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WO2015025059A1 - Cellules stromales mésenchymateuses (csm) multipotentes négatives à cd105 - Google Patents

Cellules stromales mésenchymateuses (csm) multipotentes négatives à cd105 Download PDF

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WO2015025059A1
WO2015025059A1 PCT/EP2014/067996 EP2014067996W WO2015025059A1 WO 2015025059 A1 WO2015025059 A1 WO 2015025059A1 EP 2014067996 W EP2014067996 W EP 2014067996W WO 2015025059 A1 WO2015025059 A1 WO 2015025059A1
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mscs
composition
expression
cells
disease
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Per Anderson
Francisco Martin Molina
Ana CARRILLO GALVEZ
Marién COBO PULIDO
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Fundacion Publica Andaluza Progreso y Salud
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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
    • 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
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells

Definitions

  • This invention relates to multipotent mesenchymal stromal cells. More particularly, this invention relates to multipotent mesenchymal stromal cells which lack the expression of the phenotypic marker CD105. Such multipotent mesenchymal stromal cells have greater capacity to differentiate into adipocytes and osteocytes compared to CD105+ multipotent mesenchymal stromal cells and are more efficient at inhibiting T cell proliferation in vitro.
  • MSCs Mesenchymal stromal cells
  • MSCs Mesenchymal stromal cells
  • MSCs murine MSCs
  • hMSCs hMSCs
  • immunomodulatory mechanisms could play part.
  • ex vivo cultures of MSCs contain biochemically and phenotypically distinct cell types representing mesodermal progenitors at different stages of commitment as well as cells with no differentiation capacity.
  • the analysis of the transcriptome of bone-marrow derived MSC revealed several subpopulations expressing various proteins involved in neural activity, angiogenesis and immunity suggesting that MSCs are not a homogenous population.
  • MSCs for human disease relies on the injection of a large number of cells necessitating the expansion of MSCs in vitro for several weeks before yielding enough cells (>l-2 x 106 cells/kg).
  • the culturing of MSCs is associated with several problems including (i) loss of homing capacity due to down-modulation of chemokine receptor expression and increased cell size (is) increased susceptibility to cell death and ⁇ iii) loss of differentiation potential.
  • CD105- MSCs represent a distinct multipotent MSC population. They have similar growth potential and maintain identical MSCs markers than CD105+ MSCs.
  • CD 105- mMSCs are better at T cell suppression and more prone to differentiate into adipocytes and osteocytes than CD105+ MSCs.
  • a first aspect of the present invention refers to a composition comprising MSCs, preferably a homogenous population of MSCs, wherein said MSCs lack the expression of the phenotypic marker CD 105 (endoglin).
  • the phenotypic, proliferative and clonogeneic properties of the MSCs are as follows; ⁇ i) adherence to plastic fii) capacity to differentiate into adipocytes, chondrocytes and osteocytes and (iii) optionally expression of CD73, CD90 and lack of CD31, CD34 and CD45 upon culture.
  • the MSCs are human MSCs.
  • said composition is a pharmaceutical composition which optionally comprises an acceptable pharmaceutical carrier and/or additional active ingredients,
  • a second aspect of the invention refers to a method of obtaining MSCs lacking the expression of the phenotypic marker CD105 ⁇ endoglin), comprising: obtaining at least one cell population including MSCs from at least one donor; determining the amount of the phenotypic marker CD105 (endoglin) expressed by the MSCs in each of said at least one cell population(s); and selecting MSCs which lack the expression of the phenotypic marker CD105 (endoglin).
  • the phenotypic, proliferative and clonogeneic properties of the MSCs are as follows: (i) adherence to plastic (ii) capacity to differentiate into adipocytes, chondrocytes and osteocytes and (iii) optionally expression of CD73, CD90 and lack of CD31, CD34 and CD45 upon culture.
  • the MSCs are human MSCs.
  • a third aspect of the inventions refers to a composition comprising the MSCs lacking the expression of the phenotypic marker CD105 (endoglin) cells obtained or obtainable by the method of the second aspect of the invention.
  • composition is a pharmaceutical composition which optionally comprises an acceptable pharmaceutical carrier and/or additional active ingredients.
  • a fourth aspect of the invention refers to the composition of the first or third aspect of the invention, for its use in therapy.
  • a fifth aspect of the invention refers to the composition of the first or third aspect of the invention, for its use in the treatment of autoimmune diseases, immune diseases or bone-related disorders.
  • the immune disease is selected from the list consisting of graft-versus-host disease; solid organ transplant rejection such as, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, intestine transplant rejection, and kidney transplant rejection.
