disease, Langerhans see cells for the treatment of diabetes, intestinal walls, especially the colon, Pancreas and bile as well as bile ducts, vagina, bone, bone marrow, nerve tissue, lung and bronchial / tracheal tissue, esophagus, skin and cartilage, in particular articular, prostate, testis and ovarian tissue.
  • stem cells can also be isolated from erythrocytes, in
  • the cells obtained according to the invention are also particularly suitable for examining the influence of new medications and of environmental toxins and environmental conditions on embryonic development. In this way it is possible to do without a large number of animal experiments. In addition, statements made on human cells are often more meaningful than results obtained on animals.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Oncology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Procédé permettant d'obtenir des cellules souches diploïdes pluripotentes à partir de tissu tumoral n'étant pas une tumeur de la lignée germinale. Lesdites cellules souches sont obtenues par isolation de zones tissulaires individuelles et / ou par décomposition de la liaison entre les cellules d'une manière connue et enfin par isolation des cellules ainsi obtenues. La présente invention se caractérise en ce qu'un tératome sacro-coccygien est utilisé en tant que tissu tumoral. Les cellules ainsi obtenues sont utilisées pour la fabrication d'organes, de parties d'organes, de vaisseaux sanguins et de tissus de remplacement, ainsi que pour étudier la tératogénicité de substances et de conditions de l'environnement.
PCT/DE2003/002734 2002-08-16 2003-08-14 Procede permettant d'obtenir des cellules souches diploides pluripotentes et utilisation desdites cellules WO2004018652A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003266125A AU2003266125A1 (en) 2002-08-16 2003-08-14 Method for obtaining diploid, pluripotential stem cells and use thereof
DE10393633T DE10393633D2 (en) 2002-08-16 2003-08-14 Verfahren zur gewinnung von diploiden, pluripotenten stammzellen sowie deren verwendung

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE0203006 2002-08-16
DEPCT/DE03/03006 2002-08-16

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WO2004018652A2 true WO2004018652A2 (fr) 2004-03-04
WO2004018652A3 WO2004018652A3 (fr) 2004-05-21

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DE (1) DE10393633D2 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007063198A1 (de) * 2007-12-19 2009-06-25 Eberhard-Karls-Universität Tübingen Universitätsklinikum Herstellung von pluripotenten Stammzellen

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053461A1 (fr) * 2000-01-24 2001-07-26 Mcgill University Cellules embryonnaires neuronales multipotentes issues de tissus peripheriques et utilisations correspondantes
WO2001059076A2 (fr) * 2000-02-09 2001-08-16 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Origine premeiotique et postmeiotique de teratomes: cellules souches de teratomes isolees a usages therapeutiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053461A1 (fr) * 2000-01-24 2001-07-26 Mcgill University Cellules embryonnaires neuronales multipotentes issues de tissus peripheriques et utilisations correspondantes
WO2001059076A2 (fr) * 2000-02-09 2001-08-16 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Origine premeiotique et postmeiotique de teratomes: cellules souches de teratomes isolees a usages therapeutiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANDERSON G B ET AL: "DEVELOPMENT OF BOVINE AND PORCINE EMBRYONIC TERATOMAS IN ATHYMIC MICE" ANIMAL REPRODUCTION SCIENCE, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, Bd. 45, Nr. 3, 1996, Seiten 231-240, XP001019645 ISSN: 0378-4320 *
ANDREWS P W: "TERATOCARCINOMAS AND HUMAN EMBRYOLOGY: PLURIPOTENT HUMAN EC CELL LINES" APMIS, COPENHAGEN, DK, Bd. 106, Nr. 1, Januar 1998 (1998-01), Seiten 158-168, XP001106164 ISSN: 0903-4641 *
CHOI S-J ET AL: "DEVELOPMENT OF TUMORS FROM BOVINE PRIMORDIAL GERM CELLS TRANSPLANTED TO ATHYMIC MICE" ANIMAL REPRODUCTION SCIENCE, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, Bd. 52, Nr. 1, 30. Juni 1998 (1998-06-30), Seiten 17-25, XP001027033 ISSN: 0378-4320 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007063198A1 (de) * 2007-12-19 2009-06-25 Eberhard-Karls-Universität Tübingen Universitätsklinikum Herstellung von pluripotenten Stammzellen

Also Published As

Publication number Publication date
WO2004018652A3 (fr) 2004-05-21
AU2003266125A8 (en) 2004-03-11
DE10393633D2 (en) 2005-07-07
AU2003266125A1 (en) 2004-03-11

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