WO2011029877A1

WO2011029877A1 - Separation of mesenchymal stem cells

Info

Publication number: WO2011029877A1
Application number: PCT/EP2010/063247
Authority: WO
Inventors: Wilhelm Aicher; Gregor-Alexander Pilz; Christine Ulrich
Original assignee: Eberhard-Karls-Universitaet Tuebingen Universitaetsklinikum
Priority date: 2009-09-09
Filing date: 2010-09-09
Publication date: 2011-03-17
Also published as: DE102009041885B4; DE102009041885A1

Abstract

The present invention relates to the use of a binding molecule, which binds to CD146 for isolating and/or identifying stem cells with osteogenic differentiation potential. The invention further relates to a method for isolating such stem cells using said binding molecules, and to the use of such isolated stem cells for treating and/or diagnosing and/or preventing diseases.

Description

Separation of mesenchymal stem cells

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

Mesenchymal stem cells (MSCs), 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, they can also be found in astrocytes, neurons, endothelial cells, hepatocytes, Differentiate pancreas-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 partly can also be found on endothelial, epithelial cells as well as on muscle cells. However, MSCs can be distinguished 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), but the latter are heterogeneous in their proliferation and differentiation capabilities.

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. They could therefore, for example, in the context of stem cell therapies directly the treatment of degenerative diseases of organs such as bones, cartilage, tendons, muscle, connective tissue, blood cells, etc. serve.

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 obtained in this way are poorly defined and Not only do they differentiate into heterogeneous MSC populations, but also into osteoblasts, and / or into osteoblast precursor cells, into 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.

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 isolation and / or Identification of mesenchymal stem cells (MSC) with a high expression of the cell surface marker CD146, compared to MSC, which show no or low expression of CD146, from a biological sample. 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 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 demonstrate in their own experiments that using CD146 binding binding molecules it is possible to specifically isolate mesenchymal stem cells (MSC), which subsequently differentiated into osteoblasts. The MSC, which have an osteogenic differentiation potential, are characterized by a high expression of CD146, so all MSC, which can be identified, for example, in the flow cytometry with "CD146 ^high ". It has also been shown that MSCs that do not express CD146 do not 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 so mesenchymal stem cells can be provided, which, for example, in turn, advantageously used in therapy and prophylaxis or in diagnostics and research can be. 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.

By "high CD146 expression" is meant any expression of the cell surface marker CD146 on the MSC, which is increased in comparison to MSC with no or low CD146 expression, which can be achieved, for example, by a FACS / or MACS For example, by FACS "CD146 ^high " MSC can be distinguished from "CD146 ^low " MSC, where the CD146 ^high MSCs represent those stem cells that are osteogenic in accordance with the present invention Have differentiation potential.

It is preferred if the CD146-binding binding molecule is selected from CD 146 binding antibodies, aptamers, as well as 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 depict portions or portions of the disclosed binding molecules that simultaneously exhibit and possess the functional properties, particularly the cell surface marker binding properties of the binding molecules, among 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 CD 146 can on the other hand also be correspondingly humanized, and within the scope of the invention disclosed here, can be used for the uses and / or methods of the invention, in particular also for stem cell therapy.

Humanized antibodies may, for example, be 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. The devices can optionally also consist of completely or partially absorbable materials and in addition to the antibodies have other therapeutic active substances that are commonly used in implants / grafts 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.

The inventors of the present application were able to demonstrate on the basis of the mark CD 146 that MSC from bone marrow had a higher osteogenic differentiation potential than, for example, MSC with a low CD146 expression from the placenta. These results were also verified in experiments in which the expression of alkaline phosphatase in MSC from bone marrow and in placental isolated MSC was investigated. This enzyme is a key factor in bone formation or regeneration. Thereafter, the expression of alkaline phosphatase in CD146-positive MSC from the bone marrow was comparatively higher than in CD146-positive (but CD146 ^low ) MSC from the placenta.

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 CD 146, and b) separating MSC to which the binding molecule binding CD 146 has bound to a high degree, of cells which have not or only to a limited extent bound the binding molecule, for obtaining mesenchymal stem cells with osteogenic differentiation potential.