  • the autoimmune disease is selected from the list consisting of rheumatoid arthritis, multiple sclerosis, Type I diabetes, Crohn's disease, Guillain-Barre syndrome, lupus erythematosus, myasthenia gravis, optic neuritis, psoriasis, Graves' disease, Hashimoto's disease, Ord's thyroiditis, aplastic anemia, Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome, opsoclonus myoclonus syndrome, temporal arteritis, acute disseminated encephalomyelitis . , Goodpasture's syndrome, Wegener's granulomatosis, coeliac disease, pemphigus, polyarthritis, warm autoimmune hemolytic anemia, and scleroderma.
  • FIG. 1 This figure shows that murine mesenchymal stem cells (MSCs) are heterogenous for CD105 (endoglin) expression.
  • MSCs murine mesenchymal stem cells
  • ASC Adipose Stem Cell cultures were harvested with 2 mM EDTA, TrypLE or Trypie followed by a 30 minute collagenase type 1 treatment and analyzed by flow cytometry. The expression level (MFI) of each marker was normalized to the corresponding staining on EDTA- harvested cells.
  • (G) ASCs were starved for 24 hours and then stimulated with 10 ng/ml recombinant human TGF-betal for 0, 2, 6 and 24 hours. At each time point total RNA was purified and reverse transcribed. The expression of CD105L was measured using qPCR.
  • (H) Total ASCs were cultured with or without TNF-a!pha ⁇ 10 ng/ml) and IFN-gamma (10 ng/ml) for 24 hours after which the cells were harvested and analyzed by flow cytometry for a panel of SC markers. Results are shown as mean (SE ) of 2-3 independent experiments.
  • CD105 defines two distinct MSC subpopulatioms in mouse.
  • Balb/c ASCs were double-stained for CD105 in combination with CD29, CD44, CD45, CD49f, sca-1 and MHC class I and analyzed by flow cytometry. One representative experiment out of 3 independent experiments is shown.
  • B MACS- purified CD105- and CD105+ mASCs were cultured separately for 9 passages and CD105 expression was analyzed at each passage by flow cytometry.
  • CD105- and CD 105+ mASCs were starved for 24 hours and then stimulated with 10 ng/ml recombinant human TGF- ⁇ for 0, 2, 6 and 24 hours.
  • D ASCtot, CD105- and CD105+ cells were plated at 2500 cells/cm in T25 cell culture flasks and cultured at 5% 02 until reaching 70-80% confluency. Cells were harvested, counted and reseeded at the same density for 10 passages.
  • E ASCtot, CD105- and CD105+ cells were cultured in 10 cm petri dishes ⁇ 2000 cells/plate ⁇ for 14 days for CFU-F assay as described in materials and methods. Cells were subsequently fixed with PFA and stained with cresyt violet to visualize CFU-F.
  • CD105- ASCs are more prone to differentiate into adipocytes and osteocytes.
  • Adipogenesis (A) and osteogenesis (B) were induced in ASCtot, CD105- and CD105+ mASC populations.
  • Differentiation data is shown as one representative experiment out of 3-4 independent experiments.
  • the extent of adipocyte differentiation was visualized using OilRED O and the amounts of OilRED O extracted from the different ASC populations were measured using a spectrophotometer (450 nm). Bars are shown as mean (SD) from experimental triplicates.
  • * * * p ⁇ 0.001 vs. CD105- mASCs.
  • the deposition of calcium by differentiating osteocytes was visualized using alizarin red and quantified using the ImageJ software.
  • CD105- and CD105+ mASC were stained for TGF- ⁇ receptor li (TGF-pRll) and analyzed by flow cytometry.
  • TGF-pRll TGF- ⁇ receptor li
  • D Total RNA was purified from ASCtot, CD105- and CD105+ mASCs, reverse transcribed and the expression levels of ⁇ - glycan, ALK1, AL 2 and ALK5 assayed using qPCR.
  • FIG. 4 Comparison of the immunomodulatory capacity of CD105- and CD105+ mASCs.
  • A The levels of PGE2 and TGF- ⁇ were measured in supernatants from ASCtot, CD105- and CD105+ mASCs using specific ELISAs ⁇ see materials and methods).
  • B ASCtot, CD105- and CD105+ mASCs were stimulated with TWF-ct (10 ng/ml) and IFN-y (10 ng/ml) for 12 and 24 hours. Total RNA was purified for each time point and the expression of iNOS and IL-6 was analyzed using qPCR.
