WO2013165252A1 - Culturing of mesenchymal stem cells - Google Patents

Culturing of mesenchymal stem cells

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WO2013165252A1
WO2013165252A1 PCT/NL2013/050340 NL2013050340W WO2013165252A1 WO 2013165252 A1 WO2013165252 A1 WO 2013165252A1 NL 2013050340 W NL2013050340 W NL 2013050340W WO 2013165252 A1 WO2013165252 A1 WO 2013165252A1
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cells
wnt
cell
pathway
example
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PCT/NL2013/050340
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French (fr)
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BERGE Derk TEN
Roberto NARCISI
OSCH Geertruda Johanna Victoria Maria VAN
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Erasmus University Medical Center Rotterdam
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655Chondrocytes; Cartilage
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    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662Stem cells
    • C12N5/0663Bone marrow mesenchymal stem cells (BM-MSC)
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    • C12N2500/00Specific components of cell culture medium
    • C12N2500/05Inorganic components
    • C12N2500/10Metals; Metal chelators
    • C12N2500/20Transition metals
    • C12N2500/24Iron; Fe chelators; Transferrin
    • C12N2500/25Insulin-transferrin; Insulin-transferrin-selenium
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/115Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/15Transforming growth factor beta (TGF-β)
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30Hormones
    • C12N2501/38Hormones with nuclear receptors
    • C12N2501/39Steroid hormones
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40Regulators of development
    • C12N2501/415Wnt; Frizzeled
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    • C12N2506/00Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)

Abstract

The present invention relates to a method for fast expansion of progenitor cells comprising culturing said cells in the presence of fibroblast growth factor 2 (FGF2) and Wnt3a.

Description

Title: CULTURING OF MESENCHYMAL STEM CELLS

FIELD OF THE INVENTION

The invention relates to the field of tissue engineering, more particularly to the fields of culturing, differentiating, expanding and

developing tissue for repair of cartilage or bone defects. More specifically, the invention relates to culturing, chondrogenesis, and osteogenesis in stem cells, preferably mesenchymal stem cells.

BACKGROUND Mesenchymal stem cells (MSCs) are characterized by their ability to produce daughter stem cells and also to differentiate into many distinct cell types including, but not limited to, osteoblasts, stromal cells that support hematopoiesis and osteoclastogenesis, chondrocytes, myocytes, adipocytes of the bone marrow, neuronal cells and [beta] -pancreatic islet cells. Thus, MSCs are able to provide the appropriate number of progenitor cells and stromal cells needed for tissue repair and remodelling, cartilage and bone development, bone remodeling and hematopoiesis throughout life.. Mesenchymal stem cells (MSCs) and especially bone-marrow-derived mesenchymal stem cells (BMSCs) are multipotent and self-renewing cells used to repair cartilage defects.

However, MSCs represent a heterogeneous population of cells with varying chondrogenic differentiation potential. Moreover, it has been widely reported that MSCs lose their self-renewing capacity and their pluripotency in time during the in vitro expansion. Although administration of fibroblast growth factor (FGF), in particular FGF-2, during the expansion enhances both proliferation and chondrogenic potential of these cells, MSCs fail to produce the quality and quantity of cartilage matrix that is obtained from culturing articular chondrocytes under identical conditions. SUMMARY OF THE INVENTION

The present invention relates to a method for fast expansion of progenitor cells comprising culturing said cells in the presence of fibroblast growth factor 2 (FGF2) and Wnt3a. Specifically in such a method, the progenitor cell is a mesenchymal stem cell, preferably a bone-marrow-derived mesenchymal stem cell.

In a specific embodiment of such a method, the cells are cultured in vitro. In an alternative embodiment the cells are implanted in vivo.

Further part of the invention is a culture medium comprising components for culturing progenitor cells, further comprising FGF2 and Wnt3a. Preferably in said culture medium the FGF2 is present in a

concentration of about 0.1 to about 100 ng/ml, preferably about 0.1 to about 10 ng/ml, more preferably about 0.5 to about 2 ng/ml, most preferably at about 1 ng/ml. Further preferred in said culture medium the Wnt3a is present in a concentration of about 1 to about 1000 ng/ml, preferably about 10 to about 500 ng/ml, more preferably about 100 to about 500 ng/ml, most preferably about 250 ng/ml.

In a preferred embodiment said culture medium is expansion culture medium.

The invention also relates to a matrix for culturing and/or production of chondrocytes comprising FGF2 and Wnt3a. Preferably such a matrix has been seeded with mesenchymal stem cells.