By "isolation of mesenchymal stem cells with osteogenic differentiation potential" is meant according to the invention 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 allows centrifugation of the latter with mesenchymal stem cells having osteogenic differentiation potential bound thereto and separation of the complex of binding molecule and mesenchymal stem cells with osteogenic differentiation potential from the biological sample ,

In a further development of the method according to the invention, the further step a) is also provided after step a): a) Separation of the MSC binding molecule complex from unbound components of the biological sample and of 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, ¹³ % 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 process 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 large mechanical Impairment with a high degree of enrichment, ie 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 carried out via an intermediate bacterial protein, such as protein A, protein G or protein L, which on the one hand bind the constant regions (Fe) of antibodies, and on the other hand can easily be coupled to the carrier substance 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 also initially be differentiated in vitro to osteoblasts, 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 achieved by adding low molecular weight components such as dexamethasone, beta-glycerophosphate and vitamin C (see Pittenger et al., "Multilinear Potential of Adult Human Mesenchymal Stern 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 selectively 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 found in their own experiments that using antibodies directed against CD146 in a method for the isolation / identification of mesenchymal stem cells 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 are used for the therapy and / or prophylaxis of bone damage, degenerations or diseases, in particular of the (tubule) bone in the extremities, or bone in the oral and maxillofacial region, 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. 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. 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.

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:

Figure 1 Studies on expression of CD146 on bone marrow MSC (A) and placenta (B) and (C): Expression of CD 146 is higher in bone marrow MSC (A) than in placental MSC (B) and (C);

Fig. 2 shows the division of MSC of the placenta by MACS into the fractions

CD146 ^high , CD146 ^mid and CD146 ^low ;

Figure 3 shows that CD146 ^high and CD146 ^mid cells do not lose their in vitro phenotype over multiple passages, and that in CD146 ^low population, expression of CD146 is absent for at least 4 passages; the detection of the osteogenic differentiation of CD146 ^high cells; Fig. 5 shows the evidence that the CD146 ^low population neither osteogenically differentiated spontaneously (left) nor after addition of osteogenic stimuli (right); and

Figure 6 shows evidence that MSC from bone marrow is significantly more alkaline

Phosphatase mRNA expressed as MSC from the placenta (pfMSC: placental fetal MSC; pmMSC: placental maternal MSC).

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 CD146 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-1 on chondrogenic differentiation of FCS-free expanded MSC", Osteoarthritis and Cartilage, May 2009) MSC from placenta (patients from the gynecological clinic in Tübingen) were separated from the embryonic maternal parts of the placenta, both mechanically fragmented separately with scalpels, and then enzymatically digested, undigested parts were separated by narrow sieves, the cell suspension was diluted with PBS, and The cells were then cultured in MSC expansion medium.

Separation of the CD146 positive MSC was performed by magnet sorting (Automacs, Miltenyi, Bergisch Gladbach) using the anit CD146 antibody (clone 128018, R & D Systems). For osteogenic induction, cells were inoculated with osteogenic differentiation medium (dexamethasone, beta-glycerophosphate, vitamin C).

Osteogenic differentiation was detected after 2- to 4-week cultivation of the cells by silver staining of the Ca precipitates in the cells by von Kossa staining. For this purpose, 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 the bone marrow MSCs exhibited marked osteogenic differentiation (see Figure 1, top row, left) and also prominently expressed CD146 (see Figure 1, lower row, left). In contrast, placental MSC showed weaker osteogenic differentiation (see Fig. 1, upper row, center and right) and at the same time a lower expression of CD146 (see Fig. 1, lower row, center and right).

Osteogenic differentiation of CD146 ^high and CD146 ^mid 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 ^mid 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. By means of MACS both MSC from the endometrial maternal zone (see Fig. 2, top row) can also be read from the fetal zone (lower row) of the placenta in CD146 ^high , CD146 ^mid and CD146 ^low populations.

In further experiments it was investigated to what extent the CD146 ^high and CD146 ^mid populations maintain their phenotype. The CD146 ^high and CD146 ^mid populatio They did not lose their phenotype even after at least 4 passages after separation, whereas in the CD146 ^low population the expression of this antigen was absent. These results are shown in Fig. 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 MSC stably express this antigen Figure 3, left), at the CD146 ^mid MSC

a slight trend towards higher expression / cells is detectable (see FIG. 3, center), and the CD146 ^high MSC do not express this antigen even 5 passages after MACS separation (see FIG. 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), again, the osteogenic differentiation The results showed that the CD146 ^high MSC (like the CD146 ^mid MSC, data not shown) spontaneously did not differentiate into osteoblasts (see Figure 4, left), but only after the addition of osteogenic stimuli (See Fig. 4, right.) On the other hand, CD146 ^low MSC showed neither spontaneous differentiation (see Fig. 5, left) nor differentiation after addition of osteogenic stimuli (see e Fig. 5 right).

