EP2027255A2 - Dispositif et substance destinés à isoler des cellules souches mésenchymateuses - Google Patents

Dispositif et substance destinés à isoler des cellules souches mésenchymateuses

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Publication number
EP2027255A2
EP2027255A2 EP07724981A EP07724981A EP2027255A2 EP 2027255 A2 EP2027255 A2 EP 2027255A2 EP 07724981 A EP07724981 A EP 07724981A EP 07724981 A EP07724981 A EP 07724981A EP 2027255 A2 EP2027255 A2 EP 2027255A2
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EP
European Patent Office
Prior art keywords
msc
aptamer
nucleic acid
binding
cells
Prior art date
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Application number
EP07724981A
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German (de)
English (en)
Inventor
Hans-Peter Wendel
Ketai Guo
Richard Schaefer
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Eberhard Karls Universitaet Tuebingen
Universitaetsklinikum Tuebingen
Original Assignee
Eberhard Karls Universitaet Tuebingen
Universitaetsklinikum Tuebingen
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Publication of EP2027255A2 publication Critical patent/EP2027255A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function

Definitions

  • MSO mesenchymal stem cells
  • the present invention relates to a device having at least one biological tissue and / or liquid contacting surface, which is at least partially coated with a substance that mediates the binding of mesenchymal stem cells (MSC), a method for binding and / or isolation of MSC from biological tissue and / or fluid, a nucleic acid molecule which selectively and highly specifically binds to MSC, the use of the nucleic acid molecule for binding and / or isolation of MSC from biological tissue and / or fluid, and a method for producing an aforementioned Contraption.
  • MSC mesenchymal stem cells
  • Stem cells are undifferentiated cells that have the ability to self-renew and differentiate into different effector cells. Because of these properties, they are among the most promising research objects in biomedical research and nurture the hope of being able to regenerate tissue or entire organs in the future in the context of so-called stem cell therapy.
  • embryonic stem cells and adult stem cells are differentiated.
  • Embryonic stem cells are obtained from the inner cell mass of the blastocyst stage of a mammalian or human embryo. They can divide indefinitely and theoretically develop into any cell type of the approximately 200 tissue types of humans. Embryo stem cell harvesting from blastocysts as part of stem cell research often leads to ethical conflicts, as the death of the embryo must be accepted.
  • adult stem cells In contrast, research on adult stem cells is ethically completely harmless, as they can be obtained from adult organisms.
  • Adult stem cells have been discovered in at least 20 human tissues. Their task is to form replacement cells for the corresponding tissue. Compared to embryonic stem cells, they were previously thought to be of limited distribution and development potential: It was assumed that adult stem cells of one particular tissue could not form cell types of another tissue. Recently, however, the findings that adult stem cells also have a much higher development potential than previously thought accumulate. So is now in To a large extent, experts consider that adult stem cells can also take a variety of differentiation routes.
  • the adult stem cells also include the so-called mesenchymal stem cells (MSC).
  • MSCs are found in a variety of tissues and organs, such as the liver, kidney, placenta, adipose tissue, umbilical cord blood, and bone marrow.
  • MSCs are generally characterized as multipotent CD29 + , CD44 + , DC90 + , CDlIb “, CD34", and CD45 "progenitor cells, which, because of their mesenchymal differentiation potential and good / in vitro expansion properties, provide an attractive cell population for replacement MSC can be differentiated into cartilage, bones, tendons and fat cells in vitro.Cultivated three-dimensionally on different carriers MSC have already been successfully used in many studies in small and large animal models to replace mesenchymal tissue. A clinical use of MSC in humans has also been successfully tested in pilot studies.
  • MSCs are not very suitable for tissue engineering, as they place particularly high demands on the homogeneity of the MSC populations and the reproducibility of the MSC recovery.
  • Another disadvantage of the current process for isolating MSC via plastic adherence is that it is very time consuming and takes at least two weeks.
  • the object of the present invention is therefore to provide a device and a method for binding and / or isolating MSC from biological tissue and / or fluid, which overcomes the above-mentioned disadvantages of the prior art.
  • the object is achieved by a device mentioned at the beginning, which is coated with Aptame- ren.
  • the object is further achieved by a method comprising the steps of: (1) providing MSC-containing biological tissue and / or MSC-containing fluid, (2) contacting the tissue and / or fluid with an MSC-binding substance, (3) incubation for a period of time permitting binding of the MSC to the substance, and optionally (4) isolating the MSC bound to the substance, the substance being an aptamer.