  • CD105- and CD105+ mASCs were cultured together with CFSE-labeled splenocytes (200,000 cells/well) and stimulated with anti-CD3 (1 ⁇ g/ml) for 3 days. The cells were harvested and acquired on a FACS Canto li flow cytometer and the proliferation of CD4+ splenocytes was quantified using the FlowJo software.
  • D BM-MOs were cultured with or without mASC for 48 hours and then restimuiated with LPS ⁇ 1 g/mi) for 24 hours.
  • MSCs multipotent mesenchymal stromal cells, which in vitro adhere to plastic and express a defined set of surface molecules, wherein human MSCs (h SCs) should express CD73, CD90 and lack expression of CD14, CD19, CD34, CD45 and HLA-D , and possess tri-lineage differentiation capacity being able to differentiate into adipocytes, osteocytes and chondrocytes in vitro.
  • ASCs is understood as MSCs derived from adipose tissue.
  • phenotypic marker CD105 are understood as the general usage of CD105 (endoglin) as one out of several MSC-defining surface markers.
  • the term "lack the expression of the phenotypic marker CD105" is understood as the complete absence of the CD105 molecule on the cell surface or expression levels corresponding to 25% or less to those found in CD105+ bone marrow-derived MSCs.
  • the term “expressed” is used to describe the presence of a marker within a cell.
  • a marker In order to be considered as being expressed, a marker must be present at a detectable level.
  • detectable level is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting or FACS analysis.
  • a gene is considered to be expressed by a cell of the population of the invention if expression can be reasonably detected after 30 PCR cycles, which corresponds to an expression level in the cell of at least about 100 copies per cell.
  • the terms “express” and “expression” have corresponding meanings. At an expression level below this threshold, a marker is considered not to be expressed.
  • the comparison between the expression level of a marker in an adult stem cell of the invention, and the expression level of the same marker in another cell, such as for example an embryonic stem cell, may preferably be conducted by comparing the two cell types that have been isolated from the same species.
  • this species is a mammal, and more preferably this species is human.
  • Such comparison may conveniently be conducted using a reverse transcriptase polymerase chain reaction (RT-PCR) experiment, "Fluorescence activated cell sorting (FACS)" is a method of cell purification based on the use of fluorescent labeled antibodies.
  • the antibodies are directed to a marker on the cell surface, and therefore bind to the cells of interest.
  • the cells are then separated based upon the fluorescent emission peak of the cells.
  • autoimmune diseases is understood as diseases resulting from an immune response to self-components
  • adipogenesis is understood as differentiation of preadipocytes into adipocytes resulting in the formation of fat or fatty tissue.
  • osteogenesis is understood as the differentiation of osteoprogenitors into osteocytes resulting in formation of bone
  • bone-related disorders is understood as medical conditions related to the bone.
  • pharmaceutically acceptable carrier is intended to include formulation used to stabilize, solubilize and otherwise be mixed with active ingredients to be administered to living animals, including humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.
  • the present invention confronts the problem of providing a further characterization of MSCs that could help developing improved therapeutic strategies using more standardized MSCs.
  • the authors of the present invention have identified a new CD105- MSG subpopulation with improved immunomodulatory and differentiation properties compared to total MSCs but sharing all others phenotypic, proliferative and clonogeneic properties.
  • CD105 expression on mMSCs and hMSCs is heterogeneous.
  • it was unclear at that time whether this heterogeneity in expression of CD105 was due to the presence of differentiated cells (and therefore with a limited expansion capability) or if they represent a distinct multipotent MSC subpopulation.
  • CD105- adipose tissue-derived MSCs represent a distinct MSC subpopulation.
  • Both CD105- and CD105+ ASCs had similar proliferation capacity, colony-forming unit- fibroblast (CFU-F) potential, and expression of differentiation-related genes and shared all other MSC markers analyzed, in addition the authors of the present invention surprisingly found that CD105- ASCs had greater capacity to differentiate into adipocytes and osteocytes compared to CD 105+ ASCs and that interestingly, CD105- ASCs were more efficient at inhibiting T cell proliferation in vitro.
  • CD105 (endoglin) is a high affinity co-receptor for transforming growth factor (TGF)- betal and TGF-beta3.
  • CD105 exists in two isoforms, CD105L and CD105S, which play opposite roles in modulating TGF-betal/3-induced signaling.
  • the authors of the present invention have found that mASCs only expressed the CD105L isoform which in turn inhibits ALK5/smad3 activation while it promotes ALKl/smadl and ALK5/smad2 activation.
