WO2007118658A1

WO2007118658A1 - Method of isolating postnatal and/or adult epithelial stem and precursor cells of the intestine with the aid of a monoclonal antibody

Info

Publication number: WO2007118658A1
Application number: PCT/EP2007/003224
Authority: WO
Inventors: Herwig Brunner; Petra Bareiss
Original assignee: Peter und Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik
Priority date: 2006-04-11
Filing date: 2007-04-11
Publication date: 2007-10-25
Also published as: DE102006017015A1

Abstract

The present invention relates to a method of identifying and/or isolating live postnatal and/or adult epithelial precursor and/or stem cells of the intestine using an antibody or antibody fragment, and methods of diagnosing carcinomas of the intestine and/or precursors thereof. Moreover, the invention relates to live postnatal and/or adult epithelial precursor and/or stem cells of the intestine which are obtainable by the method according to the invention, to pharmaceutical compositions comprising these precursor and/or stem cells, if appropriate in combination with growth factors, and their use for in-vitro assay systems and for the treatment of damaged tissue, in particular damages to the intestinal mucous membrane. The invention furthermore relates to a kit for identifying and/or isolating these precursors and/or stem cells comprising at least one antibody.

Description

Method for isolating postnatal and / or adult intestinal epithelial stem and progenitor cells using a monoclonal antibody

antibody

description

The present invention relates to a method for identifying and / or isolating live postnatal and / or adult epithelial progenitor and / or stem cells of the gut with an antibody or a fragment of the antibody and methods for diagnosing carcinomas of the gut and / or precursors thereof. Moreover, the invention relates to living postnatal and / or adult epithelial progenitor and / or stem cells of the intestine obtainable by the method according to the invention, pharmaceutical compositions comprising these precursor and / or stem cells, optionally in combination with growth factors, and their use for in in vitro test systems and for the treatment of tissue damage, in particular damage to the intestinal mucosa. Furthermore, the invention relates to a kit for identifying and / or isolating these precursor and / or stem cells comprising at least one antibody.

Stem cells are undifferentiated or immature cells that are able to self-renew and differentiate into various specialized cell types. Once differentiated, stem cells can be used to regenerate damaged or defective tissues or organs. Stem cells can basically be of embryonic, fetal or adult origin.

The intestinal mucosa is one of the most regenerative tissues of the organism. There is little information on the proliferation and differentiation of stem cells of the intestinal epithelium and thus on the mechanisms of regeneration in comparison to hematopoietic or neural stem cells. In the large intestine, the stem cells are located in the base of the Intestinal crypts (Bach et al., "Sternal cells: the intestinal stem cell as a paradigm", Carcinogenesis 2000 Mar; 21 (3): 469-476). A human colonic mucosa of 500 μm ² contains about 200 crypts. It is believed that there are between 1 to 36 stem cells per crypte, from which the corresponding cells of the intestinal epithelium, such as resorbing enterocytes, mucin-producing goblet cells and peptide-hormone-producing enteroendocrine cells differentiate (Pötten "Stem cells in gastrointestinal epithelium: numbers, characteristics and death ", Int J Radiat, Biol 1997 May; 71 (5): 573-579). In the small intestine, the stem cells are also found in the crypts, but their position lies on the located in Kryptengrund Paneth'schen granule cells, which also emerge from the stem cells and produce antimicrobial substances. After toxic diseases and infections of the gastrointestinal tract (eg dysentery and cholera), the epithelium can be regenerated within a few days from the crypts. The stem cells undergo a critical mitosis, which again results in a stem cell and an epithelial cell (Adami "The causation of cancerous and other new growths", The British Medical Journal, 1901 March 16, 621-628, Jones "epithelial stem cells", Bioessays, 1997, 19 (8): 683-690).

The intestine fulfills many vital functions. In addition to absorption and secretion performance, the gut has an important immunological function. Not only does the mucosal immune system play an important role as an immune organ of the body, particularly in bacterial and viral immune defense, but it is also involved in disordered immune responses. These include ulcerative colitis and Crohn's disease. In addition to these diseases, intestinal carcinomas are of great clinical importance. A common cause of intestinal carcinoma formation is a misregulation of intestinal stem cells (Wong "Cell Proliferation in Gastrointestinal Mucosa", J. Clin. Pathol 1999, 52: 321-333). Therefore, there is a great therapeutic potential in the discovery of markers in epithelial stem and / or progenitor cells of the intestine, the have higher malignant potential. This would allow the earliest possible prevention or diagnosis of intestinal carcinomas.

The inhibition and activation of the stem cell compartment and the direction of differentiation (determination) depend crucially on the growth factors and extracellular matrix in the microcompartment. Not only cells of the mucosa play a role in the differentiation of epithelial cells, but there is some evidence that neurons and fibroblasts outside the mucosa can influence the proliferation and determination of epithelial stem cells (Bjerknes "Modulation of specific intestinal epithelial progenitors by Enteric neuron ", Proc. Nadl. Acad., USA 2001, 98 (22): 12334-12336; Rizvi" Well-derived stem cells ", Surgery 2005; 127 (6): 585-590). Three important signaling pathways involved in the regulation of the intestinal stem cell compartment are the wnt, noteh and hedgehoc signaling pathways. However, the exact mechanisms of stem cell regulation are still largely unknown.

