EP2576771A2

EP2576771A2 - Isolation of mesenchymal stem cells

Info

Publication number: EP2576771A2
Application number: EP11725901.0A
Authority: EP
Inventors: Wilhelm Aicher; Brigitte Angres
Original assignee: Eberhard Karls Universitaet Tuebingen; Baden Wuerttemberg Stiftung gGmbH; Universitaetsklinikum Tuebingen
Current assignee: Eberhard Karls Universitaet Tuebingen; Baden Wuerttemberg Stiftung gGmbH; Universitaetsklinikum Tuebingen
Priority date: 2010-06-07
Filing date: 2011-06-07
Publication date: 2013-04-10
Also published as: DE102010023837A1; US20130156819A1; WO2011154403A2; WO2011154403A3

Abstract

The present invention relates to the use of a peptide selected from the group comprising laminin-1, collagen-1, collagen-3, collagen-4, tenascin, thrombospondin-1, osteopontin, fibronectin, vitronectin, or fragments or mixtures of these, for isolating and/or identifying mesenchymal stem cells (MSC), in particular of peptide fragments of these peptides. The invention furthermore relates to the use of these peptides/peptide fragments in the treatment of wounds, injuries and/or degenerated tissue.

Description

Isolation of mesenchymal stem cells

The present invention relates to the isolation, identification and / or activation of mesenchymal stem cells with proteins or peptides derived therefrom.

Mesenchymal stem cells (MSCs) are pluripotent cells and can differentiate into various mesenchymal tissues under appropriate in vitro and vzvo conditions, such as bone, adipose tissue, muscle, cartilage. MSCs have the property of adhering stably and quickly to plastic or glass surfaces. ren, and have a fibroblastoid phenotype. Although MSCs are well distinguishable from hematopoietic stem cells because they do not express specific hematopoietic surface markers. However, no specific surface antigen is known in the art for MSCs; The surface molecules they express are also found on surfaces of endothelial, mesenchymal and epithelial cells, as well as muscle cells.

The term "mesenchymal stem cells" (MSC) is not uniformly defined in the literature, but basically two types of cells can be distinguished: MSCs isolated directly from non-hematopoietic primary tissue (eg, bone marrow, adipose tissue, placenta) and cells that are being cultured and differentiate into fibroblastoid cells adherent in culture from these primary cells, where they express cell surface markers such as CD29, CD44, CD73, CD90, CD105, CD166 but are negative for the hematopoietic stem cell marker CD34 and the pan-leukocyte marker CD45. In general, the cells generated in culture are referred to as mesenchymal stem cells, since they themselves still possess a multipotent differentiation capacity according to this method.

Due to their multi-potency, ie their capacity in various mesenchymal tissues under appropriate in vitro and in vz ^{'vo-conditions} (such as. Bone, fat, muscle, cartilage, etc.) to be able to differentiate mesenchymal stem cells already for therapeutic Use used. For example, differentiated MSCs isolated from placenta, bone marrow and adipose tissue can be expanded in vitro, differentiated into osteoblasts, chondrocytes and myocytes, and then reused in vivo, for example for the regeneration of bones, cartilage, tendons, muscles, adipose tissue and stroma ,

On the one hand, the state of the art is that bone marrow mesenchymal stem cells can be isolated by means of antibodies which are directed against the low-affinity receptor for the low-affinity nerve growth factor receptor (CD271) (Quirici et al. "Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies", Exp. Hematol., 2002, 30 (7): 783-791). Furthermore, it has been described that MSC can be isolated via antibodies against SH2 (CD105), SH3 (CD73) and SH4 (CD73) (see Barry F, et al., "The SH-3 and SH-4 antibodies recognize distinct epitopes on CD 73 from human mesenchymal stem cells ", Biochem Biophys Res Commun. 2001; 289: 519-24; and Pittenger MF, et al.," Multilinear potential of adult human mesenchymal stem cells ", Science 1999; 284: 143-7). The disadvantage of the previous markers, however, is that they are all not specific for MSC, but recognize other cell populations in the bone marrow.