  • TGF-beta-mediated activation of smad2 inhibits both adipogenesis and osteogenesis.
  • CD105- ASCs have enhanced adipo- and osteogenic potential.
  • the authors of the present invention have surprisingly found that CD105- mASCs are more effective at inhibiting CD4 T cell proliferation than CD105+ cells.
  • CD105- ASCs produced slightly more iiMOS and IL-6 mRNA upon stimulation compared to CD105+ mASCs thus increasing their immunosuppressive activity on CD4 T cells.
  • CD105-MSCs subpopulation that has similar phenotype, proliferation capacity and CFU potential than CD105+ MSCs.
  • CD105- ASCs have greater capacity to differentiate into adipocytes and osteocytes compared to CD105+ ASCs and are better at inhibiting T cell proliferation.
  • a composition comprising MSCs, preferably a homogenous population of MSCs, wherein said MSCs lack the expression of the phenotypic marker CD105.
  • the phenotypic, proliferative and clonogeneic properties of the MSCs are as follows: (i) adherence to plastic (ii) capacity to differentiate into adipocytes, chondrocytes and osteocytes and (iii) optionally expression of CD73, CD90 and lack of CD31, CD34 and CD45 upon culture,
  • the MSCs are obtained by a method comprising: obtaining at least one cell population including MSCs from at least one donor; determining the amount of the phenotypic marker CD105 (endoglin) expressed by the MSCs in each of said at least one cell population(s); and selecting MSCs which lack the expression of the phenotypic marker CD105 (endog!in).
  • the phenotypic, proliferative and clonogeneic properties of the MSCs are as follows: (i) adherence to plastic (ii) capacity to differentiate into adipocytes, chondrocytes and osteocytes and (iii) optionally expression of CD73, CD90 and lack of CD31, CD34 and CD45 upon culture.
  • a third aspect of the inventions refers to a composition comprising the MSCs lacking the expression of the phenotypic marker CD105 (endoglin) cells obtainable by the method of the second aspect of the invention.
  • the MSCs of the first or third aspect of the invention are obtained from a mammal.
  • the mammal may be a primate, including human and non-human primates.
  • said composition is a pharmaceutical composition which optionally comprises an acceptable pharmaceutical carrier and/or additional active ingredients.
  • MSCs which lack the expression of the phenotypic marker CD105 inhibit the proliferation of T cell proliferation and have greater capacity to differentiate into adipocytes and osteocytes.
  • Such MSCs are particularly useful in inhibiting immune responses, and more particularly such MSCs are useful in the treatment of graft-versus-host disease; solid organ transplant rejection such as, for example, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, intestine transplant rejection, and kidney transplant rejection; and autoimmune diseases such as, for example, rheumatoid arthritis, multiple sclerosis, Type I diabetes, Crohn's disease, Guillain-Barre syndrome, lupus erythematosus, myasthenia gravis, optic neuritis, psoriasis, Graves' disease, Hashimoto's disease, Ord's thyroiditis, aplastic anemia, Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome, opsoclonus myoclonus syndrome, temporal arteritis, acute disseminated encephalomyelitis, Goodpasture's syndrome, Wegener's gran
  • a fourth aspect of the invention refers to the composition of the first or third aspect of the invention, for its use in therapy.
  • a fifth aspect of the invention refers to the composition of the first or third aspect of the invention, for its use in the treatment of autoimmune diseases, immune diseases or bone-related disorders.
  • the immune disease is selected from the list consisting of graft-versus-host disease; solid organ transplant rejection such as, heart transplant rejection, fiver transplant rejection, pancreas transplant rejection, intestine transplant rejection, and kidney transplant rejection.
  • the autoimmune disease is selected from the list consisting of rheumatoid arthritis, multiple sclerosis, Type I diabetes, Crohn's disease, Guillain-Barre syndrome, lupus erythematosus, myasthenia gravis, optic neuritis, psoriasis, Graves' disease, Hashimoto's disease, Ord's thyroiditis, aplastic anemia, Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome, opsoclonus myoclonus syndrome, temporal arteritis, acute disseminated encephalomyelitis, Goodpasture's syndrome, Wegener's granulomatosis, coeliac disease, pemphigus, polyarthritis, warm autoimmune hemolytic anemia, and scleroderma.
  • the MSCs of the invention form a homogeneous composition.
  • Homogeneous MSC compositions may be obtained by culturing adherent marrow, adipose or periosteal cells, and the MSCs may be identified by specific cell surface markers which are identified with unique monoclonal antibodies.