The isolation of epithelial stem and progenitor cells from the gut is an important methodological component for the generation of new intestinal mucosa in the living organism or in cell culture by means of tissue engineering techniques. A corresponding need for functional mucosa for clinical applications exists, for example, in short intestine syndrome (short instestin syndrome) and in intestinal ulcers.

A much greater potential could lie in the potential transdifferentiation potential of intestinal stem cells. The intestine is next to the liver, the pancreas and the lungs to the endodermal cotyledon. Developmental biology has shown that the liver, pancreas and lungs develop from the foregut (Gilbert "Developmental Biology", 7 ^m Edition, Sinauer, 510 - 513). isolated - A -

Stem cells could possibly differentiate into hepatocytes, pancreatic cells and lung epithelial cells using appropriate growth factors. This would open up enormous market potential for the use of intestinal stem cells in the field of regenerative medicine.

While recognizing the therapeutic potential of stem cells, the use of human embryonic stem cells has been the subject of ethical and social criticism, as it involves the destruction of human embryos for the isolation and production of these cells. The isolation and proliferation of adult human stem cells with characteristics similar to those of embryonic stem cells overcome this ethical dilemma.

The prerequisite for a targeted isolation of intestinal precursor and stem cells of the intestinal tract is, on the one hand, a surface epitope which is expressed by the corresponding cells, and one suitable antibody which recognizes this epitope on living cells.

In a second step, this antibody can be used to carry out various isolation methods, for example via flow cytometry (FACS) or magnetic cell separation (MACS), and thus selectively isolate and enrich the cells.

At the molecular level, embryonic stem cells express a variety of genes that are specific for the undifferentiated state of these cells. The Stage-Specific Embryonic Antigens-1, -3 and -4 (SSEA-1, SSEA-3, SSEA-4) are glycoproteins expressed in early embryonic development and therefore are markers for embryonic stem cells (Solter and Knowles, Acad., U.S.A. 75: 5565-5569; Kannagi et al., 1983, EMBO J. 2: 2355-2361). The increased expression of the enzyme alkaline phosphatase (AP) is another marker associated with undifferentiated embryonic stem cells. Further Stem cell / progenitor cell markers include the membrane glycoprotein prominin / AC133, the transcription factor Tcf-4 and the transcription factor Cdx-1. The latter two factors are also found in the stem cell compartment of the fetal and adult intestine. However, since these are intracellular markers, they are unsuitable for the identification of living cells. So far, there are hardly any suitable markers for the identification and isolation of vital stem and progenitor cells of the intestinal epithelium.

Gong et al. ("Expression of carbohydrate antigen 19-9 and stage-specific embryonic antigen 1 in non-tumorous and tumorous epithelia of the human colon and rectum", J. Natl. Cancer Inst. 1985, 75 (3): 447-454) and Hara et al , 1987, 30 (6): 440-443) describe the expression of stage-specific embryonic antigen 1 (SSEA) ("expression of stage-specific embryonic antigen 1 in the noncancerous colorectal epithelia of familial polyposis coli", Dis Colon Rectum. 1), which is also referred to as CD15, LNF III (lacto-N-fucopentaose III) or Lewis antigen (Vogel et al. ("Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells", Haematologica 2003); 88: 126-133) in the crypts of the intestine on fixed cells These publications describe that the expression of SSEA-1 is associated with neoplastic transformation and progression and that this antigen is a suitable marker for detecting neoplastic transformation in human Intestine represents.

A suitable antibody that binds to the antigen CD15 / SSEA-1 with high selectivity and with high affinity is the commercially available antibody W6D3 obtained by immunization of a Balb / c mouse with the retinoblastoma cell line WERI-RB1 and subsequent fusion of Immune spleen cells with the myeloma cell line SP2 / 0 was generated. This antibody is deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under number DSM ACC 2760 under the Budapest Treaty. So far, however, no method for isolating epithelial cells in Kryptengrund of the intestine with an antibody or antibody fragment is described in the prior art, which binds to the antigen SSEA-1. Likewise, the strong labeling of the antibody derived from the hybridoma line W6D3 on precursor and stem cells of the intestinal epithelium has not been demonstrated.

In addition, the detection of SSEA-1 with antibodies has only been described so far on fixed epithelial cells in the crypts of the intestine, but not on living cells (Gong et al., Supra). However, the selective labeling of live precursor and stem cells is the prerequisite for successful isolation and accumulation of these cell populations. Also for the identification of the antigen SSEA-1, a live marker is advantageous for performing a live diagnosis of SSEA-1 expressing intestinal tumor epithelial cells.

Against this background, therefore, there is a need to provide a method by which epithelial progenitor and / or stem cells can be identified and / or isolated.