The cell surface marker CD271 is the most specific cell surface marker for the isolation of mesenchymal stem cells, which is commercially available. Thus, for example, monoclonal antibodies to this marker are marketed by the companies BD PharMingen, San Diego, USA, and Miltenyi Biotech, Bergisch Gladbach, Germany. However, it has been found that this marker is not selective for MSC but is also expressed on other CD45-positive hematopoietic cells. Thus, not only mesenchymal cells but also hematopoietic cells are isolated in an isolation procedure with anti-CD271 antibodies.

In addition, the production of monoclonal antibodies is very time consuming and costly.

In view of the disadvantages known from the prior art, it is an object of the present invention to provide new ways to isolate mesenchymal stem cells (MSC) as pure as possible, and to separate from contaminating cells, such as fibroblasts. Although fibroblasts express the inclusion and exclusion criteria defined by Dominici and colleagues (Cytotherapy, 2006; 8: 315-7), they can not be distinguished microscopically from MSC. But they are by no means differentiation competent. Therefore, separation of MSC from fibroblasts is very important. According to the invention, this object is achieved in that a protein or a peptide derived therefrom is used for the isolation and / or identification of mesenchymal stem cells, which is selected from the group comprising lami- nin-1, collagen-1, collagen-3, collagen 4, tenascin, thrombospondin-1, osteopontin, fibronectin, vitronectin, or fragments thereof capable of binding to MSC, or mixtures thereof.

In particular, it is preferable that the protein has a sequence selected from SEQ ID Nos. 1 to 7, and more preferably, when the peptide derived therefrom has any one of SEQ ID NOS: 8 to 32 of the attached sequence listing.

The object underlying the invention is completely solved in this way.

The proteins provided or the peptides derived therefrom bind specifically and preferentially to mesenchymal stem cells, which makes it possible to isolate mesenchymal stem cells from contaminating cells such as fibroblasts and to use them for further applications. According to the invention, the proteins and peptide fragments listed herein can also be used for culturing and / or activating MSC.

The use of the disclosed proteins or peptides derived therefrom for the isolation and / or identification of mesenchymal stem cells has hitherto not been described or suggested in the prior art.

By "derived peptides" or "peptide derived therefrom" or "peptide fragment" herein is meant any peptide contained in the listed proteins, and therefore having a sequence of contiguous amino acids, as such, ie, having the same sequence the amino acid containing protein, the sequence has binding properties to MSC, preferably the same or similar to the total protein In this context, it is understood that "protein" is a gene identified as such. meant with certain functions and structures, and with "peptide" a part, or a partial sequence thereof.

Laminin is a glycoprotein found mainly in the basal lamina of epithelia and endothelia with a 14% carbohydrate content. The laminin molecule consists of an a-, a ß- and a γ-protein chain, which are assembled in heterotrimeric form to the respective laminin molecule. At present 15 different laminin isoforms are known.

Collagen is an extracellular matrix protein that is a water-insoluble, fibrous structure of skieroproteins. It is particularly involved in the formation of connective tissues such as the skin, blood vessels, ligaments, tendons and cartilage, as well as the formation of bones and teeth (dentin). There are currently about 28 different types of collagen known (types I to XXVIII), all of which have the structure of three polypeptide chains in common, referred to as collagen helices, which are wound around each other in the form of a triple helix. Collagen-1 and collagen-3, together with collagen-2, -9 and -11, are among the fibrillar collagens. Collagen-1, a trimer, consists of [al (I) ₂ a2 (I)] (alpha-1 type I collagen) or 3 [al (I)] chains; Collagen-3 is a homotrimer of 3 [al (III)] chains.