  • Alternative sources for MSCs include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium.
  • the amount of cellular CD105, that is expressed in a culture of MSCs may be determined by methods known to those skilled in the art. Such methods include, but are not limited to, quantitative assays such as quantitative ELISA assays, for example Flow cytometry, immunofluoresence, immunohistochemistry, Western blot and quantitative-PCR. It is to be understood, however, that the scope of the present invention is not to be limited to any particular method for determining the amount of CD105.
  • the amount of CD 105 expressed by a culture of MSCs is determined by an ELISA assay.
  • a cell lysate from a culture of MSCs is added to a well of an ELISA plate.
  • the well may be coated with an antibody, either a monoclonal or a polyclonal antibody(ies), against the CD105 phenotypic marker, The well then is washed, and then contacted with an antibody, either a monoclonal or a polyclonal antibody(ies), against the CDiOS phenotypic marker.
  • the antibody is conjugated to an appropriate enzyme, such as horseradish peroxidase, for example.
  • the well then may be incubated, and then is washed after the incubation period.
  • the wells then are contacted with an appropriate substrate, such as one or more chromogens.
  • Chromogens which may be employed include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the substrate(s) is (are) added, the well is incubated for an appropriate period of time.
  • CD105- MSCs of the also encompasses CDlOSlow mesenchymal stem cells with expression levels being inferior to 25% of the CD105 expression levels found on CD105+ MSCs derived from bone marrow.
  • the authors of the present invention have found a similar CD105 expression pattern in MSCs derived from either adipose tissue (AT) or bone marrow (BM) independently of the strain used ( Figure ID).
  • the present invention provides for the selection of a population of MSCs which lack the expression of the phenotypic marker CD105.
  • Such selected MSCs then may be admixed with an appropriate pharmaceutical carrier for treatment of the diseases and disorders mentioned hereinabove.
  • the MSCs may be administered as a cell suspension including a pharmaceutically acceptable liquid medium for injection.
  • the MSCs of the present invention are administered to an animal in an amount effective to treat one or more of the above-mentioned diseases or disorders in the animal.
  • the animal may be a mammal, and the mammal may be a primate, including human and non-human primates.
  • the MSCs may be administered systemically, such as, for example, by intravenous, intraarterial, or intraperitoneal administration.
  • the exact dosage of mesenchymal stem cells to be administered is dependent upon a variety of factors, including, but not limited to, the age, weight, and sex of the patient, the disease(s) or disorder(s) being treated, and the extent and severity thereof.
  • mASC mesenchymal stem cells
  • HBSS Hank ' s balanced salt solution
  • the fat tissue was weighed, cut into small pieces (2 mm 3 ) and re-suspended in 2.5 ml of HBSS containing 2 mg/ml col!agenase type I (Sigma Aldrtch, St. Louis, MO) per gram fat tissue and incubated for 30 minutes at 37°C, swirling the tube every 5 minutes.
  • the digest was washed twice with 10 ml HBSS and filtered each time through a 100- ⁇ nylon mesh. Finally, cells were re-suspended in complete MesenCult (Stem Cell, Grenoble, France) medium containing 100 units/ml penicillin/streptomycin (Gibco, Invitrogen) and 20% mouse mesenchymal supplements (Stem Cell), seeded at a density of 15,000- 30,000 cells/cm 2 and cultured at a 5% 0 2 /5% C0 2 atmosphere. Non-adherent cells were removed after 24 hours in culture. Subsequent passages were plated at 10,000 cells/cm 2 in complete MesenCult medium.
  • Bone marrow-derived MSC were derived by flushing femurs and tibiae of BALB/c mice and adding 1.5 x 10 s cells/cm2 in cell culture flasks. BM-MSC were used at passage >5.
  • CD105+ and CD 105- ASCs For purification of CD105+ and CD 105- ASCs, in vitro cultured ASC (passage 2-4) were labeled with CD105-PE Abs (eBioscience, San Diego, CA), subsequently labeled with anti-PE microbeads according to the manufacturer's instructions (Miltenyi Biotech, Bergish Gladbach, Germany) and separated using an AUTO MACSpro ⁇ Miltenyi Biotech). The acquired cell lines were generally 85-95% pure. For stimulation with TGF- betal, cells were cultured for 24 hours in Mesencult with 1% supplements and then stimulated with 10 ng/ml recombinant human TGF-betal (Peprotech, London, UK) for different time points.