This object is achieved in accordance with the invention by providing a method for identifying and / or isolating live postnatal and / or adult epithelial progenitor and / or stem cells of the intestine with an antibody or a fragment of the antibody, the antibody being available from the Deutsche Sammlung von Microorganisms and cell cultures (DSMZ) under number DSM ACC 2760 according to the Budapest Treaty deposited hybridoma cell line W6D3 is available.

Surprisingly, the inventors were able to show for the first time that it is possible with the new method according to the invention using the antibody derived from the hybridoma cell line W6D3, postnatal and / or adult progenitor and / or stem cells of the lower To stain the crypts of the intestine not only in the fixed but also in the living state (see Figure 1, 2).

More particularly, the invention relates to a method for identifying living postnatal and / or adult epithelial progenitor and / or stem cells of the intestine comprising the steps of: a) providing at least one antibody or fragment of the antibody, wherein the antibody is derived from the one of German Collection of Microorganisms and Cell Cultures (DSMZ) under the number DSM ACC 2760 under the Budapest Treaty deposited hybridoma cell line W6D3 is available. b) Providing a sample of living intestinal epithelial cells. c) contacting the at least one antibody or a fragment of the antibody from step a) with the sample from step b). d) identifying those living intestinal epithelial cells of the sample from step b) which bind to the at least one antibody or to a fragment of the antibody from step a).

According to the invention, the sample of living intestinal epithelial cells is provided by first preferably the single-layered intestinal epithelium of the underlying lamina propria by means of an enzymatic

Method using a thermolysin solution or by means

Use of calcium-free media to which calcium-binding substances can be added is separated to remove positive cell populations outside the epithelium.

The reason for the separation of the intestinal epithelial cells from the lamina propria is that in the human intestine, the antibodies originating from the hybridoma cell line W6D3, in addition to the intestinal epithelial cells, also stain granulocytes of the underlying lamina propria and of the underlying submucosa. In the enzymatic method, thermolysin, trypsin, collagenase, dispase and / or papain and particularly preferably thermolysin and papain are preferably used as the enzyme. The chemical separation of the intestinal epithelium is preferably carried out using calcium-binding substances, in particular chelating agents such as

Ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and nitrilotriacetic acid (NTA), and more preferably using EDTA.

The subsequent identification of those living intestinal epithelial cells from the sample which bind to the antibody is carried out according to the invention preferably by incubation of the intact crypts, which were carefully isolated from the lamina propria, with the antibody. The staining of the living intestinal progenitor cells of the lower crypt ground with the antibody produced from the hybridoma cell line W6D3 is clearly visible in Figure 2. Figure 2A shows an immunofluorescence image of a crypt stained with the antibody produced from the hybridoma cell line W6D3. The crypt ground (arrow) shows a clear signal. Figure 2B represents a bright field image of the same crypt from A. In a further preferred embodiment of the invention, the staining with the antibody can also be carried out on fixed cells. Thus, according to Figure 1, an immunohistological staining of fixed colon tissue with the antibody produced from the hybridoma cell line W6D3 is shown. Figure 1A is a histological illustration of antibody binding to human acetone-fixed Kyrostat sections of the colon. Clearly a specific marking of the crypts bottom (arrows) can be recognized. Figure 1 B shows proliferating progenitor and stem cells immunocytochemically stained with the KI67 antibody. The same epithelial cells were stained in the lower part of the crypts (arrows), as was the case with the antibody from the hybridoma cell line W6D3 in Figure 1 A. In Figure 1 B could thus be clearly identified with the KI-67 proliferation marker It can be shown that the antibody derived from the hybridoma cell line W6D3 stains the proliferative region in the crypts.

A further embodiment of the present invention preferably relates to a method of isolating the above-mentioned cells, comprising the steps of: a) separating the live intestinal epithelial cells from the above-mentioned sample, which consists of intact crypts. b) contacting at least one antibody or a fragment of the antibody which is known from the German

Microorganisms and cell cultures (DSMZ) under the number DSM ACC 2760 according to the Budapest Treaty deposited hybridoma cell line W6D3 is available, with the isolated intestinal epithelial cells from step a). c) isolating those isolated intestinal epithelial cells from step b) which bind the at least one antibody or a fragment of the antibody.

The separation (dissociation) of the epithelial cells mentioned under step a) is preferred for further cell isolation. The isolated epithelial cells can be labeled with antibodies and can then be separated.

The antibody obtainable from the hybridoma cell line W6D3 preferably binds to the antigen SSEA-1. This antigen is expressed in early embryonic development. In addition, expression of SSEA-1 is associated with neoplastic transformation and progression. Thus, SSEA-1 is a suitable marker for both embryonic stem cells and intestinal cells associated with neoplastic transformation and progression.

