DE102006017015A1 - Method for isolating postnatal and adult intestinal epithelial stem and progenitor cells using a monoclonal antibody - Google Patents

Abstract

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

Description

The The present invention relates to a method of identification and / or isolating live postnatal and / or adult epithelial Precursor and / or stem cells of the intestine with an antibody or a fragment of the antibody and methods for diagnosing carcinomas of the intestine and / or precursors from that. Furthermore the invention relates to living postnatal and / or adult epithelial Precursor- and / or stem cells of the intestine obtainable by the method according to the invention, pharmaceutical compositions comprising these precursors and / or Stem cells, optionally in combination with growth factors, as well as their use for in vitro test systems and for the treatment of tissue damage, in particular damage 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 renew itself and into different specialized cell types too differentiate. Once they are differentiated, stem cells can be used for regeneration from damaged or defective tissues or organs. stem Cells can in principle embryonic, fetal or of 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., "Stem 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 (Potten "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).

Of the Intestine met many vital functions. In addition to the absorption and secretion performance has the intestine has an important immunological function. The mucosal immune system not only has a significant role as an immune organ of the body, in particular in bacterial and viral immune defense, but is also on involved in misfired immune responses. These include the Ulcerative colitis and Crohn's disease. Besides these diseases have the intestinal carcinomas have great clinical significance. One frequently Reason in the intestinal carcinoma is a dysregulation of intestinal stem cells (Wong "Cell proliferation in gastrointestinal mucosa ", J. Clin. Pathol 1999, 52: 321-333) There is a big one therapeutic potential in the discovery of markers in epithelial Parent and / or progenitor cells of the intestine, which is a higher one have malignant potential. This could be as early as possible prevention or diagnosis of intestinal Carcinomas are achieved.

The Inhibition and activation of the stem cell compartment and the direction The differentiation (determination) depend crucially on the growth factors and the extracellular Matrix in the microcompartment off. Not only cells seem to be there of the mucosa in the differentiation of epithelial cells play a role but there is some evidence that even neurons and Fibroblasts outside the mucosa the proliferation and determination of the epithelial Can affect stem cells (Bjerknes' modulation of specific intestinal epithelial progenitors by enteric neuron ", Proc. Nadl. Acad. Sci. USA 2001, 98 (22): 12334-12336; Rizvi's "Good-Derived Stem Cells", Surgery 2005; 127 (6): 585-590). Three major signaling pathways involved in the regulation of the intestinal Stem cell compartments involved are the wnt, notch and hedgehoc Signal paths. The exact mechanisms of stem cell regulation are but still largely unknown.

The Isolation of epithelial stem and progenitor cells from the intestine is an important methodological component for the generation of new intestinal mucosa in the living organism or in cell culture using techniques of tissue engineering. A corresponding need for functional Mucosa for Clinical applications include, for example, the short bowel syndrome ("short instestine" syndrome) and at 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. It is known from developmental biology that the liver, pancreas and lungs develop from the foregut (Gilbert Developmental Biology, 7 ^th Edition, Sinauer, 510-513). Isolated stem cells could possibly differentiate into hepatocytes, pancreatic cells and lung epithelial cells using suitable growth factors. This would open up enormous market potential for the use of intestinal stem cells in the field of regenerative medicine.

While that The therapeutic potential of stem cells is recognized Use of human embryonic stem cells Subject of ethical and social criticism, as these involve the destruction of human embryos for isolation and generating these cells is connected. The isolation and propagation adult human stem cells with characteristics similar to those of embryonic stem cells this ethical dilemma.

requirement for one Targeted isolation of intestinal progenitor and stem cells of the darmeptihel is, on the one hand, a surface epitope that is expressed by the respective cells, and a suitable one Antibody, which recognizes this epitope on living cells.

With this antibody can in a second step, different isolation methods, for example via a Flow cytometry (FACS) or magnetic cell separation (MACS) and thus the cells are selectively isolated and enriched.