  • TGF- betal For stimulation with TGF- betal, cells were cultured for 24 hours in Mesencult with 1% supplements and then stimulated with 10 ng/ml recombinant human TGF-betal (Peprotech, London, UK) for different
  • CD105- and CD105+ cells were stimulated with 10 ng/ml TNF-alpha and 10 ng/ml IFN-gamma (Peprotech) for 12 and 24 hours for RNA extraction and 24 hours for flow cytometry.
  • Cell confluency was quantified using the Image J software and 25 microM GM6001 (Sigma) was added to the cells to inhibit MMPs.
  • Cell proliferation was assayed by seeding passage 3 ASCtot, CD105- and CD105+ ASCs in T25 culture flasks at 2500 cells/cm2. Cells were harvested at 70-80% confluency, counted and re-plated at the same density for 10 passages.
  • the CFU-F assay was performed as previously described ⁇ Jiang et at., Biomaterials 2010). In brief, total ASCs, CD105- and CD105+ ASCs were seeded in 10 cm petri dishes at 2000 cells/plate and cultured for 14 days in complete Mesencult medium. Half of the media were replaced with fresh medium every 3-4 days. Cells were subsequently washed with PBS, fixed with 3.7% paraformaldehyde (15 minutes at room temperature) and stained with 1% cresyl violet. Colonies >2mm were counted.
  • Collagenase type I digests or cultured ASCs were incubated with 7AAD (Sigma-A!drich) and 2.4G2 (eBioscience) and then stained with antibodies specific for CDllb, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD61, CD73, CD 105, CD140a, CD146, sca-1, MHC class I and MHC class II (all from eBioscience) or anti-TGF-betaRI (AL 5) and anti-
  • TGF-betaRII antibodies (R&D Systems, Minneapolis, MN). Cells were acquired and analyzed on a BD FACS Canto II using the FACS Diva software (BD Biosciences, Bedford, MA). 1.5. MSC differentiation
  • Unsorted ASCs or MACS-sorted CD105- and CD105+ ASCs were plated in 6-well plates at a density of 20,000 cells/cm2 for adipogenesis, 10,000 purseis/cm2 for osteogenesis or 0.5-1 x 106 cells/15 ml tube for chondrogenesis.
  • MSC differentiation was induced using the hMSC Adipogenic-, Osteogenic- and Chondrogenic BulletKits, (LONZA, Walkersville, MD) respectively, according to the manufacturer ' s instructions. Cells were maintained in parallel in complete mesencult as control for spontaneuous differentiation. Adipocytes were visualized on day 14 using OilRED O (Sigma-Aldrich).
  • RNA from ASC was obtained using trizol reagent (Invitrogen) according to the manufacturer ' s instructions. RNA samples were reverse-transcribed using Superscript first-strand (Invitrogen) and Q-PCR was performed using the QuantiTect SYBRGreen PCR kit (Ojagen, Valencia, CA) on a Stratagene MX3005P system. Primers: Murine CD105S and CD105L transcripts were amplified using primer pairs as previously described: CD105L FW: 5 ' -GCACTCTGGTACATCTATTCTCACACACGTGG-3 ' ; CD105L RV: 5 '-G G G CACTACG CCATG CTG CT6GTG G -3 ' ; CD105S FW: 5 ' - -
  • RV 5'- GGCTGGGGACTGAGGCTCCA-3 ' ; PPAR-g FW: 5 ' -AATCCTTGGCCCTCTGAGAT-3 ' ; PPAR-g RV: 5 -TTTTCAAGGGTGCCAGTTTC-3 ' ; LPL FW: 5 ' -TCCACCTCTTAGGGTACAGC-3 ' ; LPL RV: 5 AATGTCAACATG CGCTACTG-3 ' ; SOX-9 FW; 5 '-AAG CTCTG G G CA AG CTCTG-3 ' SOX-9 RV: 5 ' -AGATCAACTTTGCCAGCTTG-3 ' ; nanog FW: 5 ' -TTG CCTAGTTCTG AG G AAG C-3 ' ; nanog RV: 5 ' -A AC ACAGTCCG CATCTTCTG -3 ' ; ⁇ -actin FW: 5 ' - AATCGTGCGTGACATCAAAG-3 ' ; ⁇ -
  • ASCs were treated with mitomycin C (50 g/ml, Sigma-Aldrich) for 20 minutes at 37°C and washed 3 times with complete RPMI 1640 (2 m L-Glutamine, 100 U/ml penicillin /streptomycin, 50 ⁇ 2-mercaptoethanol and 10% heat-inactivated FCS, all from Invitrogen).
  • Mitomycin C-treated M5C were plated at different concentrations in flat bottomed 96-well plates and allowed to adhere for 3-4 hours.