The antibody used according to the invention, which originates from the hybridoma cell line W6D3 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the number DSM ACC 2760 in accordance with the Budapest Treaty, is preferred by immunization of a Balb / c mouse with the retinoblastoma cell line WERI -RB1 and subsequent Fusion of immune spleen cells generated with myeloma cell line SP2 / 0. It has excellent properties regarding specificity and affinity for the antigen SSEA-1 and binds in the intestine with correspondingly high selectivity to intestinal stem and / or progenitor cells.

Suitable antibodies within the scope of the present invention may be monoclonal, polyclonal, monovalent and bivalent fragments.

To produce the antibodies, various hosts, including goats, rabbits, rats, mice, humans, and other hosts may also be immunized by injection with the protein or any fragment or oligopeptide thereof having immunogenic properties. Depending on the host species, various adjuvants may be used to enhance the immunological response. Peptides, fragments or oligopeptides are preferably used for the induction of antibodies of the protein which have an amino acid sequence of at least five amino acids and more preferably of at least ten amino acids.

Particularly preferred within the scope of the present invention are monoclonal antibodies. Monoclonal antibodies can be prepared using techniques that provide for the production of antibody molecules by continuous cell lines in culture. These techniques include hybridoma, in particular human B-cell hybridoma and EBV hybridoma. The production of monoclonal antibodies by the fusion of spleen cells from immunized mice and myeloma cells was already described in 1975 by Kohler and Milstein ("Continuous cultures of fused cells secreting antibodies of predefined specificity" Nature, 1975, 256: 495-497). The techniques for the chemical selection of the hybridomas for the production of monoclonal antibodies resulting from such a fusion and the subsequent isolation of cell clones which secrete individual antibodies are also known in the art. In addition, methods for the production of genetically engineered antibodies, such as chimeric antibodies can be used. In this case, preferably constant regions in a mouse antibody are replaced by constant regions of a human antibody. Alternatively, methods for the production of single-chain antibodies may be employed to produce, for example, single-chain antibodies which in a preferred embodiment are specific for the SSEΞA-1 protein of the invention. These are obtained by engineering a construct from the gene segments for the two antibody variable regions linked by a segment for the peptide.

In the context of the present invention, instead of the complete antibody, it is also possible to use a fragment of the antibody without this being expressly mentioned in each case. By "fragment" is meant any fragment of the antibody that retains the antigen binding function, preferably for the SSEA-1 antigen of the antibody. Such fragments include Fab, F (ab ') ₂ , Fv ₁ ScFv, and other fragments such as complementarity determining region (CDR) fragments, as well as fragments produced by a Fab expression library. The fragments mentioned have the binding specificity of the antibody and can also be recombinantly produced by known methods. The F (ab ') ₂ fragments can be obtained by pepsin digestion of the antibody molecule and the Fab fragments by reducing the disulfide bridges of the F (ab') ₂ fragments or by papain digestion of the antibody. Alternatively, a Fab expression library can be constructed to allow rapid and easy identification of monoclonal antibody fragments with the desired specificity.

According to the invention, the immunoglobulins IgG, IgA, IgM, IgD and IgE can be used as antibodies. It is preferably an IgG Antibodies, and more preferably an IgGI antibody, since this is due to its smaller size, for example, against IgM antibodies particularly well suited to isolate cells by magnetic cell separation (MACS, Magnetic Cell Sorting). Furthermore, this antibody recognizes an epitope that is not recognized by other SSEA-1 antibodies.

For further cell isolation, the epithelial cells are isolated according to the invention (dissociated). It is particularly preferred to separate the intestinal epithelial cells by means of an enzyme / DNase solution. The enzymes used are preferably collagenases, trypsin, dispases, thermolysin and / or papain. Papain is particularly preferred in the context of the present invention. With a papain / DNase solution, according to extensive investigations, the inventors advantageously achieved optimal cell separation with simultaneously high cell vitality. Especially with papain as an enzyme, an improved yield of epithelial cells could be observed which maintained their ability to express antigens, in particular of the SSEA-1 antigen, which were also free of mesenchymal cells and which allows the maintenance of continuously growing epithelial cells. Thus, papain has been shown to be a particularly gentle enzyme for cell dissociation while maintaining integrity, particularly the epitope structure of the cells. Thus, Figure 3 shows a FACS analysis of dissociated human intestinal epithelial cells. A subpopulation of the labeled epithelial cells was clearly recognizable, which were labeled with the antibody produced from the hybridoma line W6D3.

For the subsequent isolation of intestinal stem and / or progenitor cells, the following methods are preferably used according to the invention: flow cytometry (Fluorescent Activated Cell Sorter (FACS)), magnetic cell sorting (MACS) or binding of cells to an antibody, which is preferred monoclonal antibody which is immobilized on a support, as for example in the Column chromatography is the case. In flow cytometry, cells are loaded with antibodies that are coupled on the one hand specifically for a surface marker and on the other hand with a fluorescent dye. Those cells that are marker-positive fluoresce while the negative cells remain dark. It can thus be determined which proportion of a cell population is marker-positive. At the same time, a flow cytometer allows to detect the size and granularity of cells. In the case of magnetic cell separation, the cells are labeled with magnetic beads, which beads may be coupled to the antibodies, for example. In the context of the present invention, the isolated intestinal epithelial cells are particularly preferably isolated by means of FACS.