On At the molecular level, embryonic stem cells express a variety of genes responsible for the undifferentiated state of these cells are specific. The Stage-Specific Embryonic antigens-1, -3 and -4 (SSEA-1, SSEA-3, SSEA-4) are glycoproteins, in the early embryonic development and therefore markers for embryonic stem cells (Solter and Knowles, 1978, Proc Natl. Acad Sci. USA 75: 5565-5569; Kannagi et al., 1983, EMBO J. 2: 2355-2361). The raised Expression of the enzyme alkaline phosphatase (AP) is another Marker associated with undifferentiated embryonic stem cells is. Other 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 also found in the stem cell compartment of the fetal and adult intestine. There but it is intracellular Markers are, these are unsuitable for the identification of living Cells. So far, there are hardly any suitable markers for 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 nontumorous and tumorous epithelia of the human colon and rectum ", J Natl. Cancer Inst. 1985, 75 (3): 447-454) and Hara et al. ( "The expression of stage-specific embryonic antigen 1 in the noncancerous colorectal epithelia of familial polyposis coli ", Dis Colon Rectum, 1987, 30 (6): 440-443) the expression of the stage-specific embryonic antigen 1 (SSEA-1), 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. In these publications is described that the expression of SSEA-1 with the neoplastic Transformation and progression is associated and that this antigen a suitable marker for detecting neoplastic transformation represents in the human intestine.

One suitable antibody, the one with high selectivity and with high affinity binds to the antigen CD15 / SSEA-1, is the commercially available antibody W6D3, by immunization of a Balb / c mouse with the retinoblastoma cell line WERI-RB1 and subsequently Fusion of immune spleen cells with the myeloma cell line SP2 / 0 was generated. This antibody is with the German collection of microorganisms and cell cultures (DSMZ) under the number DSM ACC 2760 under the Budapest Treaty.

So far However, in the prior art is not a method for isolation of epithelial cells in the crypts bottom of the intestine with an antibody or Antibody fragment described that binds to the antigen SSEA-1. Likewise, the strong Labeling of the originating from the hybridoma cell line W6D3 antibody Precursor- and stem cells of the intestinal epithelium have not been demonstrated.

Moreover So far, the detection of SSEA-1 with antibodies has only been fixed Epithelial cells in the crypts bottom of the intestine, but not on living Cells (Gong et al., Supra). The selective marker live precursor and stem cells, however, is the prerequisite for successful isolation and enrichment of these cell populations. Also for the identification of the antigen SSEA-1 is a live marker beneficial to a live diagnosis to perform SSEA-1 expressing tumor epithelial cells of the intestine.

In front Therefore, there is a need to provide a method with the epithelial precursor and / or stem cells can be identified and / or isolated.

These The object is achieved by Provision of a method for identifying and / or isolating of live postnatal and / or adult epithelial progenitor and / or stem cells of the intestine with an antibody or a fragment of the antibody, wherein the antibody from the German Collection of Microorganisms and Cell Cultures (DSMZ) under number DSM ACC 2760 under the Budapest Treaty Hybridoma cell line W6D3 available is.

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 precursor and / or stem cells of the lower crypt base of the intestine not only in the fixed, but also to dye in the living state (see 1 . 2 ).

• a) Bereitstellen mindestens eines Antikörpers oder eines Fragments des Antikörpers, wobei der Antikörper aus der bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen (DSMZ) unter der Nummer DSM ACC 2760 gemäß dem Budapester Vertrag hinterlegten Hybridomazelllinie W6D3 erhältlich ist.
• b) Bereitstellen einer Probe aus lebenden Darmepithelzellen.
• c) Inkontaktbringen des mindestens einen Antikörpers oder eines Fragments des Antikörpers aus Schritt a) mit der Probe aus Schritt b).
• d) Identifizieren derjenigen lebenden Darmepithelzellen der Probe aus Schritt b), die an den mindestens einen Antikörper oder an ein Fragment des Antikörpers aus Schritt a) binden.

In particular, 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 a fragment of the antibody, wherein the antibody is obtainable from the hybridoma cell line W6D3 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under number DSM ACC 2760 under the Budapest Treaty.
• 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 Sample from live intestinal epithelial cells provided by first preferred the monolayer epithelium of the underlying lamina propria by means of an enzymatic method using a thermolysin solution or by using calcium-free media, which are calcium-binding Substances can be added is separated to remove positive cell populations outside the epithelium.