In further experiments, MSC isolated from bone marrow with MSC isolated from the placenta were compared for their CD146 expression. It was found that strong CD146 expression could be detected in almost all MSC preparations from the bone marrow, compared to a comparatively low CD146 expression of MSC from the placenta (data not shown). In further experiments, the expression of the enzyme alkaline phosphatase in MSC was investigated, which were isolated either from bone marrow or from the placenta, and the CD146 strongly expressed (Rnochenmarks-MSC) bwz. CD146 only to a minor extent (placental MSC). Alkaline phosphatase is a key factor in bone formation or regeneration, as this enzyme releases phosphate from phosphoproteins, which is important for the precipitation of calcium in the bone material.

The enzymatic activity of alkaline phosphatase was examined cytochemically in the MSC on the one hand, using osteoblasts as a control. The cells were fixed, washed and incubated in a naphtol-AS-MX solution mixed with an alkaline diazonium solution and further processed according to the manufacturer's instructions (LAP Kit, Sigma-Aldrich). The precipitates indicative of phosphatase activity were observed microscopically. It was found that in the MSC isolated from bone marrow the alkaline phosphatase was highly active due to the strong substrate precipitation - and thus dye accumulation - in contrast to the MSC isolated from the placenta (data not shown) in which no alkaline phosphatase Activity was observed.

Furthermore, gene expression of alkaline phosphatase was investigated by RT-PCR (real-time polymerase chain reaction). To isolate the RNA, the cells were carefully harvested, washed and the RNA was purified by means of the RNeasy kit (Qiagen, Hilden, Germany). Reverse transcription into cDNA was performed with 1 μg total RNA and the corresponding kit from Clontech, USA. The gene-specific cDNA was analyzed by means of quantitative RT_PCR (qRT-PCR, LightCycler®, Roche, Mannheim, Germany) using commercially available primer pairs (AP (alkaline phosphatase), CD-RAP (cartilage-derived retinoic acid sensitive protein; cartilage derived retinoic acid-sensitive protein), OC (osteocalcin) and PPARy2 (peroxisome proliferator-activated receptor gamma-2; peroxisome proliferator-activated receptor gamma-2), all from SearchLC, Heidelberg, Germany). For quantification, GAPDH transcripts and serial dilutions of a recombinant DNA standard served as References. Expression of the target genes AP, CD-RAP, OC and PPARy2 was normalized for GAPDH expression.

The results of these experiments showed significantly higher expression of alkaline phosphatase in bone marrow isolated MSC compared to placental MSC (see Figure 6).

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

Claims

claims

1. Use of binding to the cell surface marker CD146 binding molecules for isolation and / or identification of mesenchymal stem cells (MSC) with a high CD146 expression compared to MSC with no or low CD146 expression from a biological sample.

2. Use according to claim 1, characterized in that the binding molecule is selected from at least one of the following binding to CD146 antibodies, aptamers, or functional fragments thereof.

A method of isolating and / or identifying 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, b) separation of MSC to which the CD 146 binding binding molecule has bound to a high degree, of cells that have not or only slightly bound the binding molecule, for isolation and / or Identification of mesenchymal stem cells with osteogenic differentiation potential.

4. The method according to claim 2, characterized in that after step a) the further step al) is carried out: al) Separating the MSC binding molecule complex from unbound constituents of the biological sample and optionally unbound binding molecules.

5. The method according to claim 3 or 4, characterized in that the binding molecule is selected from against / to CD146 binding antibodies, Apta- mers, or functional fragments thereof.

6. The method according to any 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.

7. The method according to any one of claims 3 to 6, characterized in that the binding molecule is coupled to a detectable marker.

8. The method according to any 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 enzyme-linked immunosorbent assays (ELISA), b ) fluorescence activated cell separation (FACS), and c) magnetic cell separation (MACS).

9. Use of mesenchymal stem cells, which have been isolated and / or identified by a method according to any one of claims 3 to 8, for the therapy or diagnosis.

10. Use of mesenchymal stem cells, which have been isolated and / or identified by a method according to any one of claims 3 to 8, for the defined generation of osteoblasts.

11. Use according to claim 11, characterized in that the mesenchymal stem cells which have been isolated and / or identified and differentiated into osteoblasts by a method according to one of claims 3 to 8 are used for therapy and / or prophylaxis.

12. Use of mesenchymal stem cells, which have been isolated and / or identified by a method according to any one of claims 3 to 8, for the therapy and / or prophylaxis of bone damage, degenerations, or diseases.