After mixing the cell suspension with the antibody, those cells preferentially bind the antibody expressing the SSEA-1 antigen, whereupon these cells can be isolated from the non-antibody bound cells by the method described above.

As can be seen from Figure 3, could with the inventive

Method using the hybridoma cell line W6D3 derived antibody or a fragment of the antibody, after a

Dissociation of intestinal crypt material a well-defined

Subpopulation be represented by means of FACS analysis. In the present

The invention has been experimentally shown to be possible with the antibody of the cell line W6D3, unfixed live cells which are the binding site for the specific antigen, in particular the SSEA-1.

Antigen even after dissociation, of the lower

To identify and isolate crypts reason.

For identification and / or isolation of postnatal and adult intestinal epithelial progenitor and / or stem cells is preferred

Mammalian intestine, more preferably human intestine.

These are according to the invention to large and / or small intestine, there Both precursor and / or stem cells are found in both the crypts of the large and small intestines.

Furthermore, the invention relates to postnatal and / or adult epithelial progenitor and / or stem cells of the intestine, which are obtainable by the identification and / or isolation method described above.

The postnatal and / or epithelial progenitor and / or stem cells of the intestine are preferably cultured in a suitable culture medium to perform cell culture experiments, for example for pharmacological and diagnostic studies.

The invention further relates to a pharmaceutical composition comprising live postnatal and / or adult epithelial progenitor and / or stem cells of the intestine obtainable by the above-mentioned method. In a preferred embodiment, the pharmaceutical composition additionally comprises suitable

Growth factors, hormones, proteins and / or a matrix. The inhibition and activation of the stem cell compartment and the direction of the differentiation (determination) depend crucially on the growth factors and the extracellular matrix in the microcompartment. Therefore, isolated stem cells can differentiate into hepatocytes, pancreatic cells and lung epithelial cells using appropriate growth factors. This represents an extensive potential for the use of stem cells in the field of regenerative medicine. Suitable growth factors for the pharmaceutical composition according to the invention include epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF) and transforming growth factor α (TGF -α), which also binds to the same receptor as TGF. These two growth factors stimulate gastrointestinal epithelial cell proliferation. Furthermore, the pharmaceutical Composition optionally optionally hormones and / or peptides. Preferred is enteroglucagon, which contains the glicentin-related pancreatic polypeptide (GRPP), glucagon, oxyntomodulin and the glucagon-like peptides GLP-1 and GLP-2. In particular, GLP-2 has been shown to be a potent stimulator of intestinal epithelial proliferation. Another preferred peptide is gastrin, which has a trophic effect on intestinal cell lines. Furthermore, prostaglandins as well as androgens and estrogens are preferred which can also stimulate cell proliferation.

The pharmaceutical composition according to the invention may further contain pharmaceutically customary carriers, adjuvants, additives and suitable buffers. Suitable buffers include, for example, TRIS, HCl, glycine and phosphate. The pharmaceutical composition may further contain suitable diluents, preferably aqueous solutions of NaCl, lactose, mannitol, water and alcohols. Suitable additives include, for example, detergents, solvents, antioxidants and preservatives. An overview of the substances usable for such compositions is e.g. in the latest issue of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

The invention further relates to the use of the precursor and / or stem cells obtained by the identification and / or isolation method according to the invention for the production of a

Drug for the treatment of tissue damage in vivo and ex vivo.

According to the invention, the precursor and / or stem cells obtained can be used for the treatment of tissue damage in the living organism or in cell culture by means of tissue engineering techniques. Tissue engineering is the production of functional artificial cell and tissue associations based on cultured cells and various artificial matrices. In the context of the present invention Tissue engineering for the treatment of tissue damage involves a number of different methodological approaches. These include the application of biologically active molecules, such as growth factors, hormones, proteins, etc., which can influence or restore intestinal functions as direct injections into the intestine (in vivo treatment). These include the injection of resorbable polymers as growth factor transporters, the transmission of transgenes that stimulate the formation of growth factors in the target organs, and the implantation of three-dimensional matrices (polymer size) that allow colonization by the body's own cells. Other tissue engineering methodologies include the transplantation of in vitro precursor and / or stem cells; artificially prepared ZeII polymer matrices for the implantation of precursor and / or stem cells or the extracorporeal (ex vivo) production of organ cells from precursor and / or stem cells.

The precursor and / or stem cells obtained by the method according to the invention are preferably used for the treatment of damage to the intestinal mucosa. The precursor and / or stem cells are particularly preferably used for clinical applications in which there is a corresponding requirement for functional mucosa, as is the case in short intestine syndrome, intestinal ulcers and / or intestinal ischaemia.