Of the reason for the separation of intestinal epithelial cells from the lamina propria is located in that the antibody derived from the hybridoma cell line W6D3 in the human intestine in addition to the intestinal epithelial cells also granulocytes the underlying lamina propria and the below lying submucosa colors.

at the enzymatic method is preferred as the enzyme thermolysin, Trypsin, collagenase, dispase and / or papain and more preferred Thermolysin and papain used. The chemical separation of the intestinal epithelium is preferably done using calcium-binding substances, in particular chelating agents such as ethylenediaminetetraacetic acid (EDTA), Ethylene glycol tetraacetic acid (EGTA) and nitrilotriacetic acid (NTA) and most 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 base with the antibody produced from the hybridoma cell line W6D3 is clearly in 2 to recognize. In 2A is 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. 2 B 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. So is according to 1 demonstrated immunohistological staining of fixed colon tissue with the antibody produced from the hybridoma cell line W6D3. 1A is a histological representation of antibody binding to human, acetone-fixed Kyrostatschnitten of the colon. Clearly a specific marking of the crypts bottom (arrows) can be recognized. 1B 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 1A the case was. In 1B Thus, it could be shown clearly with the KI-67 proliferation marker that the antibody derived from the hybridoma cell line W6D3 stains the proliferative region in the crypts.

• a) Vereinzeln der lebenden Darmepithelzellen aus der oben genannten Probe, die aus intakten Krypten besteht.
• b) Inkontaktbringen mindestens eines Antikörpers oder eines Fragments des Antikörpers, der aus der bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen (DSMZ) unter der Nummer DSM ACC 2760 gemäß dem Budapester Vertrag hinterlegten Hybridomazelllinie W6D3 erhältlich ist, mit den vereinzelten Darmepithelzellen aus Schritt a).
• c) Isolieren derjenigen vereinzelten Darmepithelzellen aus Schritt b), die den mindestens einen Antikörper oder ein Fragment des Antikörpers binden.

A further embodiment of the present invention preferably relates to a method for isolating the above-mentioned cells, which comprises the following steps:

• a) Separation of the living intestinal epithelial cells from the above sample, which consists of intact crypts.
• b) contacting at least one antibody or a fragment of the antibody which is obtainable from the hybridoma cell line W6D3 deposited under the number DSMZ under the number DSMZZ under the number DSM ACC 2760 in accordance with the Budapest Treaty 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 mentioned under step a) Separation (dissociation) of the epithelial cells is responsible for further cell isolation prefers. The isolated epithelial cells can be labeled with antibodies and can then be separated.

Of the from the hybridoma cell line W6D3 available antibodies binds preferably to the antigen SSEA-1. This antigen is in the early embryonic Development expressed. Furthermore is the expression of SSEA-1 with the neoplastic transformation and progression associated. Thus, SSEA-1 provides a suitable Markers for both embryonic stem cells and neoplastic transformation and progression associated cells of the intestine.

Of the used according to the invention Antibody, the from the at the German collection of microorganisms and cell cultures (DSMZ) under number DSM ACC 2760 under the Budapest Treaty Hybridoma cell line W6D3 is preferred by immunization a Balb / c mouse with the retinoblastoma cell line WERI-RB1 and subsequent fusion generated by immune spleen cells with myeloma cell line SP2 / 0. He points excellent properties in terms of specificity and affinity for the antigen SSEA-1 up and binds in the intestine with correspondingly high selectivity to intestinal Parent and / or progenitor cells.

suitable antibody In the context of the present invention, monoclonal, polyclonal and be monovalent and bivalent fragments.

to Preparation of antibodies can also various hosts, including goats, rabbits, rats, mice Humans and other hosts by injection with the protein or one any fragment or oligopeptide thereof which has immunogenic properties has to be immunized. Depending on the host species can different Excipients can be used to enhance the immunological response. To be favoured thereby peptides, fragments or oligopeptides for the induction of antibodies of the Protein that has an amino acid sequence of at least five amino acids and stronger preferably have at least ten amino acids.

