DE102009041885B4 - Separation of mesenchymal stem cells - Google Patents

Abstract

Use of binding molecules which bind to the cell surface marker CD146 for the in vitro isolation and / or identification of mesenchymal stem cells with osteogenic differentiation potential from a biological sample.

Description

The present invention relates to the isolation and / or identification of mesenchymal stem cells with osteogenic differentiation potential from a biological sample, as well as the use of these stem cells.

Mesenchymal stem cells (MSC), also called mesenchymal stromal cells, are multipotent cells that have the ability to differentiate into various mesenchymal tissues under appropriate in vitro and in vivo conditions. For example, they can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, and form bone, cartilage, fat, and muscle tissues. In addition, however, they can also differentiate into astrocytes, neurons, endothelial cells, hepatocytes, pancreatic-like cells and lung epithelial cells. Morphologically they are recognizable by their fibroblastoid phenotype, and are found in various adult and embryonic human tissues, including the brain, bone marrow, umbilical cord blood, blood vessels, skeletal muscle, skin, liver, gums, and placenta.

MSCs express a variety of surface markers such as CD105 (endoglin, SH2), CD73 (ecto-5'-nucleotidase, SH3, SH4), CD166 (ALCAM), CD29 (β1-integrin), CD44 (H-CAM) and CD90 (Thy-1), the z. T. also found on endothelial, epithelial cells and muscle cells. However, MSCs can be differentiated from hematopoietic stem cells because MSCs do not express the hematopoietic stem cell markers CD45, CD34 and CD133.

Mesenchymal stem cells have the property of adhering rapidly and stably to plastic or glass surfaces and forming colony-forming fibroblasts (CFU-F). The latter, however, are heterogeneous in their proliferation and differentiation abilities.

Mesenchymal stem cells with a certain differentiation potential are of great interest in medicine and research: they can be obtained in particular from the bone marrow, even from that of older people, have a high division rate and, as mentioned, can differentiate into tissue cells of mesenchymal origin. For example, in the context of stem cell therapies, they could serve directly for the treatment of degenerative diseases of organs such as bones, cartilage, tendons, muscle, connective tissue, blood cells, etc.

For the recovery of mesenchymal stem cells, unfractionated bone marrow cells are currently used as a starting material which cultivates on plastic dishes, identifies the MSC via their adhesion to the plastic surface, and discards the nonadherent hematopoietic cells from the sample. However, the cells thus obtained are poorly defined and differentiate not only into heterogeneous MSC populations, but also into osteoblasts, and / or in osteoblast precursor cells, in fat cells, reticulum cells, macrophages and endothelial cells. A specific treatment of degenerative diseases of an organ is therefore difficult with MSC without a particular differentiation potential or impossible due to possible side effects.

The isolation of mesenchymal stem cells with a very specific differentiation potential has hitherto not been possible or known in the prior art. However, such isolation would, as mentioned, offer the great advantage that stem cells identified in this way could be used specifically for the therapy / treatment of diseased, degenerated or damaged tissue, into which the thus specifically isolated stem cells differentiate.

For example, cartilage damage could be treated by adding specifically isolated mesenchymal stem cells with osteogenic differentiation potential either directly in situ to the affected tissue, where they differentiate into osteoblasts and thereby replace the damaged bone tissue (stem cell therapy). On the other hand, a differentiation into osteoblasts in vitro may be of interest, if - for example, for research / diagnostics / medicine - differentiated osteoblasts are to be obtained.

Sacchetti et al. (Self-Renewing Osteoprogenotirs in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment, Cell (2007) 131: 324-336) describe the hematopoietic microenvironment in human bone marrow, and in particular, try to characterize the cell types that make up this environment. The experiments that Sacchetti et al. disclosed that MCAM / CD146-expressing subendothelial cells in human bone marrow are able to provide such a hematopoietic microenvironment.

Tripodo et al. ("CD146 + bone marrow osteoprogenitors increase in the advanced stages of primary myelofibrosis", Haematologica (2009) 94: 127-130), examined in their publication bone marrow sections of healthy and myelofibrosis patients, and found that the number of CD146 + stromal cells in the bone marrow correlates with the degree of fibrosis, vessel density and disease progression.