  • catcher molecules can be provided in the form of aptamers which bind selectively and highly specifically to MSC. This finding was particularly surprising, since to date reliable MSC markers are missing.
  • STRO-I was discussed as a promising surface antigen for the identification of MSC, but in the meantime its importance has been relativized again; see. Dennis et al., The STRO-1 + Marrow Cell Population is Multipotential, Cells Tissues Organs 170, p. 7-82.
  • MSC marker antigen W8B2 of unknown identity shows a heterogeneous expression on MSC populations; see. Vogel et al. (2003) Heterogeneity among human bone marrow-derived mesenchymal stem cells and neuroprogenitor cells, Haematologica 88, pages 126-133.
  • the prior art discloses a variety of devices coated with peptides, such as receptors or antibodies, designed to capture biological material.
  • Aptamers are high affinity RNA or DNA oligo- or polynucleotides, i. 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 to 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.
  • aptamers of different sequences and secondary structures can be generated enzymatically Aptamers with high affinity to a target molecule, such as MSC, picked out 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.
  • An exemplary process for obtaining aptamers is described, for example, in DE 100 19 154 A1, which is incorporated by reference into the present application.
  • such devices are suitable for extracorporeal use as well as implants.
  • An extracorporeal device coated with the aptamers according to the invention may be contacted with biological tissue or fluid to be examined for the presence of MSC. Further, for targeted isolation of MSC, such device may be contacted with tissues or fluids known to contain MSCs. Examples of such MSC-containing biological tissues or fluids are bone marrow, peripheral blood or apheresis blood. After contacting the device with the tissue or fluid, the MSC, if present, binds to the aptamer. After incubation, the device is separated from the tissue or fluid with the MSC bound via the aptamer. It is advantageous that the bound MSC need not necessarily be separated from the aptamers because, depending on the storage conditions, the aptamers are completely degraded within a shorter time, for example within 2 days.
  • the device may be a simple aptamer-coated carrier, but also a tube, a pump, an oxygenator, a catheter, a vascular access, a blood component storage system.
  • An advantage of an aptamer coating is further that it is stable and sterilizable, whereby a cost-effective production is possible of the device. In contrast, peptides often lose their activity through sterilization.
  • biological tissue and / or fluid comprises any biological material of animal or human origin or any fluid that is to be examined for the presence of MSC. It may be a tissue complex, a cell suspension or organs, parts of organs or organisms.
  • 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, Plaventacells, peripheral blood, umbilical cord blood, apheresis blood.
  • a further subject of the present invention relates to a nucleic acid molecule or an aptamer which selectively and highly specifically binds to MSC, as well as its use for the binding and / or isolation of MSC.
  • nucleic acid molecule or aptamer according to the invention binds to MSC in a targeted manner and largely or completely stops interactions with other structures or moves within the scope of customary cross-reactivities.
  • the aptamer is a nucleic acid molecule which contains at least one of the sequences SEQ ID NO. 1 to SEQ ID NO. 20 from the attached sequence listing. This measure has the advantage that already the primary structure of such an aptamer is provided which binds highly specifically and selectively to MSC. The implementation of a SELEX process is therefore no longer mandatory. Rather, the desired aptamer can be prepared directly by simple and time-saving synthetic procedures.
  • sequence-specific aptamer according to the invention still binds MSC in a highly specific and selective manner, even if it contains, in addition to one of the nucleotide sequences SEQ ID NO. 1 to SEQ ID NO. 20 has one or more further nucleotides at its 5 'or 3' end.
  • the selectivity and specificity of the aptamer is retained in this embodiment because replacement occurs outside of the so-called “hair pin loops" or "bulgs,” which are the functional regions of an aptamer. These areas occupy the secondary structures responsible for binding the target structure.
  • this embodiment also encompasses such an aptamer which comprises the functional sections from the nucleotide sequences SEQ ID NO. 1 to SEQ ID NO. 20, which is modified in the non-functional sections by nucleotide substitutions or deletions. Such a modified aptamer is not or not significantly altered in its ability to bind to MSC.
  • sequence-specific aptamers in question may be modified by suitable techniques so that they are protected and do not lose their efficacy in the biological environment, for example they are not digested by nucleases.
  • Protective mechanisms that are suitable for this purpose are well known in the art and include, for example, LNA (locked nucleic acids) technologies with furanose [see, for example, Wahlestedt et al. (2000), Patent and non-toxic antisense oligonucleotides containing locked nucleic acids, Proc. Natl. Acad. Be. USA 97 (10), Pages 5633 to 5638)], or the Spiegelmer® technology from Noxxon, Berlin, Germany.