In a further embodiment of the present invention, preferably isolated intestinal stem cells are transdifferentiated using suitable growth factors in hepatocytes, pancreatic cells and lung epithelial cells. Transdifferentiation is the transformation of one cellular phenotype into another. Transdifferentiation is associated with a change in cell morphology associated with a change in the program of gene expression. Suitable growth factors for converting intestinal stem cells into hepatocytes, pancreatic cells and lung epithelial cells are preferably Fibroblast Growth Factor (FGF), Dexamethasone, Insulin, Nicotinamide, Epidermal Growth Factor (EGF), Hydrocortisone, Transferrin, Glutamycin, Amphotericin, Progesterone. For the purposes of the present invention, dexamethasone and insulin are particularly preferably used for conversion into hepatocytes and nicotinamide for conversion into pancreatic cells. The therapeutic potential of this tissue reprogramming due to the transdifferentiation potential of intestinal stem cells offers far-reaching opportunities for new treatment strategies in the field of regenerative medicine.

Therefore, it is particularly preferred to use the precursor and / or stem cells obtained by the method according to the invention for the treatment of tissue damage to the liver, pancreas and / or lung, particularly preferably in combination with growth factors.

Further preferred is the use of the precursor and / or stem cells or a culture of such cells for the preparation of an agent for the diagnosis of carcinomas of the intestine and / or precursors thereof.

The use of the precursor and / or stem cells and / or the culture of these cells for the preparation of an agent for characterizing cellular reactions to biological, chemical or pharmacological agents, is provided in the context of the present invention, in particular the use in a screening - Assay for the identification of drugs for the prevention and / or treatment of intestinal-associated diseases is intended.

The diseases associated with the intestine are preferably selected from the group consisting of damage to the intestinal mucosa, short bowel syndrome, intestinal ulcer and / or carcinoma of the intestine and / or precursors of carcinoma of the intestine. Another embodiment of the present invention relates to the use of the above-described antibody or a fragment of the antibody of this antibody to identify and / or isolate live postnatal and / or adult epithelial progenitor and / or stem cells of the crypt base of the gut.

Furthermore, the present invention relates to the use of the antigen SSEA-1 for identifying and / or isolating live postnatal and / or adult epithelial progenitor and / or stem cells of the crypts base of the intestine. So far, only a few suitable markers for stem cells of the crypts reason of the intestine are known. The Stage Specific Embryonic Antigen-1 (SSEA-1) is in the context of the present invention, a highly suitable marker, since it is expressed both in the crypts of the gut in healthy tissue and in larger quantities in pre- and / or carcinogenic tissues of the intestine. Expression of SSEA-1 has been found to be associated with neoplastic transformation and progression in the gut. Thus, the SSEA-1 antigen is an ideal marker for both isolating healthy postnatal and / or adult intestinal epithelial progenitor and / or stem cells, as well as for diagnosing intestinal cancers and / or precursors thereof.

Therefore, the invention also relates to a method for the diagnosis of carcinomas of the intestine and / or precursors thereof, which comprises the following steps: a) providing at least one antibody and / or a fragment of the antibody which is known from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under the number DSM ACC 2760 according to the Budapest Treaty deposited hybridoma cell line W6D3 is available. b) providing a sample of living intestinal epithelial cells of a subject. c) contacting the at least one antibody or a fragment of the antibody from step a) with the sample from step b). d) identifying those living intestinal epithelial cells of the sample from step b) which bind to the at least one antibody or a fragment of the antibody from step a). e) determining the extent of the reaction of the at least one antibody or a fragment of the antibody from step a) with the identified living intestinal epithelial cells from step d), as a measure of the diagnosis.

For example, the detection of living cells to which the above-mentioned antibody specifically binds provides a significant advantage in diagnostic procedures during endoscopy in a patient's intestinal lumen, e.g. for tumor diagnosis.

The method according to the invention is particularly suitable for the diagnosis of such carcinomas of the intestine and / or precursors thereof selected from the group consisting of colorectal carcinoma, familial adenomatous polyposis coli, hereditary non-polyposis colorectal cancer, ulcerative colitis, Chron's disease, chronic ulcerative colitis, benign adenoma of the colon.

The invention further relates to a kit for identifying and / or isolating living postnatal and / or adult epithelial progenitor and / or stem cells of the intestine which comprises at least one antibody or a fragment of this antibody as described above.

The following examples illustrate the stated advantages of the method according to the invention for identifying and / or isolating living postnatal and / or adult epithelial progenitor and / or stem cells of the intestine with an antibody or a fragment of the antibody derived from the hybridoma cell line W6D3. pictures:

Figure 1: Immunohistological staining of fixed colon tissue with the antibody produced from the hybridoma line W6D3.

Figure 1 A shows the histological picture of antibody binding to human acetone-fixed cryostat sections of the colon. Clearly a specific marking of the crypts bottom (arrows) can be recognized. Figure 1 B shows proliferating progenitor and stem cells immunocytochemically stained with the KI67 antibody. The same epithelial cells were stained in the lower part of the crypt (arrows), as was the case with the antibody from the hybridoma cell line W6D3 in Figure 1A.

Figure 2: Live staining of intestinal crypt cells with the antibody produced from the hybridoma cell line W6D3.