  • Spleens from Balb/c mice were homogenized and erythrocytes lysed using ACK buffer (0.15 M NH 4 CI, 10 mM KHC0 3; 0.1 mM Na 2 EDTA at pH 7,4).
  • Splenocytes were labeled with 5 ⁇ CFSE in PBS for 7 minutes at room temperature (company: molecular probes?) and 2 x 10 s cells were added to wells with or without ASCs.
  • Anti-CD3 (1 pg/ml, BD Biosciences) was added to cultures as a mitogenic stimulus of T cells. Cells were harvested on day 3, stained with CD4-APC and analyzed on a FACS Canto II flow cytometer (BD Biosciences). Cell division was analyzed using the FlowJo software.
  • BM-MFs Bone marrow-derived macrophages
  • BM-MFs Bone marrow-derived macrophages
  • ASCtot, CD105- ASC and CD105+ ASC were added to 24 well plates ⁇ 40,000 cells/well) with macrophages ⁇ 0,15 x 106 cells/well) and cultured for 48 hours at normoxia.
  • macrophages and ASCs were added to wells separately as controls.
  • the cells were then cultured for another 24 hours with or without LPS (B4:ll; 1 Sigma-Aldrich) after which the supernatants were harvested and stored at -20C for the quantification of IL-10 and 1L-12 by ELISA.
  • LPS B4:ll; 1 Sigma-Aldrich
  • Nonsorted ASCs, CD105- and CD105+ ASCs (20,000 cells/cm2) were cultured with or without TNF-alpha, IFN-gamma and the supernantant were collected after 24 and 48 hours. Cytokine content and PGE2 levels were analyzed using Ready-SET-go ELISA kits for TGF-betal, IL-10, IL-12 (eBioscience) or PGE2 (Cayman Chemicals, Ann Arbor, Ml), according to the manufacturer ' s instruction. For measuring TGF-betal, complete Mesencult was added to cell free wells and collected and frozen in parallel with supernatants from ASC containing wells.
  • Example 2 MSCs are heterogenous for CD105-Long (endoqlin-L) expression
  • the authors of the present invention were interested in characterizing the early phenotype of mASCs.
  • the authors stained cell suspensions from collagenase type 1-dtgested adipose tissue (CD-AT) before and early after plastic adherence (1, 2 and 6 days of culture; before passage 1) for several MSC markers.
  • CD-AT collagenase type 1-dtgested adipose tissue
  • Figure 1A the percentage of cells expressing CD44 and CD105 was substantially lower (7% and 5% respectively)
  • Figure 1A Upon in vitro culture, 30-40% of all cells adhered to the plastic and this adherent fraction was nearly 100% positive for CD29+, CD44+ and sca-l+ after 2 days in culture.
  • CD105 expression reached a maximum of 30-40% after 6 days.
  • the authors found some contaminating CD45+ cells that decreased rapidly and had disappeared completely by day 6.
  • the absence of CD44 on the CD-AT was due to its removal by the enzymes used to disaggregate the tissue ( Figure IB).
  • CD105 is resistant to the action of these enzymes ( Figure IB) and therefore, its absence from the CD-AT could indicate that CD105 is induced upon in vitro culture in a sub-population of mASCs.
  • CD105-Long CD105L
  • CDIOS-Short CD105S
  • the authors therefore analyzed by RT-PCR which isoform is expressed in the studied ASCs.
  • Figure IE clearly shows that mASCs only express the CD105L isoform.
  • the differences observed in CD105 expression could be due to the influence of culture conditions.
  • the authors therefore investigated whether the expression of CD 105 on mASCs could be modulated by culture time in vitro (passage number), confluence, oxygen pressure or cytokines. They found a small but continuous reduction of CD105 expression during ASCs expansion (Figure IF, open circles).
  • CD105 define two MSC subpopulations.
  • the CD 105- and CD105+ MSCs could represent different stages of activation and/or differentiation or could also unveil different subpopulations inside the MSC- preparations with distinct properties.
  • the authors of the present invention stained mASCs for CD105 in combination with several markers for MSC and found that CD105- and CD105+ cells shared a similar expression pattern of the characteristics MSCs markers: negative for CD45 and positive for CD29, CD44, CD49f, sca-1 and MHC class I ⁇ Figure 2A). The authors then separated the mASCs into CD105- and CD105+ populations using magnetic activated cell sorting (MACS).
  • MCS magnetic activated cell sorting
  • CD105- cells represent differentiated cells they should have lower proliferation and clonogenic potential than CD105+.