  • LNA locked nucleic acids
  • the aptamer has a detectable and / or selectable marker.
  • a marker means any compound by means of which localization and identification of the aptamer is possible in vitro, in vivo or in situ.
  • 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.
  • FITC fluorocein isothiocyanate
  • AMPPD rhodamine
  • CSPD chemiluminescent properties
  • 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.
  • the method according to the invention can be used in the context of established fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • MSC can be isolated from a cell suspension particularly well.
  • the MSC-containing cell suspension is incubated with the fluorescence-labeled aptamers.
  • the aptamers bind to the MSC.
  • the cell suspension is then passed through a thin cannula whose jet is finally resolved by vibration into individual tropics.
  • the fluorescent marker is excited to fluoresce by a laser beam.
  • This fluorescence can be measured with a light detector and used to separate and thus isolate the MSC.
  • the bound MSC are charged by means of an electrical pulse depending on the fluorescence intensity and deflected and sorted when passing an electric field.
  • Suitable selectable markers are magnetic particles, which are preferably very small in the order of 50 nm. These can be over in the state couple the technique known to the art to the aptamers.
  • Such magnetic aptamers can also be used for the isolation of MSC, in the context of the so-called. Magnetic cell sorting (MACS).
  • MSC Magnetic cell sorting
  • the magnetic aptamers are added to the cell suspension. After incubation, the aptamers have bound to the MSC.
  • the cell mixture is separated by means of 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 MSC, to which the magnetic aptamers are bound, are held 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 MSC can also be rinsed out of the matrix. This method allows a fast separation of MSC without great mechanical impairment with a high degree of enrichment, ie even a very small population of MSC can be isolated almost pure.
  • the device according to the invention is an implant.
  • this is a device which is used for a certain time or permanently in the human or animal body.
  • prostheses for example.
  • stents are often used in vascular surgery, these being made of different plastics or materials.
  • vascular prostheses accesses, ports or conduits
  • the surface (s) are locally coated with different aptamers according to the invention, so that different MSC populations are bound.
  • the invention advantageously enables the colonization of the implants with the body's own MSC. This ensures on the one hand that an autologous functional interface is generated on the implant, which is no longer recognized as foreign by the body, and the implant, the functional physiological properties of each site or organ, eg. As a bone substitute, dental implant, etc. take over.
  • the colonization of the device according to the invention with MSC can be carried out intracorporeally, extracorporeally or else in a separate bioreactor in which the biological tissue and / or the liquid is contained.
  • patches or films which are to be coated with MSC.
  • a patch consists, for example, of poly-N-isopropylacrylamide (PIPAAm), as described in Miyahara et al. (2006), Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction, Nature Medicine, Online Publication, pages 1-7.
  • PIPAAm poly-N-isopropylacrylamide
  • the authors describe transplantation of MSC-populated patches into a myocardially-compromised heart, which showed surprisingly good regeneration by the MCS introduced. However, the authors had to colonize the patch first with previously isolated MSC, and only the populated patches were then implanted in the patient.
  • an aptamer-coated patch can now be introduced directly into the heart, with the affinity of the aptamers subsequently providing itself for colonization with MSC. Keeping patches populated with MSC and the costly prior isolation and populating of patches with MSC is no longer required. A needy patient can be helped faster.
  • a material selected from the group consisting of: polytetrafluoroethylene, polystyrene, polyurethane, polyester, polylactide, polyglycolic acid, polysulfone, polypropylene, polyethylene, polycarbonate, polyvinyl chloride is used as the surface for the device according to the invention , Polyvinyl difluoride, polymethyl methacrylate, hypoxyl apatite, isoproparyl acrylamide, texin or copolymers thereof, nylon, silanized glass, ceramics, metals, in particular titanium, or mixtures thereof.
  • Such materials have proven themselves in the specialist fields, for example in tissue engineering, and are used in various embodiments.
  • the shape of the surface can be selected arbitrarily.
  • the aptamers are attached either directly and / or via a linker molecule to the surface of the device according to the invention.
  • linker molecule or “linker” is here to be understood any substance with which an aptamer can be attached to the surface.
  • aptamers like all nucleotides (for example after coupling with amino or biotin groups), can be attached to the surface of the device via suitable linker molecules or spacers.
  • linker molecules or spacers suitable linker molecules or spacers.
  • a functional anchor SiO 2 TiO 2 , -COOH, HfO 2 , -Au, -Ag, N-hydroxysuccinimide, -NH 2, epoxide, maleimide, acid hydrazide, hydrazide, azide, diazirine, benzophenone, etc.