A: Immunofluorescence image of a crypt stained with the antibody produced from the hybridoma cell line W6D3. The crypt ground (arrow) shows a clear signal. B: bright field image of the same crypt from A.

Figure 3: FACS analysis of dissociated human intestinal epithelial cells. Significantly, a subpopulation of the labeled epithelial cells can be recognized, which were labeled with the antibody produced from the hybridoma cell line W6D3. example 1

Method for cell separation (dissociation) of the intestinal epithelium

The colon tissue was washed three times with a cold 1% penicillin / streptomycin HBSS (HBBS = Hanks Basal Salt Solution) buffer solution. Thereafter, the cautious mechanical removal of the epithelial layer, including the underlying lamina propria, from the submucosa located below the lamina propria, took place by means of two preparation tweezers.

Subsequently, the lamina propria, on which the epithelial layer is located, was incubated for further separation of the crypts in a 2 mM EDTA-HBSS solution without Ca ² VMg ^2+, and the detaching crypts were rinsed out. Alternatively, instead of the EDTA solution, the submucosa was first incubated for 1 h in a 0.05% thermolysin solution at room temperature, and the crypts were rinsed from the lamina propria. Thereafter, the centrifugation (Eppendorf 5810 R) of the crypts was carried out at 8 g for 4 min and the inclusion of the pellet in 10 ml of HBSS solution. The centrifugation step was repeated twice to remove single cells from the suspension.

Part of the intact crypt material was used for immunocytochemistry with the hybridoma cell line antibody W6D3.

The remaining crypts were incubated in a papain solution (papain 0.2% / 0, 05% Dnase / 5 mM L-cysteine; SIGMA) for 1, 5 - incubated for 3 h at 37 ⁰ C and then separated with a 2 ml pipette (dissociated). The enzymatic digestion was stopped by means of a trypsin inhibitor solution (10 mg / ml trypsin inhibitor, SIGMA). Subsequently, the cells were centrifuged at 350 g for 4 min, the pellet was taken up in PBS and the cell suspension was filtered through a filter with a pore diameter of 40 μm. The single suspension could then be used for cytometric analysis. Example 2

Staining the cells for flow cytometry

For the cytometric analyzes, the dissociated epithelial cells were taken up in 20 μl of polyglubin per 10 ⁶ cells and then incubated with 25 μl of the

Antibody incubated at 4 ⁰ C for 15 min in reaction tubes. After one

Washing in FACS buffer (PBS, 0.1% BSA, 0.025% NaN ₃ ) was used

Cells with a goat anti-mouse IgG1-phycoerythrin antiserum (1:15) for

15 min at 4 ⁰ C incubated. After another wash, the cells were submerged with a flow cytometer FACSCalibur, Becton Dickinson

Using the Cell Quest software (Becton Dickinson).

Example 3 Immunohistological staining

For histological examinations, first human intestinal tissue was frozen and 12 μm thick cryostat sections were made in the cryotome. After a 10 minute fixation with ice-cold acetone followed by three washes, the tissue was blocked with 10% pig serum and the subsequent incubation of the antibody overnight at 4 ^° C. After three washes for 5 min each, the tissue was treated with 3% H ₂ O ₂ blocked and after three further washes with a biotinylated goat anti-mouse IgG antibody incubated for 30 min. Visualization was performed using an avidin-biotin enhancer kit (DAKO) and a diaminobenzidine (DAB) stain. The binding of the antibody was additionally performed on intact crypts. For this purpose, the crypts were previously carefully separated by means of an EDTA buffer solution by gentle rinsing with a pipette from the lamina propria. After several washes, detection of the first antibody produced by the hybridoma cell line W6D3 was done with a goat anti-mouse IgG-Cy3 labeled fluorescent second antibody. The analysis of the labeled cells was carried out with a fluorescence microscope (Zeiss).

Claims

claims

A method for identifying and / or isolating live postnatal and / or adult epithelial precursors and / or adults

5 stem cells of the intestine with an antibody or a fragment of the

Antibody, wherein the antibody is obtainable from the hybridoma cell line W6D3 deposited under the number DSMZ of the German Collection of Microorganisms and Cell Cultures (DSMZ) under the Budapest Treaty.

10

2. A method for identifying the cells according to claim 1, comprising the following steps: a) providing at least one antibody or a fragment of the antibody as defined in claim 1. i5 b) Providing a sample of living intestinal epithelial cells. c) contacting the at least one antibody or a fragment of the antibody from step a) with the sample from step b). d) identifying those living intestinal epithelial cells of the sample from step b) which are linked to the at least one antibody or to a fragment

20 of the antibody from step a) bind.

3. A method for isolating the cells of claim 1 or 2, comprising the following steps: a) separating the intestinal epithelial cells from step b) of claim 2. 5 b) contacting the at least one antibody or a

Fragment of the antibody from step a) of claim 2 with the isolated intestinal epithelial cells from step c) of claim 2. c) isolating those isolated intestinal epithelial cells from step b) containing the at least one antibody or a fragment of the antibody 0 from step a) of claim 2 bind.