Finally, tenascin is an oligomeric extracellular matrix glycoprotein involved in interactions between epithelial and mesenchymal cells. In vertebrates, three different types of tenascin have been described so far (tenascin-R, -C and -X), which are inter alia expressed in the number of specific domains, namely the EGF (epidermal growth factor) and fibronectin type III-like domains, differ. Tenascin is involved in the regeneration of nervous tissue in the adult organism. In adult skin, tenascin is induced during wound healing. Tenascin directs the migration of cells in these processes, while it can stimulate or inhibit cell adhesion across different protein domains. Osteopopntin is a glycoprotein in all higher mammals involved in the maintenance of bone tissue and some immune processes. It binds hydroxyapatite and forms the basic structure for bones. Synonymous names of the protein are sialoprotein I and 44K BPP (bone phosphoprotein).

Thrombospondin-1 belongs to a family of proteins involved in different biological processes. The protein family consists of thrombospondin-1 to -5, distinguishing subgroups: Subgroup A consists of TSP1 and TSP2, which are homotrimers; Subgroup B consists of TSP3, TSP4 and TSP5. TSP1 is involved in many different biological processes such as angiogenesis, apoptosis, activation of tissue hormone TGF and immune regulation.

Fibronectin (from Latin: fibra for "fiber", nexus for "linkage") is an extracellular glycoprotein that plays an important role in many physiological processes. It is a heterodimer of two rod-shaped polypeptide chains held together close to the C-terminal end by disulfide bridges. So far, more than 20 different isoforms have been found that are generated by alternative splicing of the mRNA of a single gene. A single fibronectin polypeptide chain (~ 230 kDa) consists of a large number of domains (about 40-90 amino acids) which, due to their homology, are classified into structural types I, II, and III.

Vitronectin is a glycoprotein present in the serum and the extracellular matrix and provides a secreted protein which is either in the form of a single chain or a double chain linked by a di-sulphide bridge. The reference number for this protein in the NCBI (National Center for Biotechnology Information) database (GenBank®) is NP_000629).

It is particularly preferred if the protein or the peptide derived therefrom has a sequence which is selected from SEQ ID Nos. 1 to 32 of the attached sequence listing, or that contained therein, a binding-relevant sequence.

The sequence with the ID no. Figure 1 illustrates the sequence of the human laminin-1 alpha-1 chain and is listed, for example, in the GenBank® of the National Center for Biotechnology Information under the number NP_005550; the sequence with the ID no. Figure 2 is the sequence of the beta-l-chain of human laminin-1 (GenBank® # NP_002282), the sequence with the ID-No. Figure 3 shows the sequence of the human laminin-1 gamma 1 chain (GenBank® # NP_00284).

Similarly, the SEQ ID NO. 4 represents the sequence of the human collagen alpha (I) chain (GenBank® No. P02452), the sequence with the ID no. FIG. 5 shows the sequence of the human collagen alpha-2 (I) chain (GenBank® No. P08123), and the sequence with the ID no. Figure 6 shows the sequence of the human collagen alpha (III) chain (GenBank® # P02461). The three sequences with the SEQ ID Nos. 4, 5 and 6 therefore represent sequences for the trimer collagen 1. The sequence with the SEQ ID NO. 6 is also part of the homotrimer collagen-3.

The sequence with the ID no. Figure 7 depicts the sequence of human tenascin-C and has the identification CAA55309 in GenBank®.

In a preferred embodiment, in the use according to the invention, one of the following binding-specific peptide fragments is used: a) a peptide having one of the SEQ ID numbers SEQ ID Nos. 8 to 32 listed in the sequence listing, or b) fragments of the sequences according to a) having substantially the same biological activity of the peptide according to a) in a test for the binding of mesenchymal stem cells, c) a peptide fragment having a sequence which is at least 80%, preferably at least 80% to 99%, identical to one of the sequences listed in a) and b).