  • Another suitable substance for attaching an aptamer to the surface of a device according to the invention is a hydrogel which is marketed by Schott, Mainz, Germany under the name Nexterion®, to which then, for example, amino-modified aptamers are covalently bound.
  • a coating of the device according to the invention with a blood-compatible hydrogel for example from the group of PEGs or Star-PEGs which, for example, have a free carboxyl group, to which then, for example, an amino-modified aptamer can be covalently bound.
  • a blood-compatible hydrogel for example from the group of PEGs or Star-PEGs which, for example, have a free carboxyl group, to which then, for example, an amino-modified aptamer can be covalently bound.
  • a further method for the immobilization of oligonucleotides on surfaces is the photolinking dar.
  • the NH 2 -coupled oligonucleotide (aptamer) is first provided with a so-called photolinker molecule (eg anthraquinone), which later with UV activation photochemical reactions can enter a plastic surface and thus the oligonucleotide covalently binds to the surface.
  • Kits and substances for carrying out this method are commercially available, for example, under the name AQ-Link TM and DNA Immobilizer TM from Exiqon (Vedbaek, Denmark).
  • linker molecule is N-succinimidyl-3- (2-pyridyldithio) propionate.
  • N-succinimidyl-3- (2-pyridyldithio) propionate has been shown to be useful in the immobilization of a regulator of the complement system on certain surfaces of biomaterials (see Andersson et al. "Binding of a model regulator of complement activation (RCA) to a biomaterial surface: surface-bound factor H inhibits complement activation ", Biomaterials 22: 2435-2443, 2001). With the use of this linker, the biological activity of the regulator was not impaired.
  • the device according to the invention may additionally be coated with growth factors.
  • This embodiment has the advantage that the bound MSC can be differentiated by means of specific growth factors in the desired direction, thereby further improving the functionality of the device according to the invention.
  • the growth factors are selected from the group consisting of: Platelet derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Colony Stimulating Factor (CSF), Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF), Fibroblast Growth Factor (FGF) and / or growth factor from the Transforming Growth Factor (TGF) superfamily.
  • PDGF Platelet derived Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • CSF Colony Stimulating Factor
  • EGF Epidermal Growth Factor
  • NGF Nerve Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF Transforming Growth Factor
  • This measure has the advantage that already suitable growth factors are provided.
  • BMP differentiation of the bound MSC is effected in osteocytes, which favors the ingrowth of a bone replacement implant according to the invention.
  • the invention further relates to a method for producing a device having at least one biological tissue and / or liquid contacting surface, which is at least partially coated with a substance that mediates the binding of mesenchymal stem cells (MSC), comprising the following steps: (1) providing nucleic acid molecules, and (2) binding the nucleic acid molecules of step (1) to the surface of a device, said nucleic acid molecules having aptamers described above.
  • MSC mesenchymal stem cells
  • Figure 1 shows part of Figure (A) the characterization and identification of adult MSCs (aMSC); 'AB' shows an osteogenic staining of aMSC according to Von Kossa at 100X, 'A' shows the control; 'CD' shows a staining for osteogenic alkaline phosphatase and hematoxylin of aMSC at 200x magnification, 'C represents the control; 'EF' is an adipogenetic staining of aMSC with Red OiI and hematoxylin at 400x magnification, E represents the control.
  • Panel (B) shows the epitope identification of aMSC.
  • the adult porcine aMSC are CD29 ⁇ CD44 + , CD90 + , SL-class I + , SLA-class II DQ ", SLA-class II DR- (curve 1 represents the isotype control).
  • Fig. 2 shows the binding of a selected aptamer (G-8) to aMSC by means of
  • Panel 1 shows binding of aptamer G-8 to bone marrow
  • panel 2 shows the binding of aptamer G-8 to peripheral blood (curve 1 represents the cell-incubated aptamer G-8, curve 2 represents cell control)
  • FIG. 3 shows in part illustration (A) the aptamer-based cell sorting.
  • the left picture shows the control, single balls incubated with total bone marrow, with very few cells growing on the culture dish.
  • the right image shows total bone marrow incubated with the aptamer (fixed to the magnetic microspheres), where much more cells were accumulated and grew (x100).
  • Fig. 4 shows the phenotype identification of the isolated aMSC. part Figure
  • (A) shows the subpopulation Rl of the isolated aMSC stained with PE-labeled antibodies immediately after isolation.
  • the results shown were CD4 " , CD8 “ , CD29 “ , CD44 + , CD90, - the subpopulation R2 of the isolated aMSC was stained with PE-labeled antibodies immediately after isolation, showing CD4 " , CD8 " , CD29, CD44 + , CD90 + .