  • CFU-F colony forming units-fibroblast
  • CD105- ASCs are more prone to differentiate into adipocytes and osteocytes.
  • MSCs One of the defining characteristics of MSCs is their ability to differentiate into adipocytes, osteocytes and chondrocytes.
  • the authors of the present invention thus set out to investigate the differentiation capacity of CD105- and CD105+ ASCs. They found that CD105- cells generated significantly more adipocytes and osteocytes compared to total and CD105+ ASCs (figure 3A). However, they did not find any difference in chondrogenesis between the different subpopulations (Data not shown).
  • TGF-beta inhibits adipogenesis and osteogenesis and therefore a reduction in TGF-beta signaling will enhance differentiation towards these lineages. Thus a reduction of TGF- beta receptors could therefore favor adipo- and osteogenesis.
  • the authors first set out to study the expression pattern of different TGF- beta receptors on CD105- and CD105+ ASCs and showed that both mASC subsets expressed similar levels of beta-glvcan, ALKl, ALK5 ( Figure 3D) and TGF-beta receptor 11 (Figure 3C), while ALK2 was not expressed by either cell population (Data not shown).
  • IL-11 interleukin-11
  • IL-11 is a TGF- betal-inducible gene, dependent upon the activation of smad2/3/4. Stimulation of total mASCs, CD105- and CD105+ mASCs revealed a higher and more sustained induction of IL-11 in CD105+ mASCs compared to CD105- mASCs ( Figure 3E). The lower IL-11 expression by CD105- mASCs upon TGF-betal stimulation could partly explain the increased adipogenic capacity of this subset.
  • CD105- ASCs have improved immunomodulatory capacity compared to CD 105+ ASCs.
  • Mesenchymal stromal cells are known to inhibit the immune response in vitro and in vivo through multiple mechanisms. In this sense, the authors of the present invention have found that CD105- ASC produced slightly more TGF-betal and less PGE2 compared to CD105+ ASCs but the differences were not statistically significant (Figure 4A). However, they found that CD105- ASCs were significantly better at inhibiting splenocyte proliferation compared to CD105+ ASCs ( Figure 4C).

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Abstract

Le présent brevet décrit une méthode basée sur l'expression de CD105 qui permet d'isoler et de conserver des CSM ayant des propriétés immunomodulatrices et différenciatrices améliorées. Dans ce sens, les auteurs de la présente invention montrent pour la première fois que les CSM CD105- représentent une population de CSM multipotentes distincte. Elles ont un potentiel de croissance similaire et conservent des marqueurs CSM identiques aux CSM CD105+. Cependant, les CSM CD105- sont meilleures en termes de suppression de lymphocytes T et plus promptes à la différenciation en adipocytes et ostéocytes que les CSM CD105+. L'utilisation de ces CSM CD105- multipotentes, expansibles en tant qu'alternative aux CSM CD105+ et/ou totales pour le traitement des maladies autoimmunes et des troubles osseux est en outre décrite.
PCT/EP2014/067996 2013-08-23 2014-08-25 Cellules stromales mésenchymateuses (csm) multipotentes négatives à cd105 Ceased WO2015025059A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007108003A2 (fr) * 2006-03-23 2007-09-27 Pluristem Ltd. Procedes de developpement cellulaire et utilisations therapeutiques des cellules et des milieux conditionnes produits de cette maniere
US20090110668A1 (en) * 2007-10-30 2009-04-30 The University Of Louisville Research Foundation, Inc. Subpopulations of bone marrow-derived adherent stem cells and methods of use therefor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007108003A2 (fr) * 2006-03-23 2007-09-27 Pluristem Ltd. Procedes de developpement cellulaire et utilisations therapeutiques des cellules et des milieux conditionnes produits de cette maniere
US20090110668A1 (en) * 2007-10-30 2009-04-30 The University Of Louisville Research Foundation, Inc. Subpopulations of bone marrow-derived adherent stem cells and methods of use therefor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEYVA-LEYVA M ET AL: "Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential", STEM CELLS AND DEVELOPMENT 20130415 MARY ANN LIEBERT INC. USA, vol. 22, no. 8, 15 April 2013 (2013-04-15), pages 1275 - 1287, XP002730027, ISSN: 1547-3287 *
LV XIAO-JIE ET AL: "In vitro proliferation and differentiation of adipose-derived stem cells isolated using anti-CD105 magnetic beads", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, vol. 30, no. 4, October 2012 (2012-10-01), pages 826 - 834, XP002730028 *

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