The sequences having SEQ ID NOS: 8 to 32 are preferred peptides with which mesenchymal stem cells can be specifically bound. The peptides having SEQ ID Nos. 8 (GF-Orn-GER containing ornithine at position 3) and 9 (GEFYFDLRLKGDK) are derived from collagen-1 and -4, SEQ ID Nos.10 to 13 of laminin (LRE Laminin; AASIKAVAVSADR, laminin alpha chain; LAIKNDNLVYVY, DVISLYNFKHIY (SEQ ID NO: 23), each laminin alpha4 chain; RYVVLPRPVLFEK, laminin betal chain), SEQ ID Nos. 14 and 15 of thrombospondin (ELTGAARKGSGRRLVKGPD, thrombospondin l) KKTRGTLLALERKDHS, thrombospondin-1), SEQ ID NO: 16 of osteopontin (SVVYGLR), and SEQ ID NOS: 17 to 20 and 32 of fibronectin (YIIR; GSKS; TYSSPEDGIHE; WQPPRARITGY; DELPQLVTLPHPNLHGPEILDVPST).

It will be clear to the person skilled in the art that apart from the abovementioned proteins / peptides, proteins / peptides can also be used for the purposes of the inventive use, which, for example, are functionally identical to the disclosed peptides because of sequence homologies, so that, for example, proteins can also be used , which may be possible in comparison to the mentioned proteins / peptides deletions, substitutions, insertions, etc., but which nevertheless have the same function as the mentioned proteins / peptides. Thus, for example, only binding-relevant fragments of said proteins / peptides can be used.

In this context, "a substantially identical biological activity (of a peptide) in a test concerning the binding of mesenchymal stem cells" means that variants or derivatives of the peptides with SEQ ID Nos. 8 to 32 in the context of the present invention for the In this context, it is understood that by "substantially" an activity is meant that is nearly the same with the values for the peptides specifically disclosed with their sequences. The person skilled in the art will be aware of this or it will be apparent from the disclosed sequences by reasonable, simple experiments which sequence variants are still possible in order to achieve such a similar binding specificity and effectiveness.

The term "hybridization under stringent conditions" in the context of this invention means that the hybridization is performed in vitro under conditions that are stringent enough to ensure specific hybridization. The term "specific hybridization" refers to the fact that a molecule preferentially binds under stringent conditions to a particular nucleic acid sequence, the target sequence, if this is part of a complex mixture of e.g. DNA or RNA molecules is, but not or at least substantially reduced to other sequences. The exact conditions for stringency depend on appropriate circumstances, for example with regard to the material used. Typically, stringent conditions are those in which hybridization between said nucleotide sequences occurs under conventional conditions, especially at 20 ° C below the melting point of said nucleotide sequences. Preferred hybridization conditions are, for example, those in which a solution of 5 × or 6 × SSPE (or SSC), 1% or 0.5% SDS, 1 × Denhardts solution is used, and the hybridization temperatures between 35 ° C. and 70 ° C., preferably at 65 ° C. After hybridization, washing is preferably carried out first with 2 × SSC, 1% SDS and then with 0.2 × 10 × SSC at temperatures between 35 ° C. and 70 ° C., preferably at 65 ° C. (for the definition of SSPE, SSC and Denhardts See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)).

The term "sequence homology" or "sequence identity" denotes the proportion of the bases that match two nucleic acid sequences or the proportion of the amino acids that match two amino acid sequences. If the sequence homology is expressed as a percentage, e.g. B. 90%, called the Percent the proportion of matches over the length of the sequence compared to another sequence.

It is further understood that on the one hand proteins / peptides derived therefrom of human origin can be used, but on the other hand also functionally and structurally similar / identical proteins from other mammals can be used, as well as artificially synthesized or recombinantly produced peptides / proteins the sequence of said proteins / peptides - or parts thereof - have.

With the mentioned peptides it could be shown that a specific binding of mesenchymal stem cells is possible.

With the use according to the invention it is, for example, possible to coat culture bottoms with the proteins or the peptides derived therefrom and to keep the MSC binding to them in the undifferentiated state, whereby larger batches of differentiation-competent MSC can be produced.

On the other hand, it is also possible to use the proteins or the peptides derived therefrom for the direct isolation of MSC from primary tissue, such as bone marrow, umbilical cord blood, etc.