  • Curve 1 shows the isotype control
  • FIG. 5 shows the adipogenetic and osteogenic differentiation of
  • Aptamer-isolated porcine aMSC passage 0 (A) adipogenetic differentiation after 14 days of treatment with hydrocortisone, isobutylmethylxanthine and indomethacin. Staining with oil red O, hematoxylin counterstain (x100). (B) Control (normal medium, staining with OiI red O, hematoxylin counterstaining (x100)). (C) Osteogenetic differentiation after 14 days of treatment with dexamethasone, ascorbic acid and ß-glycerol phosphate. Staining for alkaline phosphatase, hematoxylin counterstain (x100). (D) Control (normal medium, staining for alkaline phosphatase, hematoxylin counterstain (x100)).
  • Fig. 6 shows the plasma stability. Analysis of the stability of aptamer G-8 in human blood plasma by agarose gel electrophoresis. Samples were taken at different times between 0 hours to 6 hours. The result shows that the aptamer can withstand at least 6 hours of degradation.
  • Figure 7 shows the adipogenetic (A) and osteogenic (B) differentiation of the aptamer-isolated porcine aMSC (passage 0) versus the plastic adherence method for the isolation of aMSC (passage 0).
  • Mononuclear cells were isolated from fresh whole bone marrow by density gradient centrifugation and plated at a density of 500 cells / well (a + c). After 24 hours, the medium was changed to remove non-adherent cells. Then an a-dipogenetic or osteogenic or normal medium was added. Aptamer-isolated aMSC were plated at a density of 500 cells per well (b + d). After 24 hours, the medium was changed and adipogenetic or osteogenic or normal medium was added.
  • (B) osteoogenic differentiation: a: total bone marrow - adherence 24 hours, osteogenetic medium; b: aptamer-isolated aMSC - 24 hour adherence, osteogenetic medium; c: total bone marrow - 24 hours adherence, control (normal medium); d: Aptamer-isolated aMSC - 24 hours adherence, control (normal medium). Staining for alkaline phosphatase, hematoxylin counterstaining.
  • the animals (pigs, German Landrace, 50 kg, male) were kept and treated according to the animal welfare requirements of the University of Tübingen.
  • Porcine AMSC were isolated according to known modification procedures; see. Ponomarev et al. (2003), Preliminary Results of Enhanced Osteogenesis by Fibrogammin and Mesenchymal Stem Cells on ChronOS Cylinders, European Cells and Materials 5, page 80. Briefly, mononuclear cells (MNCs) were derived from bone marrow aspirate Centrifugation isolated over a Ficoll-Histopaque layer (30 min, 300g, density 1.077).
  • the cells were cultured under standard culturing conditions with low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibcol) supplemented with 10% fetal calf serum, penicillin (50 U / ml) and streptomycin (50 ⁇ g / ml) , The medium was changed after 24 hours and then twice a week. When the cells reached 80% confluency, they were detached by 0.25% trypsin-EDT A solution and replated for SELEX preparation and differentiation potential scores.
  • DMEM low-glucose Dulbecco's modified Eagle's medium
  • rat and human aMSC were isolated and characterized for specificity tests (FACS with aptamer).
  • the animals (Sprague Dawley rats) were kept and treated according to the animal welfare regulations of the University of Tübingen.
  • the human bone marrow was removed during orthopedic surgery and approved by the local ethics committee of the University of Tübingen according to the Helsinki Declaration.
  • Mouse P19 cells were purchased from ATCC (Manasas, VA, United States of America).
  • aMSC The potential of aMSC to differentiate into adipogenetic and osteogenic lines was tested as follows.
  • the aMSC were cultured in an osteogenic culture medium containing 0.2 mM L-ascorbic acid, 2-phosphate magnesium salt n-hydrate and 0.01 ⁇ M dexamethasone (Dex) (Sigma-Aldrich Co.), 10 mM ⁇ - Glycerophosphate contained.
  • the subcultured cell layers were washed with phosphate-buffered saline PBS and fixed with 4% paraformaldehyde and stained according to the alkaline phosphatase staining kit (Sigma Kit # 85).
  • the cells were washed with PBS, fixed with 10% formalin for 10 minutes, washed with distilled water, rinsed with 60% isopropanol and covered with a 3% oil red O solution (Sigma-Aldrich) in 60% isopropanol. After 10 minutes, the cultures were rinsed briefly in 60% isopropanol and washed thoroughly with distilled water and allowed to dry at room temperature.