Especially in the context of the present invention, monoclonal are preferred Antibody. Monoclonal antibodies can be made using techniques that are continuous Cell lines in culture for the production of antibody molecules. These techniques include hybridoma, especially human B-cell hybridoma technique as well as the EBV hybridoma technique. The production of monoclonal antibodies by the Fusion of spleen cells from immunized mice and myeloma cells was made as early as 1975 by Kohler and Milstein ("Continuous cultures of fused cells secreting antibody of predefined specificity "Nature; 1975, 256: 495-497). The techniques for the chemical selection of hybridomas for the production of monoclonal antibodies which result from such a merger, and the subsequent isolation cell clones secreting individual antibodies are as well known in the art.

Furthermore can Process for the production of genetically engineered antibodies, such as chimeric antibodies be used. It prefers constant regions in one antibody the mouse replaced by constant regions of a human antibody. Alternatively you can Procedure for the production of single-chain antibodies can be used to For example, to produce single-chain antibodies in a preferred embodiment specific for the SSEA-1 protein of the invention. These are genetically engineered Expression of a construct from the gene segments for the two obtained antibody variable regions, passing through a segment for the peptide are linked.

In the context of the present invention, it is also possible to use a fragment of the antibody instead of the complete antibody, without this being expressly mentioned in each case. By "fragment" is meant any fragment of the antibody which retains the antigen binding function, preferably for the SSEA-1 antigen of the antibody. Such fragments include, for example, Fab, F (ab ') ₂ , Fv, 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 provide fast and allow easy identification of monoclonal antibody fragments with the desired specificity.

According to the invention can as antibody the immunoglobulins IgG, IgA, IgM, IgD and IgE are used. It is preferably an IgG antibody and particularly preferred to an IgG1 antibody, because of its smaller size, it is especially resistant to IgM antibodies, for example is well suited to cells by magnetic cell separation (MACS, Magnetic Cell Sorting). Furthermore, this antibody recognizes an epitope, that of other SSEA-1 antibodies is not recognized.

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 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. So is according to 3 presented a FACS analysis of dissociated human intestinal epithelial cells. Significantly, a subset of the labeled epithelial cells can be recognized, which were labeled with the antibody produced from the hybridoma cell line W6D3.

to subsequent isolation of intestinal stem and / or progenitor cells are preferred according to the invention following methods used: flow cytometry (fluorescent activated cell sorter (FACS)), magnetic cell separation (magnetic cell sorting (MACS)) or binding of cells to an antibody that preferably a monoclonal antibody is that on a carrier immobilized, as e.g. in column chromatography. In flow cytometry, cells are loaded with antibodies, on the one hand specific for a surface marker and on the other hand coupled with a fluorescent dye. Those cells that are marker-positive fluoresce while the negative cells remain dark. It can thus be stated which proportion of a cell population is marker-positive. simultaneously allows a flow cytometer, the size and granularity of cells capture. In the case of magnetic cell separation, the cells become labeled with magnetic beads, these beads, for example coupled to the antibodies could be. Particular preference is given in the context of the present invention the isolated intestinal epithelial cells 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. How out 3 As can be seen, with the method according to the invention using the antibody derived from the hybridoma cell line W6D3 or a fragment of the antibody, after a dissociation of the intestinal crypt material, a clearly defined subpopulation could be demonstrated by means of FACS analysis. In the present invention, it has been experimentally demonstrated that it is possible with the antibody of the cell line W6D3, unfixed living cells, which have the binding site for the specific antigen, in particular the SSEA-1 antigen even after dissociation, the lower crypts reason identify and isolate.

to Identification and / or isolation of postnatal and adult Epithelial precursor and / or Intestinal stem cells are preferably mammalian intestines, more preferred human intestine, used. According to the invention, these are thick and / or small intestine, in both the crypts of the large and small intestines Precursor and / or stem cells are located.

Farther The invention relates to postnatal and / or adult epithelial progenitor and / or stem cells of the intestine, which according to the above-described identification and / or isolation methods are available.