Against this background, there is a great interest in mesenchymal stem cells with osteogenic differentiation potential, in particular to use the stem cells thus obtained in appropriate uses.

The object of the present invention is therefore to provide new ways in which mesenchymal stem cells with osteogenic differentiation potential can be isolated.

According to the invention, this object is achieved by the use of a binding molecule which binds to the cell surface marker CD146, for the in vitro isolation and / or identification of stem cells with osteogenic differentiation potential. CD146 is also referred to in the literature as "Melanoma Cell Adhesion Molecule" (MCAM, Mel-CAM), MUC-18, S-endo or Gicerin.

The object is further achieved by a method for the in vitro isolation and / or identification of mesenchymal stem cells with osteogenic differentiation potential, in which CD146 binding binding molecules are used.

Furthermore, the invention relates to the stem cells isolated in this way and their use, in particular in therapy.

The object underlying the invention is completely solved in this way. The inventors of the present application have been able to show in their own experiments that using CD146-binding binding molecules it is possible to specifically isolate mesenchymal stem cells which subsequently differentiated into osteoblasts. It has also been shown that MSCs which do not express CD146 neither spontaneously differentiate into osteoblasts under the usual osteogenic induction conditions, which are further described below.

Thus, a tool is provided with which mesenchymal stem cells can be obtained, which differentiate specifically into osteoblasts. This was not possible in the prior art.

Due to the new use and the new method, it is therefore possible to provide mesenchymal stem cells which, for example, in turn can advantageously be used in therapy and prophylaxis or else in diagnostics and research. Thus, the thus isolated stem cells in particular for the treatment of diseases that are characterized by a degenerated, injured or damaged tissue, are used, for example. In the context of a stem cell therapy: isolated by the method according to the invention stem cells are transplanted into the affected tissue (eg also in connection with certain implants), and differentiate there into the corresponding tissue. This regenerates the degenerated tissue and makes it functional again.

It is preferred if the binding molecule binding to CD146 is selected from antibodies binding to CD146, aptamers, and from fragments of said antibodies, aptamers.

Suitable antibodies are, for example, the monoclonal antibodies clones 128018 (R & D Systems, 614 McKinley Place, NE, Minneapolis, USA) or N1238, P1H12 and c264 (all from abcam, 1 Kendall Square, Suite 341, Cambridge, MA, USA). It will be understood that any other antibody directed against CD146 is also suitable for the purposes of the present application.

The terms "directed against CD146", "anti-CD146", "binding to CD146", "recognizing CD146" are used interchangeably herein to mean that binding molecules can specifically recognize and bind this marker.

Aptamers are high-affinity RNA or DNA oligo- or polynucleotides, ie nucleic acid molecules which, due to their specific spatial structure, have a high affinity for a target molecule. Aptamers typically have a length of up to 100 nucleotides, which, while having comparable antigen binding properties as antibody fragments, are often much more specific and significantly more stable. With their relatively large and flexible surface, they can potentially interact with more target molecules in a highly specific and selective way. Aptamers, when introduced into an organism, show little immunogenic or toxic effects but a rapid clearance.

By means of "SELEX" (Systemic Evolution of Ligands by Exponential Enrichment), large amounts of aptamers of different sequences and secondary structures can be generated enzymatically. From this amount, such aptamers with high affinity for a target molecule, such as, for example, MSC, are then selected and enriched. The primary structure of this aptamer can be elucidated by sequencing methods known in the art so that they can subsequently be synthesized in vitro.

As used herein, the terms "functional fragments" or "functional fragments" as used in the application are intended to include portions or portions of the disclosed binding molecules and simultaneously show and possess the functional properties, in particular the cell surface marker binding properties of the binding molecules from which they are derived. These fragments can be used either as such or in combination with other fragments. For the purposes of the present invention, the latter also means, for example, modified anti-CD146 antibodies which have been adapted, for example humanized, for appropriate uses and uses in humans. The antibodies suitable for the purposes of the present invention are preferably monoclonal.