In addition, the proteins or the peptides derived therefrom can be used to modify or stabilize the specific differentiation of the mesenchymal stem cells into the mature mesenchymal cell types, such as chonrocytes, osteoblasts, adipocytes, myocytes.

In addition, the proteins or the peptides derived therefrom are also suitable for use in sztw applications: For example, support structures such as implants or stents or the like can be used. be coated with the peptides. The MSCs bind to the carrier structures via these peptides, as a result of which the MSC concentrates locally on the carrier materials. be rung. By differentiating the thus concentrated mesenchymal stem cells into the corresponding tissue, it is possible, for example, to regenerate injured or damaged tissue or to form new tissue at the location of the carrier structure. Advantageously, the peptides are of human origin, so that no immune reactions occur.

In a further embodiment it is preferred if the proteins or the peptides derived therefrom are used for the selection of MSC from a sample containing MSC and other cell types.

Thus, for example, as already mentioned above, MSC can be isolated directly from a primary culture by means of the use of the disclosed proteins or the peptides derived therefrom, or else from an already cultivated cell population.

Further, in another embodiment of the use of the invention, the peptide can be used to label MSC.

In this embodiment, the protein to be used or the peptide derived therefrom can be modified or labeled, for example by fluorescent groups, so that after binding of the protein / peptide to the MSC they can be easily and quickly identified on the basis of the label.

In another embodiment, it is preferred if the protein or the peptide derived therefrom is used for enrichment of MSC. In this case, for example, an enrichment can be achieved in vitro or in situ, for example by coating suitable materials / structures with the peptides, as mentioned above.

Further, in another embodiment, it is preferred that the protein or peptide derived therefrom be used to induce the proliferation or differentiation of MSC. In this embodiment, the proteins or the peptides derived therefrom can be used specifically to modify the differentiation of the MSC, for example into osteoblasts, chondrocytes, adipocytes, etc.

In yet another embodiment, it is preferred that the protein or peptide derived therefrom is selected from the group consisting of laminin-1, collagen-1, collagen-3, collagen-4, tenascin, thrombospondin-1, osteopontin, fibronectin , or binding-specific fragments or mixtures thereof, for the treatment of wounds, injuries and / or degenerated tissue.

This measure has the advantage that it is possible to resort to protein / peptide structures which are of human origin and therefore cause little or no immune reactions when, for example in connection with a carrier structure on which they are immobilized, they enter the body a patient are introduced.

The tissue to be treated can be any bone, cartilage and / or muscle tissue, preferably of humans.

In this case, it is particularly preferred if the drug is an implant coated with at least one peptide, in particular a stent.

A stent is an endoprosthesis, various materials that generally serve to hold bodily vessels open, and that are tubular and / or meshed with or without a sheath. Stents are introduced in compressed form via a suitable delivery system in the vessels and deployed at the destination for local whereabouts. In the use according to the invention, it is provided that the stent to be used is coated with the protein or the peptide derived therefrom and subsequently introduced into the vessel to be treated. On the other hand, implants, for example for skin, cartilage or bone replacement or regeneration, can be coated with the peptides and implanted in the body region to be treated. By way of example, implants for the treatment of knee and disc damage should be mentioned here, it being clear to the person skilled in the art that any other implant which is implanted in a body for the purpose of regeneration or replacement of tissue can be correspondingly coated. After implantation into the site to be treated in the body of a patient, the MSC are concentrated there via the peptides present on the carrier, thereby creating a type of "wound plaster".

Accordingly, the invention also relates to the use of a protein or a peptide derived therefrom which is selected from the group comprising laminin-1, collagen-1, collagen-3, collagen-4, tenascin, thrombospondin-1, osteopontin, fibronectin , or binding-specific fragments or mixtures thereof, for the manufacture of an implant.

The invention further relates to a method for isolating, identifying, culturing and activating mesenchymal stem cells, comprising the step of selecting the stem cells from a sample containing stem cells with a protein or derived peptide selected from laminin-1, collagen-1 , Collagen-3, collagen-4, tenascin, thrombospondin-1, osteopontin, fibronectin, or binding-specific fragments or mixtures thereof, in particular with a peptide of SEQ _. ID Nos. 1 to 32.