The postnatal and / or epithelial progenitor and / or stem cells of the intestine are preferably cultured in a suitable culture medium, in order to carry out 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. Further, if desired, the pharmaceutical composition may preferably comprise 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 according to the invention Composition may further pharmaceutically customary carriers, excipients, additives as well as suitable buffers. Suitable buffers include, for example TRIS, HCl, glycine and phosphate. The pharmaceutical composition beyond that suitable diluents, preferably aqueous NaCl, lactose, manitol solutions as well as water and alcohols. Suitable additives include for example, detergents, solvents, Antioxidants and preservatives. An overview of such Compositions useful substances is e.g. in the newest Edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

The The invention further relates to the use of the invention according to the identification and / or Isolation method obtained precursor and / or stem cells for the manufacture of a medicament for the treatment of tissue damage in vivo and ex vivo.

According to the invention can received precursor and / or stem cells for the treatment of tissue damage in the living organism or in cell culture using techniques of tissue engineering. Under Tissue Engineering One understands the production of functional artificial cell and tissue associations the base of cultured cells and various artificial ones Matrices. In the context of the present invention, the tissue comprises Engineering for the treatment of tissue damage a number of different methodical approaches. These include the Application of biologically active molecules, such as growth factors, Hormones, proteins etc., which are called direct injections into the intestine Can influence or restore bowel functions (in vivo treatment). These include also the injection of resorbable polymers as transporters of growth factors, the transfer of transgenes that promote the formation of growth factors in the target organs stimulate, as well as the implantation of three-dimensional matrices (polymer size), the a colonization with the body's own Allow cells. Other tissue engineering methodologies include the transplantation of in vitro precursor and / or stem cells; artificially produced cell polymer matrices for Implantation of precursor and / or stem cells or extracorporeal (ex vivo) production of organ cells from precursor and / or stem cells.

Prefers are the precursor and / or stem cells obtained by the method according to the invention for the treatment of damage the intestinal mucosa used. Particularly preferred are the precursor and / or stem cells for clinical Applications where there is a corresponding need for more functional Mucosa, as in short bowel 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 that the precursor and / or stem cells, with the method according to the invention to treat tissue damage of the liver, pancreas and / or the lung, particularly preferably in combination with growth factors use.

Farther 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.

Also the use of the precursor and / or Stem cells and / or the culture of these cells for production a means of characterizing cellular responses to biological, chemical or pharmacological agents, is within the scope of the present Invention, in particular the use in one Screening Assay for the identification of medicines for prevention and / or treatment Intestinal diseases is intended.

The gut associated diseases are preferably selected from the group consisting of injuries intestinal mucosa, short bowel syndrome, intestinal ulcer and / or Carcinoma of the intestine and / or precursors of carcinoma of the intestine.

A another embodiment The present invention relates to the use of the above-described antibody or a fragment of the antibody this antibody for identifying and / or isolating live postnatal and / or adult epithelial precursor and / or stem cells of the crypts base of the intestine.

Farther The present invention relates to the use of the antigen SSEA-1 for identifying and / or Isolating live postnatal and / or adult epithelial progenitors and / or Stem cells of the crypt base of the intestine. So far, only a few suitable markers for Stem cells of the crypts reason of the intestine known. The Stage Specific Embryonic antigen-1 (SSEA-1) is within the scope of the present invention an excellent suitable marker, as it is both in the crypts of the intestine is expressed in healthy tissue as well as in larger quantities in pre- and / or carcinogenic Tissues of the intestine. It has been found that the expression of SSEA-1 with neoplastic transformation and progression in the Intestine goes along. Therefore, the SSEA-1 antigen is an ideal marker both for isolating healthy postnatal and / or adult epithelial Precursor- and / or stem cells of the intestine as well as for the diagnosis of carcinomas of the intestine and / or precursors thereof.