4. The method according to any one of the preceding claims, characterized that the antibody binds to the antigen SSEΞA-1.

5. The method according to any one of the preceding claims, characterized in that one uses intestine of mammals, preferably human intestine.

6. The method according to any one of the preceding claims, characterized in that one uses large intestine and / or small intestine.

7. The method according to any one of the preceding claims, characterized in that one uses as antibodies a monoclonal antibody or a fragment of a monoclonal antibody.

8. The method according to any one of the preceding claims, characterized in that the antibody is an IgG antibody.

9. The method according to any one of the preceding claims, characterized in that the antibody is an IgGI antibody.

10. The method according to claim 1 or 3 to 9, characterized in that isolating the isolated intestinal epithelial cells by flow cytometry (FACS), magnetic cell separation (MACS) and / or column chromatography.

11. The method according to claim 1 or 3 to 10, characterized in that one separates the intestinal epithelial cells by means of an enzyme / DNAse solution.

12. The method according to claim 11, characterized in that one selects the enzyme from the group consisting of collagenases, trypsin, dispases, thermolysin and / or papain.

13. Postnatal and / or adult epithelial progenitor and / or stem cells of the intestine, obtainable by a method according to claim 1 or 3 to 12.

14. Culture of postnatal and / or epithelial progenitor and / or stem cells of the intestine according to claim 13.

A pharmaceutical composition comprising live postnatal and / or adult epithelial progenitor and / or stem cells of the intestine according to claim 13, optionally in combination with growth factors, hormones, proteins and / or a matrix.

16. Use of precursor and / or stem cells according to claim 13 for the manufacture of a medicament for the treatment of

Tissue damage in vivo and ex vivo.

17. Use of the precursor and / or stem cells according to claim 13 or 16 for the manufacture of a medicament for the treatment of damage to the intestinal mucosa.

18. Use of the precursor and / or stem cells according to claim 13 or 16 to 17 for the manufacture of a medicament for the treatment of the short bowel syndrome ("short-intestine" syndrome) and / or the intestinal ulcer etc.

19. Use of the precursor and / or stem cells according to claim 13 or 16 to 18 for the manufacture of a medicament for the treatment of Tissue damage to the liver, pancreas and / or lungs.

20. Use of the precursor and / or stem cells according to claim 13 and / or the culture according to claim 14 for the preparation of an agent for the diagnosis of carcinomas of the intestine and / or precursors thereof.

21. Use of the precursor and / or stem cells according to claim 13 and / or the culture according to claim 14 for the preparation of an agent for characterizing cellular reactions to biological, chemical or pharmacological agents.

Use according to claim 21 in a screening assay for the identification of drugs for the prevention and / or treatment of intestinal-associated diseases.

23. Use according to claim 22, characterized in that the diseases associated with the intestine are selected from the group consisting of damage to the intestinal mucosa, short bowel syndrome, intestinal ulcer and / or carcinomas of the intestine and / or precursors of carcinomas of the intestine.

24. Use of the antibody according to any one of claims 1 to 3 or 7 to 9 or a fragment of the antibody for identifying and / or isolating live postnatal and / or adult epithelial progenitor and / or stem cells of the crypts reason of the intestine.

25. Use of the antigen SSEA-1 for identifying and / or isolating live postnatal and / or adult epithelial progenitor and / or stem cells of the crypts base of the intestine.

26. A method for in vitro and / or in vivo diagnosis of carcinomas of the intestine and / or precursors thereof, comprising the following steps: a) providing at least one antibody as defined in claim 1 and / or a fragment of the antibody. b) providing a sample of living intestinal epithelial cells of a subject.

5 c) contacting the at least one antibody or a

Fragment of the antibody from step a) with the sample from step b). d) identifying those living intestinal epithelial cells of the sample from step b) which bind to the at least one antibody or a fragment of the antibody from step a). lo e) determining the extent of the reaction of the at least one

Antibody or a fragment of the antibody from step a) with the identified live intestinal epithelial cells from step d), as a measure of the diagnosis.

i5

27. The method according to claim 26, characterized in that carcinomas of the intestine and / or their precursors are selected from the group consisting of colorectal carcinoma, familial adenomatous polyposis coli, hereditary non-polyposis Colorectal

20 Cancer, ulcerative colitis, chronic ulcerative colitis, Morbus

Chron, benign adenoma of the colon etc.

A kit for identifying and / or isolating live postnatal and / or adult epithelial progenitor and / or stem cells of the intestine comprising at least one antibody according to one of

Claims 1 to 3 or 7 to 9 or a fragment of the antibody.

29. A method of treating tissue damage in vivo and ex vivo, comprising administering to a pharmaceutically effective amount of a pharmaceutical composition, the living intestinal postnatal and / or adult epithelial progenitor and / or stem cells of claim 13, optionally in combination with growth factors, Hormones, proteins, and / or a matrix, to a patient.