WO2004018652A2

WO2004018652A2 - Method for obtaining diploid, pluripotential stem cells and use thereof

Info

Publication number: WO2004018652A2
Application number: PCT/DE2003/002734
Authority: WO
Inventors: Ulrich Drews; Paul Schweizer; Christian Busch
Original assignee: Ulrich Drews; Paul Schweizer; Christian Busch
Priority date: 2002-08-16
Filing date: 2003-08-14
Publication date: 2004-03-04
Also published as: AU2003266125A1; DE10393633D2; WO2004018652A3; AU2003266125A8

Abstract

The invention relates to a method for obtaining diploid, pluripotential stem cells from tumour tissue which is not a germ tract tumour. Said stem cells are obtained by insulating the individual tissue areas and/or dissolving the cell cluster in a manner known per se and subsequently insulating the cells thus obtained. The invention is characterised in that a coccyx atom is used as tumour tissue. Said cells thus obtained are used for producing organs, organ parts, blood vessels and replacement tissue, and for studying the teratogenicity of substances and environmental conditions.

Description

Process for obtaining diploid, pluripotent stem cells and their use

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.

In the early stages of division of the embryo, an entire organism can arise from each cell (totipotency). This ability is lost when the embryo is divided into embryoblast and trophoblast. The 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.

In principle, 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) have their origin in embryonic germ cells. These migrate as primary germ cells from the yolk sac into the gonadal system. This happens in the 3rd and 4th week of human embryonic development. As soon as they have reached the gonadal system, they enter a proliferation phase. 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.

In addition, 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. In this way, for example, the human stem cell line Terra II was isolated from the lung metastasis of a human seminoma (Andrews in APMIS 106, 158-168, 1998).

Finally, stem cell lines (TC) 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.

Furthermore, 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. Finally, neural adult stem cells (NAS) can also be obtained from the matrix zone of the diencephalon of adult donors.

A large number of attempts have already been made to treat diseases with such cells, such as, for. B. the replacement of dead nerve tissue, for example after a stroke or paraplegia or the replacement of nerve cells in Parkinson's or muscle cells after a heart attack.

The 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.

Up to now, the source of such cells has usually not been transplanted, fertilized egg cells that are left in the in vitro fertilization or the umbilical cord and fetuses of abortions. Another source for their extraction are germline tumors such as the teratoms already mentioned. Teratomas of this type, in particular teratoms of the ovary and testis, do not only arise spontaneously, but can also be induced in the animal model as embryonic carcinomas or teratocarcinomas. However, such cells have been shown to be aneuploid, i.e. that individual chromosomes are not present in the normal number in these cells.

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. However, 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.

The extraction of stem cells from teratoms is known per se. So z. B. by G. R. Martin PNAS (1981), Vol. 78, No. 12, pages 7634-7638 describes the manner in which teratocarcinomas and teratomas develop by injection of mouse embryonic cell lines in mice, from which in turn pluripotent embryonic stem cells can be developed. However, such cells are also derived from the germline and, as is stated in this document, also show all the essential properties of carcinoma cells.

P. W. Andrews describes in 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.

However, the development of tissue from embryonic stem cells as well as from pluripotent cells that are derived from germline cells are ethically controversial due to the special protection of embryos and for this reason at least currently prohibited in many countries.

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.

This goal is achieved by the method defined in the claims.

Surprisingly, it has been found that the coccyx is not a teratoma that develops from parts of the germline. In addition, it was surprisingly found that - although 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.