• a) Bereitstellen mindestens eines Antikörpers und/oder eines Fragments des Antikörpers, der aus der bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen (DSMZ) unter der Nummer DSM ACC 2760 gemäß dem Budapester Vertrag hinterlegten Hybridomazelllinie W6D3 erhältlich ist.
• b) Bereitstellen einer Probe aus lebenden Darmepithelzellen einer Testperson.
• c) Inkontaktbringen des mindestens einen Antikörpers oder eines Fragments des Antikörpers aus Schritt a) mit der Probe aus Schritt b).
• d) Identifizieren derjenigen lebenden Darmepithelzellen der Probe aus Schritt b), die an den mindestens einen Antikörper oder ein Fragment des Antikörpers aus Schritt a) binden.
• e) Bestimmen des Ausmaßes der Reaktion des mindestens einen Antikörpers oder eines Fragments des Antikörpers aus Schritt a) mit den identifizierten lebenden Darmepithelzellen aus Schritt d), als Maßstab für die Diagnose.

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 obtainable from the hybridoma cell line W6D3 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the number DSM ACC 2760 under the Budapest Treaty.
• 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.

The Detection of live cells to which the above-mentioned antibody is specific binds, for example, offers a decisive advantage in diagnostic Procedure during an endoscopy in the intestinal lumen of a patient, e.g. for tumor diagnosis.

there is the inventive method in particular for the diagnosis of such carcinomas of the intestine and / or precursors suitable for it, the selected are 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 The invention further relates to a kit for identifying and / or Isolate live postnatal and / or adult epithelial Precursor and / or Stem cells of the intestine, which at least one as described above antibody or a fragment of this antibody includes.

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

pictures:

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

In 1A the histological demonstration of antibody binding to human acetone-fixed cryostat sections of the colon is shown. Clearly a specific marking of the crypts bottom (arrows) can be recognized.

1B 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 1A the case was.

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

• A: Immunofluorescence image of a crypte with the the hybridoma cell line produced W6D3 antibody was stained. The crypts reason (Arrow) shows a clear signal. B: bright field image of the same Crypts from A.

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 treated three times with a cold 1% penicillin / streptomycin HBSS (HBBS = Hanks Basal Salt Solution) buffer solution. After that took place the careful mechanical removal of the epithelial layer including the underlying lamina propria of which, in turn, under the Lamina propria located submucosa 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⁺ / Mg 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.

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

The remaining crypts were in a papain solution (0.2% papain / 0.05 dnase / 5 mM L-cysteine; SIGMA) for 1.5-3 h Incubated at 37 ° C and subsequently isolated (dissociated) with a 2 ml pipette. The stopping of the enzymatic digestion was carried out by means of a trypsin inhibitor solution (10 mg / ml trypsin inhibitor; SIGMA). Subsequently, the cells were added 350 g for Centrifuged for 4 min, the pellet was taken up in PBS and the cell suspension over a Filter filtered with a pore diameter of 40 microns. The single suspension could then for cytometric analyzes are used.

Example 2

Staining the Cells for the 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 at 4 ° C. for 15 min in reaction vessels. After a wash in FACS buffer (PBS, 0.1% BSA, 0.025% NaN ₃ ), the cells were incubated with goat anti-mouse IgG1-phycoerythrin antiserum (1:15) for 15 min at 4 ° C. After another wash step, the cells were analyzed 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 10 minutes of 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 blocked with 3% H ₂ O ₂ and after three further washes, incubated with a biotinylated goat anti-mouse IgG antibody for 30 min. Visualization was done using an avidin-biotin enhancer kit (DAKO) and a diaminobenzidine (DAB) staining. 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 second fluorescent antibody. The analysis of the labeled cells was carried out with a fluorescence microscope (Zeiss).

Claims (29)