In the present case, however, also fragments of such antibodies, such as, for example, Fab, F (ab) ' ₂ or scFv fragments, and other fragments such as CDR ("complementarity-determining region", hypervariable region) are meant as antibodies within the meaning and scope of the present invention as long as they have their functionality, ie the specific binding properties, like the "whole" antibodies from which they are derived. For example, such antibody fragments can also be produced recombinantly using methods known in the art.

It is therefore also understood that the antibodies directed against CD146 on the other hand can also be correspondingly humanized, and in the context of the invention disclosed herein for the uses and / or methods of the invention, in particular for a stem cell therapy can be used.

Humanized antibodies can be, for example, chimeric antibodies in which the constant regions of the animal antibodies (for example of mouse or rabbit antibodies) have been replaced by the corresponding regions of human antibodies, for example the Fc fragments (Sharon et al., Nature, (1984), 309: 364-367). Alternatively, the CDR of the animal antibody can also be linked to human antibodies, this process being called antibody "reshaping". In yet another method, human antibodies are produced in transgenic animals.

Furthermore, in a use according to the invention, the antibodies, for example in humanized form, or functional fragments thereof, can be introduced or introduced onto appropriate implantable medical devices and implanted together with the device in the patient to be treated on the sites / tissue defects to be treated , At the sites to be treated, mesenchymal stem cells are then recruited via the antibodies, which attach themselves to the implant, differentiate and thus form new tissue. Suitable medical devices here are any biocompatible implants, endoprostheses, for example. Stents, etc., of any kind, which are either permanently or temporarily introduced into the patient. Optionally, the devices can also consist of completely or partially resorbable materials and, in addition to the antibodies, have further therapeutic active substances which are usually used in implants / transplants to be introduced into a body.

The biological sample in the context of the present invention may be any biological tissue and / or biological fluid, and includes any biological material of animal or human origin or any fluid that is to be assayed for the presence of mesenchymal stem cells with osteogenic differentiation potential , It may be a tissue complex, a cell suspension or organs, parts of organs or organisms. Examples of biological tissues and fluids are bone marrow tissue, bone marrow cells, cartilage cells, bone cells, adipose tissue, fat cells, liver tissue, liver cells, placental tissue, peripheral blood, umbilical cord blood, apheresis blood. It is preferred if the biological sample is a bone marrow sample.

• a) in-Kontakt-Bringen einer biologischen Probe, die mesenchymale Stammzellen (MSC) enthält, mit einem Bindemolekül, das an CD146 bindet, zur Bildung von MSC-Bindemolekül-Komplexen, und
• b) Separieren der MSC-Bindemolekül-Komplexe von Zellen, die das Bindemolekül nicht gebunden haben, zur Gewinnung von mesenchymalen Stammzellen mit osteogenem Differenzierungspotential.

As already mentioned above, the present invention also relates to a method for isolating and / or identifying mesenchymal stem cells with osteogenic differentiation potential, comprising the following steps:

• a) contacting a biological sample containing mesenchymal stem cells (MSC) with a binding molecule that binds to CD146 to form MSC binding molecule complexes, and
• b) Separating the MSC-binding molecule complexes from cells that did not bind the binding molecule to obtain mesenchymal stem cells with osteogenic differentiation potential.

Isolation of mesenchymal stem cells with osteogenic differentiation potential is understood according to the invention to mean that a complex of binding molecule and mesenchymal stem cells with osteogenic differentiation potential is separated from the biological sample. If desired, the complex can subsequently be separated into mesenchymal stem cells with osteogenic differentiation potential and binding molecule. This is done by means known in the art. For example, the binding molecule may be coupled to a support material, which facilitates handling of the binding molecule, and centrifugation of the latter, optionally with mesenchymal stem cells having osteogenic binding thereto Differentiation potential and the separation of the complex of binding molecule and mesenchymal stem cells with osteogenic differentiation potential of the biological sample allows.

• a1) Separieren des MSC-Bindemolekül-Komplexes von ungebundenen Bestandteilen der biologischen Probe und von ggf. ungebundenen Bindemolekülen.