  • Surface marker identification with the cultured MSC was by FITC-labeled monoclonal antibodies to CD29, CD44, CD45, CD90, SLA class I, SLA class DQ and SLA DR (Becton Dickinson, Germany, Heidelberg). To control the isotypes, non-specific mouse IgG were used in place of the primary antibody.
  • the DNA oligonucleotide library contains a 40 nucleotide central randomized sequence flanked by primer sites on each side (for the porcine MSC aptamers: 5'-GAATTCAGTCGGACAGCG-N40 -GATGGACGAATATCGTCTCCC- 3 '; for the human MSC -Aptamers: S '-GGGAGCTCAGAATAAACGCTCAA-NSO- TTCGACATGAGGCCCGAAAC- 3 • ).
  • the size of the library is about 10 15 .
  • the FITC-labeled forward primer (5 '-C 12 -FITC-GAATTCAGTCGGACAGCG-S' and the biotin-labeled reverse primer (5 '-Bio-GGGAGACGATATTCGTCCATC-3') were used in the PCR to obtain double-stranded DNA and the single-stranded DNA were separated by streptavidin-coated magnetic beads (M-280 Dynabeads, Dynal, Hamburg, Germany)
  • the library and all primers were synthesized by Operon Technologies (Cologne, Germany).
  • the selection of DNA aptamers against porcine aMSC was performed as follows. 4 nmol ssDNA pools were denatured in selection buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM KCl, 100 mM NaCl, 1 mM MgCl 2 and 0.1% NaN3 for 10 minutes at 80 0 C and then heat renatured at 0 ° C. for 10 minutes. To reduce background binding, a five-fold molar excess of yeast tRNA (Invitrogen, Düsseldorf, Germany) and bovine serum albumin (BSA, Sigma-Aldrich, Kunststoff, Germany) was added.
  • yeast tRNA Invitrogen, Düsseldorf, Germany
  • BSA bovine serum albumin
  • the mesenchymal stem cells (passage 2, 10 6 cells for the first round and 10 s cells for the further rounds) were incubated with the ssDNA at 37 ° C for 30 minutes in the selection buffer. Partitioning of bound and unbound ssDNA sequences was by centrifugation. After centrifugation and washing three times with 1 ml of selection buffer (with 0.2% BSA), cell-bound ssDNA was amplified by PCR (Master Mix from Promega, Mannheim, Germany). FITC- and biotin-labeled primers were used for PCR amplification (25 cycles of 1 min at 94 ° C, 1 min at 48 ° C and 1 min at 72 ° C, followed by 10 min at 72 ° C).
  • FITC-labeled ssDNA was prepared as described above. Using unmodified primers, aptamers obtained from the tenth round of selection were PCR amplified and cloned into Escherichia coli using the TA cloning kit (Invitrogen). Plasmids from individual clones were isolated by the plasmid extraction kit (Qiagen, Dusseldorf, Germany) and the inserts were PCR amplified and sequenced with ABI PRISM® 377 DNA sequencer (Applied Biosystems, Darmstadt, Germany). Individual FITC aptamers were prepared to perform the affinity binding assays.
  • FITC-labeled aptamer 200 pmol was incubated with 10 5 aMSC at 37 ° C for 30 min, washed 3 times and analyzed by FACS (BD, Heidelberg, Germany), the same amount of mouse P19 cells, rat MSC, the was incubated with the aptamer was used in the control.
  • the secondary structure of the aptamer was analyzed by DNASYS software (version 2.5, Hitachi Software Engineering Co).
  • Biotinylated aptamers were synthesized by OPERON and incubated with 10 5 aMSC for 30 min at 37 ° C, washed three times and incubated with anti-biotin microspheres (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 0 ° C. The same number of aMSC without aptamer was incubated with anti-biotin microspheres and served as a negative control. The mixture was washed three times and filtered through a magnetic column. Subsequently, the column was removed from the magnet holder and the beads were placed in cell culture medium.
  • FACS 10 ml of fresh bone marrow blood was lysed with ammonium chloride and incubated with FITC-labeled aptamer (200 pmol) for 30 min at 37 ° C. After washing three times, the cells were analyzed by FACS. The same amount of peripheral blood was treated identically to serve as a control.