Method for identifying and / or isolating of live postnatal and / or adult epithelial precursors and / or Stem cells of the intestine with an antibody or a fragment of the Antibody, wherein the antibody from the German Collection of Microorganisms and Cell Cultures (DSMZ) under the number DSM ACC 2760 under the Budapest Treaty deposited hybridoma cell line W6D3 available is. A method of identifying the cells of claim 1, comprising the following steps: a) Provide at least one according to claim 1 defined antibody or a fragment of the antibody. b) Providing a sample of living intestinal epithelial cells. c) Contacting the at least one antibody or fragment of the antibody from step a) with the sample from step b). d) Identify those living intestinal epithelial cells of the sample from step b), to the at least one antibody or to a fragment of the antibody from step a) bind. A method of isolating the cells of claim 1 or 2, comprising the following steps: a) Separate the Intestinal epithelial cells from step b) of claim 2. b) bring into contact of 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) Isolate those isolated Intestinal epithelial cells from step b) containing the at least one antibody or a fragment of the antibody from step a) of claim 2 bind. Method according to one of the preceding claims, characterized characterized in that the antibody binds to the antigen SSEA-1. Method according to one of the preceding claims, characterized characterized in that one intestine of mammals, preferably human Intestine used. Method according to one of the preceding claims, characterized characterized in that one uses large intestine and / or small intestine. Method according to one of the preceding claims, characterized characterized in that the antibody is a monoclonal antibody or a fragment of a monoclonal antibody is used. Method according to one of the preceding claims, characterized characterized in that the antibody an IgG antibody is. Method according to one of the preceding claims, characterized characterized in that the antibody an IgG1 antibody is. A method according to claim 1 or 3 to 9, characterized characterized in that the isolated intestinal epithelial cells by means of Flow cytometry (FACS), magnetic cell separation (MACS) and / or column isolated. A method according to claim 1 or 3 to 10, characterized characterized in that the intestinal epithelial cells are separated by means of an enzyme / DNAse solution. Method according to claim 11, characterized in that that the enzyme from the group consisting of collagenases, trypsin, Dispasen, Thermolysin and / or Papain selects. Postnatal and / or adult epithelial precursors and / or Stem cells of the intestine, available through a method according to claim 1 or 3 to 12. Culture of postnatal and / or epithelial precursors and / or Stem cells of the intestine according to claim 13. 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. Use of precursor and / or stem cells according to claim 13 for the preparation of a medicament for the treatment of tissue damage in vivo and ex vivo. Use of precursor and / or stem cells according to claim 13 or 16 for the manufacture of a medicament for Treatment of damage to the Intestinal mucosa. Use of precursor and / or stem cells according to claim 13 or 16 to 17 for the preparation of a medicament for the treatment of short-bowel syndrome ("short-intestine" syndrome) and / or intestinal Ulcers etc. Use of 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 the liver, pancreas and / or lungs. Use of precursor and / or stem cells according to claim 13 and / or the culture according to claim 14 for the production an agent for the diagnosis of carcinomas of the intestine and / or precursors thereof. Use of precursor and / or stem cells according to claim 13 and / or the culture according to claim 14 for the production a means of characterizing cellular responses to biological, chemical or pharmacological agents. Use according to claim 21 in a screening assay for the identification of medicines for prevention and / or treatment intestinal diseases. Use according to claim 22, characterized that the diseases associated with the intestine are selected from the group consisting of injuries intestinal mucosa, short bowel syndrome, intestinal ulcer and / or Carcinoma of the intestine and / or precursors of carcinoma of the intestine. 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 the crypts bottom of the intestine. Use of the antigen SSEA-1 for identification and / or isolating live postnatal and / or adult epithelial Precursor- and / or stem cells of the crypts base of the intestine. Method for in vitro and / or in vivo diagnosis of carcinomas of the intestine and / or precursors thereof, comprising following steps: a) providing at least one according to claim 1 defined antibody and / or a fragment of the antibody. b) Provide a sample of living intestinal epithelial cells of a subject. c) Contacting the at least one antibody or fragment of the antibody from step a) with the sample from step b). d) Identify those living intestinal epithelial cells of the sample from step b), to the at least one antibody or a fragment of the Antibody off Bind step a). e) determining the extent of the reaction of the at least an antibody or a fragment of the antibody from step a) with the identified living intestinal epithelial cells from step d), as a yardstick for the Diagnosis. Method according to claim 26, characterized in that 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 cancer, ulcerative colitis, chronic ulcerative colitis, Chron's disease, benign adenoma of the Colon etc. Kit for identifying and / or isolating living postnatal and / or adult epithelial progenitor and / or stem cells of the intestine comprising at least one antibody according to any one of claims 1 to 3 or 7 to 9 or a fragment of the antibody. Process for the treatment of tissue damage in vivo and ex vivo, comprising the administration of a pharmaceutical effective amount of a pharmaceutical composition, the living 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, to a patient.