In a further development of the method according to the invention, the further step a1) is also provided after step a):

• a1) Separating the MSC-binding molecule complex from unbound components of the biological sample and optionally unbound binding molecules.

Depending on the sample, better isolation and / or identification of the mesenchymal stem cells with osteogenic differentiation potential may be achieved with this step preceding step b). By this measure, namely, the isolation of the complex is realized and separated contaminants, such as unbound binding molecules, unbound other components of the biological sample and possibly unbound mesenchymal stem cells of the desired mesenchymal stem cells with osteogenic differentiation potential.

In particular, it is preferred if the binding molecule, as in the inventive use, is selected from antibodies binding to CD146, aptamers, or functional fragments of these antibodies and aptamers.

As mentioned above, these binding molecules are particularly suitable tools which, when used in the context of the method according to the invention, can be used successfully for isolating and / or identifying mesenchymal stem cells with osteogenic differentiation potential.

Furthermore, it is preferred in the method and the use according to the invention if the binding molecules have a detectable and / or selectable marker.

With this measure, mesenchymal stem cells can be detected and selected particularly easily. According to the invention, a marker means any compound by means of which localization and identification of the binding molecule in vitro, in vivo or in situ is possible. These include color indicators with fluorescent, phosphorescent or chemiluminescent properties, such as fluorocein isothiocyanate (FITC), rhodamine, AMPPD, CSPD, radioactive indicators such as ³² P, ³⁵ S, ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H, non-radioactive indicators, such as biotin or dioxigenin, alkaline phosphatase, horseradish peroxidase, etc.

If a fluorescently labeled binding molecule is used, the method according to the invention can be used in the context of established fluorescence-activated cell sorting (FACS). By means of FACS, mesenchymal stem cells can be isolated particularly well from a cell suspension. For this purpose, the MSC-containing cell suspension is incubated with the fluorescence-labeled binding molecules, wherein the binding molecules bind to the mesenchymal stem cells. The cell suspension is then passed through a thin cannula whose jet is finally resolved by vibration into individual tropics. If a drop contains a mesenchymal stem cell to which the binding molecule has bound, the fluorescent marker is excited by a laser beam to fluoresce. This fluorescence can be measured with a light detector and used for separation and thus isolation of the mesenchymal stem cell. For this purpose, the bound mesenchymal stem cells are charged by means of an electrical pulse depending on the fluorescence intensity and deflected and sorted accordingly when passing an electric field.

Suitable selectable markers are also magnetic particles, which are preferably very small in the order of 50 nm. These can be coupled to the binding molecules by methods known in the art. Such magnetic binding molecules can also be used for the isolation of mesenchymal stem cells, as part of the so-called. Magnetic cell sorting (MACS). For this, the magnetic binding molecules are added to the cell suspension. After incubation, the binding molecules bind to the mesenchymal stem cells. The cell mixture is separated by a column whose ferromagnetic matrix consists of metal spheres or wires. For this purpose, the column is placed in a homogeneous magnetic field in which the mesenchymal stem cells, to which the magnetic binding molecules are bound, are retained on the matrix surface. The remaining cells and components of the mixture are washed out of the column. After removing the magnetic field, the separated mesenchymal stem cells can also be rinsed out of the matrix. This method allows a rapid separation of mesenchymal stem cells without great mechanical impairment with a high degree of enrichment, d. H. even a very small population of mesenchymal stem cells can be isolated almost pure.

In a further embodiment, the isolation / identification takes place by means of immunological methods, such as the ELISA ("enzyme-linked immunosorbent assay", enzyme-linked immunosorbent assay).

This method has become established as a particularly effective and easy-to-perform separation method of antigen-antibody complexes from an incubation medium. In this method, either the antibody or the antigen is labeled with an enzyme that allows colorimetric detection, usually by alkaline phosphatase or peroxidase.

In one embodiment of the method according to the invention, the binding molecule is coupled to a carrier substance which is selected from the group consisting of: agarose, sepharose.

Such materials have been found to be particularly suitable for the immobilization of binding molecules. The coupling is preferably via an intermediate bacterial protein, such as protein A, protein G or protein L, which on the one hand bind the constant regions (Fc) of antibodies, and on the other hand can be easily coupled to the carrier agarose or Sepharose.