  • Capture experiment 20 ml of fresh bone marrow was lysed with ammonium chloride and resuspended with PBS (2% FBS, 1 mM EDTA). FcR-blocking antibody and 1 nmol aptamer were added to the bone marrow solution at room temperature for 30 min. added to tur. EasySep biotin selection cocktail (cellsystems, St. Katharinen, Germany) was added and incubated for 15 min. Subsequently, EasySep magnetic nanoparticles were added and incubated for 10 min. The mixture was placed in the magnet and set aside for 5 min. The supernatant was withdrawn and the magnetically labeled cells were washed twice with buffer and cultured further.
  • EasySep biotin selection cocktail cellsystems, St. Katharinen, Germany
  • a 12-well cell culture plate (Greiner, Nürtingen, Germany) was coated with streptavidin overnight at 4 ° C, then washed several times with PBS-T (0.05% Tween-20).
  • the plate was washed with PBS-T and incubated with aMSC at 37 ° C for 30 min with slow shaking. Subsequently, the medium was removed from the plate and the non-adherent cells were discarded. Cell adhesion was observed under an inverse microscope (Zeiss Axiovert 135, Zeiss, Oberkochen, Germany).
  • the isolated aMSC were cultured in osteogenic culture medium and adipogenic culture medium. Alkaline phosphatase staining and OiI red staining were performed as described.
  • adipogenetic and osteogenic differentiation potential were compared.
  • Mononuclear cells were isolated from fresh whole bone marrow of the pig by density gradient centrifugation and plated at a density of 500 cells / well. After 24 hours, the medium was changed to remove non-adherent cells. Subsequently, adipogeneti- beautiful or osteogenic or normal medium added. Aptamer-isolated aMSC were plated at the same density (500 cells / well). After 24 hours, the medium was changed and adipogenetic or osteogenic or normal medium was added. After 5 weeks, after the aptamer-isolated cells reached confluence, the adipogenetic and osteogenetic staining procedure was started.
  • Fresh human plasma was prepared by centrifuging (3000 g) whole blood for 20 minutes. 8 nmol of the aptamer were incubated at 37 0 C in a final volume of 0.5 ml of freshly prepared heparinized human plasma. Samples of 50 ⁇ l were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3 and 6 hours. The reactions were terminated by adding 10 ⁇ l of loading buffer and subsequent storage on ice. Full-length and digested oligonucleotides were separated on a 2% agarose gel and photodocumented.
  • Porcine and human aMSC were successfully isolated from bone marrow by gradient centrifugation, expanded in a monolayer culture, and evaluated for osteogenic differentiation potential.
  • Bipolar to polygonal spindle fibroblast cells were observed 4 days after the first plating. The cells reached confluence after 12 days of culture. After the first pass, the cells showed a uniform monolayer.
  • the aMSC cultured in osteogenic medium showed ALP positive and von Kossa positive (calcium mineral precipitation) after 8 days and 28 days, respectively.
  • the aMSCs cultured in adipogenetic differentiation medium were positive for red oil staining, whereas all controls were negative ( Figure 1 (A)).
  • the surface chenmarkerfärbung demonstrated that the adherent cells were CD29 +, CD44 +, CD45 "+ CD9O, SLA Class I +, SLA-DQ" and SLA-DR "(Fig. 1 (B)).
  • Porcine and human bone marrow-derived aMSC were used as targets for the in vitro selection of aptamers from a randomized pool of DNA molecules.
  • the starting library consisted of 79-mer single-stranded DNA molecules containing randomized 40-nucleotide inserts. This library was applied to a number of cultured cells in the same passage, minimizing non-specific interaction.
  • SELEX pools of the second and subsequent rounds were analyzed by FACS after incubation with aMSC. In each round of selection, the concentration of competitor DNA was increased to select for a higher affinity and higher specificity aptamer pool.
  • FACS Assays The fluorescence of a binding of an exemplary aptamer comprising the nucleotide sequence SEQ ID NO. 6 (G-8) to aMSC is shown in Figures 2 (A) to 2 (C), which show the specific binding of aptamers to aMSC.
  • aMSC bound to the biotinylated aptamer can be isolated and enriched using anti-biotin microspheres.
  • aMSCs Via the biotinylated aptamer, aMSCs can be fixed when they are filtered through a magnetic column.
  • the anti-biotin microspheres alone can not isolate aMSC, so that there is no in vivo the culture dish of growing cells are observed (left picture, negative control).
  • the anti-biotin microspheres with the biotinylated aptamer fixed on the surface can bind to aMSC so that growing cells can be detected (right picture). This result shows that the aptamer is able to isolate MSC from the cell solution.
  • FACS Assa ⁇ The aptamer G-8 shows almost no binding to peripheral blood cells as compared to whole bone marrow (Fig. 1 (B)).