According to the invention, 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. From the surgical site to the preparation of the tumor, 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. For preparation, it is carefully disassembled using a sharp knife, such as a scalpel, and tissue areas are preferably visually separated from one another under a microscope according to their differentiation. Alternatively, you can make a quick cut from precisely defined tissue pieces and then stain one side of the cut while isolating and cultureing the other side as described above. The target tissue has a typical morphology that corresponds to that of an embryonic blastema tissue. Among the tissue parts thus obtained, 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. Common enzymes used for this purpose are, for example, collagenase, hyaluronidase, pancreatin and streptokinase. 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. 2001, 169-174), Nestin for neuronal stem cells (H Zulewski et al.; Diabetis, March 2001, 521-533), SSEA-1 (stage specific embryonic antigen-3), SSEA-3 and SSEA-4 for pluripotent stem cells (SA Przyborski; Stem-Cells, 2001, 19 (6): 500-4, CD34 for hematopoietic stem cells (H. Klaasen et al.; Neuroscience Letters, Oct. 26, 2001, 312 (3): 180-2), p63 for keratinocytes and stem cells (G. Pellegrini et al.; Proceedings of the National Academy of Sciences of the United States of America, March 13, 2001, 98 (6): 3156-61), AC133 for hematopoietic stem cells (M. Peichev et al.; Blood, 1. Feb. 2001, 95 (3): 952-8, Betal Integrin for stem cells (UB Jensen et al.; Development, June 1999, 126 (11): 2409-18, AA4 for stem cells (vascular endothelial cells, aorta -associated hematopoietic clusters, primitive fetal liver hematopoietic progenitors) (0. Petrenko et al. ; Immunity, June 1999, 10 (6): 691-700, AP (alkaline phosphatase) for stem cells (S. Nagafuchi et al.; FEBS-Letters, July 16, 1999, 455 (1-2): 101-4 , FGF-4 for stem cells (MA Lane et al.; Proceedings of the National Academy of Sciences of the USA, Nov. 9, 1999, 96 (23): 13524-9, Thy-1 for hematopoietic stem cells (BE Petersen et al .; Hepatology, Feb. 1998, 27 (2): 433-45, TIE, TEK for hematopoietic stem cells (M. Yano et al.; Blood, June 15, 1997, 89 (12): 4317-26, OC .3 (oval cell antigen.3) for bile duct, liver stem cells (SH Sigal et al.; Hepatology, April 1994, 19 (4): 999-1006, CK 8 mRNA (cytokeratin 8 mRNA) for prostate stem cells (X. Wang et al.; Chinese Medical Sciences Journal, Dec. 1994, 9 (4): 237-41, Bcl-2 for skin Stem cells (RR Polakowska et al.; Developmental Dynamics, March 1994, 199 (3): 176-88, PSCA (Prostate Stem Cell Antigen) for prostate stem cells (Tetsuro Watabe et al.; Proceedings of the National Academy of Sciences of the USA, Jan 8, 2002, 99 (1): 401-6. By means of these cell markers and antibodies directed against them, it is readily possible to selectively isolate the respective cell from a mixture, such as a suspension with other cells Isolation carried out by means of antibodies, but it is also possible to carry out the isolation using other specific surface markers and thus immobilized receptors capable of binding.

Exact histological examinations within the scope of the invention have shown that a wide variety of tissues develop from colon crypts, which are often also found in coccyx teratoms, such as B. hair, epithelia, pancreas with islet cells, etc. Therefore, these colon structures are also the target of selective cell isolation and cell cultivation.

According to the invention, 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 mucosa. Likewise, 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.

In principle, it is also possible to preserve such cells frozen. Such frozen preservations are also well known to the person skilled in the art and require no further explanation. In individual cases, it has proven to be useful to use special nutrient media. Such media can be determined by a person skilled in the art on the basis of simple experiments.

The pluripotent stem cells obtained with the method according to the invention are particularly suitable for the production of replacement tissues and replacement organs. For example, 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 CKδmRNA 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. According to the invention, it was also found that such stem cells can also be isolated from healthy tissue close to the spine, in particular the coccyx.

However, 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.

Claims

Process for obtaining diploid, pluripotent stem cells and their use

1. A method for obtaining diploid, pluripotent stem cells from a tumor tissue, the tumor tissue not being a germline tumor, by isolating individual tissue areas and / or dissolving the cell assembly in a manner known per se and isolating the cells thus obtained and optionally subsequent cultivation, characterized in that that a coccyx teratoma is used as tumor tissue.

2. The method according to claim 1, characterized in that the tumor tissue and / or the cells isolated therefrom are free of CD3 and CD4 and have no tumor properties.

3. The method according to any one of the preceding claims, characterized in that tissue close to the spine is used.

4. The method according to any one of the preceding claims, characterized in that the cell structure is dissolved by means of one or more enzymes.

5. The method according to any one of the preceding claims, characterized in that cultivation is carried out on feeder cells, in particular fibroblast feeder cells.

6. The method according to any one of the preceding claims, characterized in that colony-forming cell clones are isolated.

7. The method according to any one of the preceding claims, characterized in that the stem cell is a precursor cell for pancreas, islet, neuronal, serous and / or mucous glands, epithelium, bone, bone marrow, cartilage, liver, Is muscle and / or muscle stem cell.

8. The method according to any one of the preceding claims, characterized in that the stem cells are isolated by means of suitable surface markers by FACS and / or magnetic beads in a cell sorter or are cultivated and propagated by means of selective media.

9. The method according to any one of the preceding claims, characterized in that for isolation surface markers selected from CEP-68, Nestin, SSEA-3, SSEA-4, CD34, p63, AC133, Betal Integrin, AA4, AP, FGF-4, Thy-1, TIE, TEK, OC.3, CK 8 mRNA, Bcl-2, PSCA used.

10. Use of cells obtained according to any one of claims 1-9 for the extraction of organs, organ parts, blood vessels and replacement tissue, and for the investigation of the teratogenicity of substances and environmental conditions.