The stem cells obtained by means of the methods according to the invention can then be used either directly in the context of stem cell therapy (autologous or allogeneic therapy), where they can differentiate into osteoblasts in situ and thus regenerate degenerated or damaged cartilage tissue.

On the other hand, the mesenchymal stem cells obtained with the method according to the invention with osteogenic differentiation potential can also be first differentiated into osteoblasts in vitro, and then used for the treatment of diseased or degenerated tissue.

In this embodiment, the isolated mesenchymal stem cells can be differentiated by means of specific growth factors, it being preferred if the growth factors are selected from BMPs (bone morphogenetic proteins) such as BMP-2 and BMP-7. Alternatively, osteogenic differentiation of the MSC can be accomplished by the addition of low molecular weight components such as dexamethasone, beta-glycerophosphate and vitamin C (see Pittenger et al., "Multilinear Potential of Adult Human Mesenchymal Stem Cells", Science, 1999, 284: 143-147 ).

This measure has the advantage that already suitable growth factors are provided. In the case of the use of BMP differentiation of the bound MSC is effected in osteoblasts, which favors the ingrowth of a bone replacement implant according to the invention.

With the method according to the invention it is now possible for the first time to specifically isolate stem cells with, for example, osteogenic differentiation potential from the tissue of a patient and a fast, efficient and targeted cultivation of osteoblast tissue for subsequent transplantation into the sample donor (autologous transplantation ) or another recipient (allogeneic transplant).

It is preferred if in the method according to the invention a binding molecule is used which is selected from CD146 binding antibodies or aptamers, or functional fragments of these antibodies and aptamers.

The inventors have determined in their own experiments that using antibodies directed against CD146 in a method for the isolation / identification of mesenchymal stem cells, a targeted enrichment of mesenchymal stem cells with osteogenic differentiation potential could be obtained.

On the other hand, the mesenchymal stem cells separated from the mesenchymal stem cells with osteogenic differentiation potential can be used for non-osteogenic applications in a variety of regenerative therapies in which an ossification must be avoided, such as in the regeneration of elastic tissue (tendon, muscle, fat, etc.) because the CD146 negative MSC does not differentiate osteogenic.

The present invention further relates to the use of osteogenic meesenchymal stem cells isolated and / or identified by the methods of the invention for therapy, diagnostics or prophylaxis.

In one embodiment, it is preferable if the stem cells obtained by the methods according to the invention are used for the defined generation of osteoblasts, specifically in vivo or in vitro.

Furthermore, in a further embodiment it is preferred if the stem cells isolated and / or identified by the methods according to the invention, which have been differentiated into osteoblasts, are used for the therapy and / or prophylaxis of degenerated or susceptible tissue.

In particular, it is preferred if the stem cells obtained by the method according to the invention for the therapy and / or prophylaxis of bone damage, degeneration or disease, in particular the (tubule) bone in the extremities, or bone in the oral jaw and Facial area, spine and other diseases in which the stem cells for bone tissue replacement (so-called "tissue repair") are to be used.

The binding molecules can be provided in a kit and / or a pharmaceutical composition for the isolation and / or identification of mesenchymal stem cells with osteogenic differentiation potential.

Furthermore, the invention relates to a pharmaceutical composition containing stem cells which have been isolated and / or identified according to the methods of the invention, and at least one pharmaceutically acceptable carrier and / or adjuvant, and optionally therapeutically active substances.

By "pharmaceutically acceptable excipients or excipients" herein is meant any substance / composition to be administered to a patient in pharmacy that does not adversely affect the efficacy of the cells / antibodies, and / or that pharmacologically supports the use of the pharmaceutical composition or can facilitate.

By "therapeutically active substance" herein is meant any substance used for the purpose of treating or ameliorating a clinical picture of a patient.

