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EP2576771A2 - Isolation de cellules souches mésenchymales - Google Patents

Isolation de cellules souches mésenchymales

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Publication number
EP2576771A2
EP2576771A2 EP11725901.0A EP11725901A EP2576771A2 EP 2576771 A2 EP2576771 A2 EP 2576771A2 EP 11725901 A EP11725901 A EP 11725901A EP 2576771 A2 EP2576771 A2 EP 2576771A2
Authority
EP
European Patent Office
Prior art keywords
peptide
collagen
msc
stem cells
mesenchymal stem
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11725901.0A
Other languages
German (de)
English (en)
Inventor
Wilhelm Aicher
Brigitte Angres
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eberhard Karls Universitaet Tuebingen
Baden Wuerttemberg Stiftung gGmbH
Universitätsklinikum Tübingen
Original Assignee
Eberhard Karls Universitaet Tuebingen
Baden Wuerttemberg Stiftung gGmbH
Universitätsklinikum Tübingen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eberhard Karls Universitaet Tuebingen, Baden Wuerttemberg Stiftung gGmbH, Universitätsklinikum Tübingen filed Critical Eberhard Karls Universitaet Tuebingen
Publication of EP2576771A2 publication Critical patent/EP2576771A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to the isolation, identification and / or activation of mesenchymal stem cells with proteins or peptides derived therefrom.
  • MSCs Mesenchymal stem cells
  • Mesenchymal stem cells 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.
  • MSCs are well distinguishable from hematopoietic stem cells because they do not express specific hematopoietic surface markers.
  • 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.
  • MSC meenchymal stem cells
  • primary tissue eg, bone marrow, adipose tissue, placenta
  • 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.
  • 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.
  • 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 ,
  • 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).
  • CD271 low-affinity nerve growth factor receptor
  • 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 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.
  • MSC mesenchymal stem cells
  • 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.
  • 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.
  • 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.
  • 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.
  • the proteins and peptide fragments listed herein can also be used for culturing and / or activating MSC.
  • 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
  • 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 ⁇ -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).
  • connective tissues such as the skin, blood vessels, ligaments, tendons and cartilage, as well as the formation of bones and teeth (dentin).
  • 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.
  • tenascin is an oligomeric extracellular matrix glycoprotein involved in interactions between epithelial and mesenchymal cells.
  • 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.
  • NCBI National Center for Biotechnology Information
  • 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.
  • 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).
  • 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.
  • Figure 7 depicts the sequence of human tenascin-C and has the identification CAA55309 in GenBank®.
  • 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.
  • thrombospondin EHTGAARKGSGRRLVKGPD, thrombospondin l) KKTRGTLLALERKDHS, thrombospondin-1
  • SEQ ID NO: 16 of osteopontin SEQ ID NOS: 17 to 20 and 32 of fibronectin (YIIR; GSKS; TYSSPEDGIHE; WQPPRARITGY; DELPQLVTLPHPNLHGPEILDVPST).
  • 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.
  • 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.
  • binding-relevant fragments of said proteins / peptides can be used.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • proteins or the peptides derived therefrom for the direct isolation of MSC from primary tissue, such as bone marrow, umbilical cord blood, etc.
  • 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.
  • proteins or the peptides derived therefrom are also suitable for use in sztw applications:
  • support structures such as implants or stents or the like can be used.
  • 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.
  • the peptides are of human origin, so that no immune reactions occur.
  • proteins or the peptides derived therefrom are used for the selection of MSC from a sample containing MSC and other cell types.
  • 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.
  • the peptide can be used to label MSC.
  • 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.
  • the protein or the peptide derived therefrom is used for enrichment of MSC.
  • an enrichment can be achieved in vitro or in situ, for example by coating suitable materials / structures with the peptides, as mentioned above.
  • the protein or peptide derived therefrom be used to induce the proliferation or differentiation of MSC.
  • 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.
  • 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.
  • 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.
  • the stent to be used is coated with the protein or the peptide derived therefrom and subsequently introduced into the vessel to be treated.
  • 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.
  • 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.
  • the MSC are concentrated there via the peptides present on the carrier, thereby creating a type of "wound plaster".
  • 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.
  • 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) ,
  • 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)
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition may also contain buffers, diluents and / or additives.
  • 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.
  • 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.
  • 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 TM 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.
  • Fig. 1 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).
  • FN hulps fibronectin
  • CVI collagen-6
  • VN vitronectin
  • VN vitronectin
  • CIV EHS collagen-4
  • laminin-10 LN huplc
  • PLL protein poly-L-lysine
  • BSA bovine serum albumin
  • the adhesion of cells to peptides was also measured by MSA TM technology (see Kuschel et al., 2006) as in Example 1 above.
  • MSA TM technology see Kuschel et al., 2006
  • 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.
  • 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
  • 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:

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Abstract

L'invention concerne l'utilisation d'un peptide sélectionné dans le groupe comprenant les laminine-1, collagène-1, collagène-3, collagène-4, ténascine, thrombospondine-1, ostéopontine, fibronectine, vitronectine ou bien des fragments ou des mélanges de ceux-ci, pour isoler et/ou identifier des cellules souches mésenchymales (MSC), notamment des fragments peptidiques de ces peptides. L'invention porte également sur l'utilisation de ces peptides/fragments peptidiques pour le traitement de plaies, de blessures et/ou de tissus dégénérés.
EP11725901.0A 2010-06-07 2011-06-07 Isolation de cellules souches mésenchymales Withdrawn EP2576771A2 (fr)

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DE102010023837A DE102010023837A1 (de) 2010-06-07 2010-06-07 Isolierung von mesenchymalen Stammzellen
PCT/EP2011/059393 WO2011154403A2 (fr) 2010-06-07 2011-06-07 Isolation de cellules souches mésenchymales

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EP3429360A4 (fr) * 2016-03-16 2019-08-28 Cell Medicine, Inc. Cellules souches mésenchymateuses présentant une efficacité améliorée

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US4978352A (en) * 1989-06-07 1990-12-18 Fedorov Svjatoslav N Process for producing collagen-based cross-linked biopolymer, an implant from said biopolymer, method for producing said implant, and method for hermetization of corneal or scleral wounds involved in eye injuries, using said implant
EP1853328A2 (fr) * 2005-02-23 2007-11-14 SurModics, Inc. Articles medicaux implantables a revetement de laminine et methodes d'utilisation
WO2006116793A1 (fr) * 2005-05-04 2006-11-09 Australian Stem Cell Centre Limited Selection, culture et creation de cellules souches hematopoietiques determinees pour une lignee
DE102005021435A1 (de) * 2005-05-04 2006-11-09 Universitätsklinikum Freiburg Verfahren zur serum-/proteinfreien Kultur von Stamm- und Progenitorzellen

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