The stem cells obtained by this method according to the invention, which are likewise encompassed by the invention, can in turn, as mentioned above, be used for the treatment of, for example, degenerated or injured tissue, for example for the treatment of bones, cartilage and / or muscles.

The present invention therefore also relates to the use of mesenchymal stem cells which have been isolated, identified, cultured or activated according to one of the methods according to the invention or which have been prepared according to the method according to the invention. have been enriched for the manufacture of a medicament for treating wounds, injuries and / or degenerated tissue, in particular cartilage, bones, muscles, vessels, or for immunotherapeutic purposes, as trophic cells, which then need not necessarily be detectable in repair tissue , or as a vehicle for recombinant (gene) therapy.

The invention further relates to a synthetic, isolated or recombinant peptide comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence according to SEQ ID Nos. 8 to 32, b) fragments of the sequences according to a), the biological activity of the peptide c) fragments having a sequence which is at least 80%, preferably at least 80% to 99%, identical to one of the sequences listed in a) and b) ,

These peptides specifically bind mesenchymal stem cells, as the experiments of the inventors have shown. Thus, the peptides provide a means to isolate and / or enrich mesenchymal stem cells.

The peptides can also be expressed by genetic engineering methods and expressed as structures both membrane-bound on cells, as well as in dissolved form and in combination with other proteins. (e.g., as peptide + collagen-1 fiber for augmented biomaterial)

In particular, the peptides according to the invention can thus also be used directly for the treatment of wounds, injuries and / or degenerated tissue. Therefore, the invention also relates to pharmaceutical compositions comprising at least one of the peptides of the invention and a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active substance in the composition.

It is further understood that in a further embodiment, the pharmaceutical composition contains further therapeutically and / or pharmaceutically active substances, which is additionally administered in the pharmaceutical composition depending on the disease (s) to be treated.

The disease to be treated is preferably a human disease.

The pharmaceutical composition may be administered systemically, i. for example, orally, subcutaneously, intravenously, rectally, parenterally, intramuscularly, interperitoneally, transdermally, or topically, the mode of administration depending on the nature of the disease, the clinical picture, as well as the condition of the patients. Likewise, the administration can take place repeatedly or once, wherein the administration in the former case can take place once or several times a day, and / or over a longer period of time.

In addition to the active substances, the pharmaceutical composition may also contain buffers, diluents and / or additives. Suitable buffers include, for example, Tris-HCl, glycine and phosphate, and suitable diluents, for example, aqueous NaCl solutions, lactose or mannitol. Suitable additives include, for example, detergents, solvents, antioxidants and preservatives. For an overview of such additional ingredients, see, for example, A. Kibbe: Hand Book of Pharmaceutical Excipients, 3rd ed., 2000, American Pharmaceutical Association and Pharmaceutical Press. Further advantages and features will become apparent from the following description and the accompanying drawings.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

An embodiment of the invention is illustrated in the drawings and will be described in more detail below with respect to these. It shows:

Fig. 1 is a comparative analysis of the adherence of human mesenchymal stem cells (MSC; Samples 5, 6, red / blue columns, right) compared to human fibroblasts (Samples 1 to 4, green / yellow columns, left center).

FIG. 2 Measurement examples of laminin, collagen-1 and collagen-4 (A), FIG.

Fibro-nectin (B) or osteopontin (C) -derived peptides compared to the positive control (PLL in A) for negative control (Mal-BSA in A) or other peptides with MSC.

3 shows examples of laminin, collagen-1 and collagen-4 (A),

Fibro-nectin (B) or osteopontin (C) -derived peptides compared to the positive control (PLL in A) for negative control (Mal-BSA in A) or other peptides with fibroblasts as controls.

example 1

In a comparative experiment, the adherence of human mesenchymal stem cells (MSC, Samples 5 and 6 in Figure 1) and human fibroblasts (Samples 1 to 4 in Figure 1) to matrix proteins was examined. The investigation took place under Using a multiple substrate array (MSA®) chip on which the proteins were immobilized. The procedure was as in Kuschel et al., "Cell adhesion profiling using extracellular matrix protein microarrays", Biotechniques 40: 523-531 (2006).