The pharmaceutical compositions can be administered systemically, i. H. For example, subcutaneously, intravenously, parenterally, intramuscularly, transdermally, or topically, the mode of administration will depend on the nature of the disease, the clinical picture and 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, and protective colloids, such as homologous albumin or biocompatible hydrogels, with. For an overview of such additional ingredients, see, for example, A. Kibbe: "Handbook of Pharmaceutical Excipients," 3rd ed., 2000, American Pharmaceutical Association and Pharmaceutical Press.

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

The invention will be explained in more detail in the following description and the attached figures.

Show it:

1 Studies on the expression of CD146 on bone marrow MSC (A) and placenta (B) and (C): Expression of CD146 is higher in bone marrow (A) MSC than in placental MSC (B) and (C) ;

2 the division of MSC of the placenta by MACS into the fractions CD146 ^high , CD146 ^medium and CD146 ^low ;

3 the expression of the absent evidence that the CD146 and CD146 ^{high medium} not lost their phenotype cells in vitro for several passages and that in the ^low CD146 CD146 population of at least 4 passages;

4 the detection of the osteogenic differentiation of CD146 ^high cells; and

5 Evidence that the CD146 ^low population neither spontaneously (left) nor after the addition of osteogenic stimuli (right) osteogenic differentiated.

Examples

Detection of CD146 expression on MSC from bone marrow and placenta

The present inventors have investigated in initial experiments the expression of the cell surface marker CD 146 on mesenchymal stem cells (in the following also: MSC) from the bone marrow and from the placenta. For this purpose, MSC were isolated from the bone marrow of patients (patients of the BG Trauma Hospital Tübingen, who were treated surgically and endoprotected): For this purpose, the bone marrow was diluted with PBS to separate the mononuclear cells by gradient centrifugation. The mononuclear cells were washed and expanded in suitable MSC expansion medium (see Felka et al., "Hypoxia reduces the inhibitory effect of IL-1β on chondrogenic differentiation of FCS-free expanded MSC", Osteoarthritis and Cartilage, May 2009). To isolate MSC from placenta (patients from the Frauenklinik Türbingen), the maternal parts of the placenta were separated from the embryonic. Both tissue portions were mechanically minced separately with scalpels and then enzymatically disrupted. Undigested parts were separated by close-meshed sieves. The cell-containing suspension was diluted with PBS and purified by gradient centrifugation. The cells were then cultured in MSC expansion medium.

The separation of the CD146 positive MSC was carried out by magnet sorting (Automacs, Miltenyi, Bergisch Gladbach) using the anti-CD146 antibody (clone 128018, R & D Systems). For osteogenic induction, the cells were inoculated into specific osteogenic differentiation medium (dexamethasone, beta-glycerophosphate, vitamin C).

The osteogenic differentiation would be detected after 2- to 4-week cultivation of the cells by silver staining of the Ca precipitates in the cells by means of Kossa staining, then cells were fixed and incubated with silver nitrate solution. Excess silver nitrate solution was washed off intensively. The Ag ²⁺ was reduced to metallic silver by carbonate and thiosulfate and fixed. The cells were stained with eosin (see Romeis, Microscopic Techniques, 2009, Spektrum Akademischer Verlag, Heidelberg). Furthermore, expression of CD146 was detected (mAK clone 128018, R & D Systems). The results of these experiments showed that MSC from bone marrow showed marked osteogenic differentiation (see 1 , upper row on the left), and CD146 also prominently expressed (see 1 , lower row on the left). In contrast, placental MSC showed weaker osteogenic differentiation (see 1 , upper row centered and right) and at the same time a lower expression of CD146 (see 1 , bottom row, center and right).

Osteogenic differentiation of CD146 ^high and CD146 ^medium populations

In the following, CD146 expression in MSC was examined immediately after magnetic cell sorting. Only placental MSCs were used to exclude the contamination of osteoblasts (which may be present in bone marrow). For this purpose, the placental MSC were separated by means of MACS in CD146 ^high , CD146 ^medium and CD146 ^low fractions (mAK clone 128018, R & D Systems) and immediately examined by FACS on the expression of CD146 to investigate the quality of the separation. Using MACS both MSC from the endometrial maternal zone (see 2 , upper row) as well as from the fetal zone (lower row) of the placenta in CD146 ^high , CD146 ^medium and CD146 ^low populations are separated.