  • Bone marrow mononuclear cells were harvested with FITC-labeled aptamer G-8 by high-speed FACS and analyzed with PE-labeled antibodies. The result shows two subpopulations of isolated cells.
  • the first subpopulation (RI) containing small granular cells was CD4 "(82.2%), CD8 '(80.5%), CD29" (70.7%), CD44 + (90.9%), CD45 + ( 86.4%) and CD90 '(77.6%).
  • the second subpopulation (R2) containing small and non-granular cells was CD4 ⁇ (98.9), CD8 "(98.9%), CD29” (83.7%), CD44 + (87.7%), CD45 + (99.2%) and CD90 + (91.8%).
  • the isolated cells were cultured for 14 days (passage 0) and also stained with PE-labeled antibodies. The results showed that these were CD29 + (98.0), CD44 + (99.6%), CD90 + (99.5) and CD45 '(87.6%), consistent with previously described markers of aMSC in Culture ( Figure 4). In contrast to the freshly isolated cells, no defined subpopulation could be detected and the cultured cells upregulated CD29 and lost the CD45 antigen.
  • the adipogenetic and osteogenic differentiation of the aptamer-isolated porcine aMSC in passage 0 showed that the isolated aMSCs have a high potential for differentiation in adipocytes and osteoblasts (FIG. 5).
  • the aptamers should be resistant to rapid degradation by exo- and endonucleases.
  • Human plasma mainly contains high 3 'exonuclease activity.
  • the G-8 aptamer was able to withstand degradation by nucleases for 6 hours, as detected by agarose gel analysis ( Figure 6), so no additional modification was required to increase stability again.

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Abstract

L'invention concerne un dispositif comprenant une surface qui vient en contact avec un tissu biologique et/ou avec un liquide, ladite surface étant recouverte partiellement d'au moins une substance qui permet la médiation de la fixation des cellules souches mésenchymateuses (MSC). L'invention concerne également un procédé de liaison et/ou d'isolement des MSC à partir de tissus biologiques et/ou de liquides, ainsi qu'une molécule d'acide nucléique qui se fixe à la MSC de manière sélective et hautement spécifique. L'invention concerne, en outre, l'utilisation de ladite molécule d'acide nucléique dans la fixation et/ou l'isolement de la MSC à partir de tissus biologiques et/ou de liquides, ainsi qu'un procédé de production d'un dispositif de ce type.
EP07724981A 2006-05-26 2007-05-08 Dispositif et substance destinés à isoler des cellules souches mésenchymateuses Withdrawn EP2027255A2 (fr)

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WO2009135198A1 (fr) * 2008-05-02 2009-11-05 Biotex, Inc. Acides nucléiques biomimétiques
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WO2014018230A2 (fr) * 2012-07-25 2014-01-30 Albert Einstein College Of Medicine Of Yeshiva University Procédés pour isoler des cellules souches mésenchymateuses humaines
BR102013021701A2 (pt) * 2013-08-26 2015-07-28 Univ São Paulo Usp Polinucleotídeos quimicamente modificados e processo de produção de polinucleotídeos quimicamente modificados
CN106483289A (zh) * 2016-12-30 2017-03-08 魏乃东 一种用于人成熟骨髓间充质干细胞检测的试剂盒
CN106771191A (zh) * 2016-12-30 2017-05-31 魏乃东 一种用于人脐血间充质干细胞检测的试剂盒
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US5567588A (en) * 1990-06-11 1996-10-22 University Research Corporation Systematic evolution of ligands by exponential enrichment: Solution SELEX
US5763566A (en) * 1990-06-11 1998-06-09 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: tissue SELEX
US5837539A (en) * 1990-11-16 1998-11-17 Osiris Therapeutics, Inc. Monoclonal antibodies for human mesenchymal stem cells
US6287765B1 (en) * 1998-05-20 2001-09-11 Molecular Machines, Inc. Methods for detecting and identifying single molecules
ES2306876T3 (es) * 2002-06-14 2008-11-16 Cartela Ab Marcador para celulas madre y su uso.
DE10242338A1 (de) * 2002-09-09 2004-03-18 Eberhard-Karls-Universität Tübingen Universitätsklinikum Antikörper zur Isolierung und/oder Identifizierung mesenchymaler Stammzellen und Verfahren zur Isolierung und/oder Identifizierung mesenchymaler Stammzellen
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DE10358407A1 (de) * 2003-12-11 2005-07-14 Cytotools Gmbh Antiapoptorisch wirksame Aptamere (zur Therapie von Herz-Kreislauf Erkrankungen)

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