MSC were enriched from bone marrow by density gradient centrifugation, cultured in MSC expansion medium as an adherent population and harvested. The differentiation potential was tested on an aliquot and adipogenic, chondrogenic and osteogenic differentiation confirmed. Thus, the cells fulfill the physiological requirements for MSC (Dominici M. et al., 2006, Cytotherapy, 8: 315 ff). It is understood that MSC can also be used from other tissues, such as fatty tissue or any other tissue in which MSC is present.

Fibroblasts were isolated from the synovial membrane and expanded as described (Aicher et al 1994 J Immunol 152: 5940 et seq.). The cells were harvested, washed, counted, and each 20,000 / assay chamber was assayed for adherence to proteins using MSA ™ chip technology. Briefly, the cells were each incubated on a microarray of 12 different proteins in a chamber initially for uniform distribution with shaking (two hours), then without shaking (two hours) under standard culture conditions. Non-adherent cells were washed off by rinsing the arrays after this incubation. Cells adhering to the proteins were chemically fixed and their nuclei labeled with dye to quantify the number of adherent cells per microsphere protein. Instead of the fibroblasts used here, for example. Skin fibroblasts, chondrocytes, osteoblasts, meniscal cells, o.a. Cells are used.

The results of this experiment are shown in Fig. 1, wherein adherence to the proteins laminin-1 (LN EHS), collagen-3 (CHI) and collagen-1 (CI) and tenascin-C (TN) with the eighth, tenth , eleventh and twelfth protein in the diagram in Fig. 1 is shown. The other proteins tested were: fibronectin (FN hulps), collagen-6 (CVI), vitronectin (VN), collagen-4 (CIV EHS), and laminin-10 (LN huplc). As a positive control, the protein poly-L-lysine (PLL) was used, as a negative control bovine serum albumin (BSA). Both populations, MSC and fibroblasts, do not bind to thrombospondin.

It can be seen from the results shown in FIG. 1 that the binding of MSC to laminin-1, collagen-1 and collagen-3 and tenascin-C was stronger than the binding of fibroblasts to these proteins. Therefore, these proteins are particularly suitable for the present uses of the invention.

Example 2

The adhesion of cells to peptides was also measured by MSA ™ technology (see Kuschel et al., 2006) as in Example 1 above. However, instead of the extracellular matrix proteins, peptides coupled to bovine serum albumin were printed on the nitrocellulose layer in the form of microarrays (8 x 8 microspots), and then the remaining surface was sealed. For incubation with cells, small silicone chambers were placed on the coated slide and incubated with MSC or fibroblasts. After two hours of incubation, the cells that did not attach were rinsed off.

Adherent cells were visualized by Coomassie blue staining on the white nitrocellulose film and evaluated in a motorized photomicroscope: the number of blue cells or the color intensity per spot is measured and transferred to tables for evaluation

The evaluation of such arrays by means of the semi-automatic photomicroscope revealed the PLL (normalized to 100%, positive control) and BSA (normalized to 0%, negative control) relative binding strength for the peptides tested in each case.

Figure 2 shows the results obtained with different peptides and MSC. The results shown in FIG. 2 show that laminin-derived peptides No. 9, # 9-COOH (SEQ ID No. 10), No. 15 and No. 16, collagen-derived peptides No. 1 and No. 5 (SEQ ID Nos. 8 and 9), vitronectin-derived peptide No. 37b , and the fibronectin-derived peptides No. 11, No. 20, No. 21, NR. 21-COOH, # 22, # 22-COOH, # 34, # 34-COOH, and # 30 allow adhesion of MSC (Figure 2) but not fibroblasts (Figure 3). The sequences of the MSC-binding peptides are shown in Table 1:

Table 1

No. Sequence SEQ ID NO.