In further experiments it was investigated to what extent the CD146 ^high and CD146 ^medium populations maintain their phenotype. The CD146 ^high and CD146 ^medium populations did not lose their phenotype even after at least 4 passages after separation, whereas in the population the expression of this antigen did not occur. These results are 3 in which the expression of CD146 in the MACS-separated subpopulations in the 2nd passage (black histograms) and in the 6th passage (gray histograms) were examined. CD146 ^high MSCs express this antigen stably 3 , left), in the CD146 ^medium MSC a slight trend towards higher expression / cells is detectable (see 3 , Middle), and the CD146 ^high MSC do not express this antigen even 5 passages after MACS separation (see 3 , right)

The osteogenic differentiation of the populations was investigated in the following experiments: MACS sorted MSC in expansion medium (DMEM medium, human plasma, platelet extract, according to Müller et al., Animal Serum-Free Culture Conditions for Isolation and Expansion of Multipotent Mesenchymal Stromal Cells from Human Bone Marrow ", Cytotherapy, 2006; 8: 437-444) or in osteogenic induction medium (DEMEM medium, dexamethasone, beta-glycerophosphate, vitamin C, according to Pittenger et al., supra). Here, too, the osteogenic differentiation was detected by means of von Kossa staining (see above). The results showed that the CD146 ^high MSC (like the CD146 ^medium MSC, data not shown) spontaneously did not differentiate into osteoblasts (see 4 left), but only after the addition of osteogenic stimuli (see 4 right). In contrast, the CD146 ^low MSC did not show any spontaneous differentiation (see 5 left) nor a differentiation after addition of osteogenic stimuli (see 5 right).

These results indicate that the expression of CD 146 correlates with the osteogenic differentiation potential and, therefore, that the cell surface marker CD146 is an excellent tool for the identification and isolation of mesenchymal stem cells with osteogenic differentiation potential.

Claims (12)

Use of binding molecules binding to the cell surface marker CD146 for the in vitro isolation and / or identification of mesenchymal stem cells with osteogenic differentiation potential from a biological sample. Use according to claim 1, characterized in that the binding molecule is selected from at least one of the following CD146-binding antibodies, aptamers, or functional fragments thereof. A method for in vitro isolation and / or identification of mesenchymal stem cells having osteogenic differentiation potential from a biological sample containing cells, characterized by comprising the steps of: a) contacting the biological sample containing mesenchymal stem cells (MSC) , with a binding molecule that binds to CD146, to form MSC binding molecule complexes, b) separating the MSC binding molecule complexes from cells that did not bind the binding molecule, for isolation and / or identification of mesenchymal stem cells with osteogenic differentiation potential , A method according to claim 2, characterized in that after step a) the further step a1) takes place: a1) Separating the MSC-binding molecule complex from unbound components of the biological sample and optionally unbound binding molecules. A method according to claim 3 or 4, characterized in that the binding molecule is selected from CD146-binding antibodies, aptamers, or functional fragments thereof. Method according to one of claims 3 to 5, characterized in that the binding molecule is coupled to a carrier substance which is selected from the group consisting of: agarose, sepharose. Method according to one of claims 3 to 6, characterized in that the binding molecule is coupled to a detectable marker. Method according to one of claims 3 to 7, characterized in that the binding molecule-MSC complex is identified and / or isolated by means of one of the following methods: a) immunological methods, in particular by means of the enzyme-linked immunosorbent assay (ELISA), b) fluorescence activated cell separation (FACS), and c) magnetic cell separation (MACS). Use of stem cells isolated and / or identified by a method according to any of claims 3 to 8 for therapy or diagnostics. Use of stem cells isolated and / or identified by a method according to any of claims 3 to 8 for the defined generation of osteoblasts. Use according to claim 11, characterized in that the stem cells which have been isolated and / or identified by a method according to one of claims 3 to 8 and differentiated into osteoblasts are used for therapy and / or prophylaxis. Use of stem cells isolated and / or identified by a method according to any of claims 3 to 8 for the therapy and / or prophylaxis of bone damage, degeneration or disease.

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