I GF-Orn-GER-OH 8

5 GEFYFDLRLKGDK-OH 9

9 LRE-OH 10

9-COOH LRE-Doa-A-OH 21

I GRGDSO-OH 22

15 LAIKNDNLVYVY-OH 12

16 DVISLYNFKHIY-OH 23

20 DRVPHSRNSIT-OH 24

21 KREDVY-OH 25

21- COOH KREDVY-COOH 26

22 EILDV-OH 27

22- COOH EILDV-COOH 28

30 WTPPRAQITGYRLTVGLTRR-OH 29

34 YIIR-OH 17

34-COOH YIIR-DOA-A-OH 30

37b KRSR-DOA-A-OH 31

41 SVVYGLR-OH 16

Claims

claims

Use of a peptide selected from the group consisting of laminin-1, collagen-1, collagen-3, collagen-4, tenascin, thrombospondin-1, osteopontin, fibronectin, vitronectin, or peptide fragments thereof which are described in are capable of binding to mesenchymal stem cells, or mixtures thereof, for in vitro isolation and / or identification of mesenchymal stem cells (MSC) from a sample.

2. Use according to claim 1, characterized in that one of the following peptide fragments is used: a) a peptide having one of the listed in the sequence listing SEQ ID Nos. SEQ ID Nos. 8 to 32, or b) fragments of the sequences according to a) having a substantially similar biological activity of the peptide according to a) in a test concerning the binding of mesenchymal stem cells, c) a peptide fragment having a sequence which is at least 80%, preferably at least between 80% and 99%, is identical to one of the sequences listed in a) and b).

3. Use according to one of claims 1 or 2, characterized in that the peptide or peptide fragment is used for the selection of MSC from a sample containing MSC and other cell types.

4. Use according to one of claims 1 or 2, characterized in that the peptide or peptide fragment is used for labeling of MSC.

5. Use according to one of claims 1 or 2, characterized in that the peptide or peptide fragment is used for enrichment of MSC.

6. Use according to claim 5, characterized in that the enrichment is in vivo, in situ or in vitro.

7. Use according to one of claims 1 or 2, characterized in that the peptide or peptide fragment is used for the differentiation of MSC.

8. A peptide having any one of the SEQ ID Nos. SEQ ID Nos. 8 to 32 listed in the Sequence Listing, or mixtures thereof, for the treatment of wounds, injuries and / or degenerated bones, cartilages or tissues.

9. A peptide according to claim 8, characterized in that the treatment is carried out with a peptide-coated implant or stent.

10. A peptide which has one of the SEQ ID Nos. SEQ ID Nos. 8 to 32 listed in the Sequence Listing, or mixtures thereof, for the production of an implant.

A method of isolating and / or identifying mesenchymal stem cells, comprising the step of selecting the stem cells from a stem cell-containing sample in vitro with a peptide selected from laminin-1, collagen-1, collagen-3, collagen-4 , Tenascin, thrombospondin-1, osteopontin, fibronectin, or MSC-binding-specific fragments or mixtures thereof.

12. mesenchymal stem cells which have been isolated or identified according to a method of claim 11, or which have been enriched according to a use according to claim 5, for the treatment of wounds, injuries and / or degenerated tissue.

13. Use according to claim 8 or 12, characterized in that the tissue is selected from cartilage, bone, muscles.

14. Synthetic, isolated or recombinant peptide, comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence according to SEQ ID Nos. 8 to 32, b) fragments of the sequences according to a), the biological activity of the peptide according to a) c) fragments having a sequence which is at least 80%, preferably at least 80% to 99%, identical to one of the sequences listed in a) and b) in a test relating to the binding of mesenchymal stem cells;

15. A peptide according to claim 14 for use in the treatment of wounds, injuries and / or degenerated tissue.

A pharmaceutical composition comprising at least one peptide according to claim 14 or 15 and a pharmaceutically acceptable carrier.