Also part of the invention is a method as described above wherein the cells are cultured in a matrix as described above. DETAILED DESCRIPTION

The present disclosure is based at least in part on the discovery that a combination of FGF-2 and a Wnt3a protein, if applied during expansion to a culture of mesenchymal stem cells (MSCs) enhances the proliferation rate of the MSCs, and increased chondrogenesis after expansion.

Fibroblast growth factors, or FGFs, are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The FGFs are heparin-binding proteins and interactions with cell-surface-associated heparan sulfate proteoglycans have been shown to be essential for FGF signal transduction. FGFs are key players in the processes of proliferation and differentiation of wide variety of cells and tissues. FGF2 also known as basic fibroblast growth factor, bFGF, or FGF-β, is believed to be a factor that inhibits differentiation in stem cell cultures.

The Wnt proteins are a group of secreted lipid-modified (palmitoylation) signaling proteins of 350-400 amino acids in length. Following the signal sequence, they carry a conserved pattern of 20-24 cysteine residues, on which palmitoylation occurs on a cysteine residue. These proteins activate various pathways in the cell that can be categorized into the canonical and

noncanonical Wnt pathways. Through these signaling pathways, Wnt proteins play a variety of important roles in embryonic development, cell

differentiation, and cell polarity generation. The human Wnt3a gene is a member of the WNT gene family. It encodes a protein showing 96% amino acid identity to mouse Wnt3A protein, and 84% to human WNT3 protein, another WNT gene product. The Wnt3a gene is clustered with WNT 14 gene, another family member, in chromosome lq42 region.

Disclosed herein is a new approach towards differentiation of MSCs into fibrochondrocytes, or fibrochondrocyte-like cells, using chemical factors, such as by the treatment of FGF-2 and Wnt3a. Such fibrochondrocytes or

fibrochondrocyte-like cells can serve as a source of therapeutic cells for the regeneration of fibrocartilage tissues. Also disclosed herein is the generation and regeneration of fibrocartilage tissues.

As demonstrated herein, MSCs treated with FGF-2 and Wnt3a showed increased chondrogenic differentiation. The inducement of in vivo or ex vivo differentiation of hMSCs into fibrochondrocytes, or fibrochondrocyte-like cells , as described herein, can be applied to fibrocartilage tissue engineering and fibrocartilage tissue regeneration.

In contrast to conventional methods, the present invention provides, in some embodiments, methods of differentiation and culturing MSCs based at least in part, or substantially, on chemical factors. Such approaches provide for larger scale differentiation and expansion, useful for tissue engineering or tissue regeneration.

A progenitor cell, as that term is used herein, is a precursor to a

fibrochondrocyte or fibrochondrocyte-like cell and can differentiate in the presence of CTGF and TGF 3. A progenitor cell can be a multipotent cell. A progenitor cell can be self-renewing. For example, a progenitor cell can be a mesenchymal stem cell (e.g., a human, horse or other animal mesenchymal stem cell, more preferably a human bone-marrow-derived mesenchymal stem cell). It can further be an adipose-derived MSC, a synovial-derived MSC, dental pulp-derived MSC or a tendon-derived MSC. The progenitor cell can be substantially less differentiated than a fibrochondrocyte or fibrochondrocyte- like cell. Progenitor cells basically can be defined as any cell with chondrogenic potential, thus also would include chondrocytes (articular, auricular and nasal) and MSCs from the umbilical cord.

Progenitor cells such as MSCs can be isolated, purified, and/or cultured by a variety of means known in the art. Methods for the isolation and culture of tissue progenitor cells are discussed in, for example, Vunjak-Novakovic and Freshney (2006) Culture of Cells for Tissue Engineering, Wiley-Liss, ISBN 0471629359. For example, mesenchymal stem cells can be isolated from bone marrow and culture-expanded (see e.g., Example 1).

In various embodiments, a progenitor cell is a precursor to a

fibrochondrocyte or fibrochondrocyte-like cell and differentiates under culture conditions including sequential or concurrent provision of FGF2 and Wnt3a as described herein.

The tissue progenitor cells can be derived from the same or different species and or the same or different individual as the transplant recipient. For example, the progenitor cells can be derived from an animal, including, but not limited to, mammals, reptiles, and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human.

In various embodiments, a fibrochondrocyte or fibrochondrocyte-like cell differentiates from a progenitor cell that was cultured under conditions including sequential or concurrent provision of FGF2 and Wnt3a as described herein. In some embodiments, a fibrochondrocyte or fibrochondrocyte-like cell displays a fibrocartilaginous matrix.

If desired a progenitor cell or a fibrochondrocyte or fibrochondrocyte-like cell can be transformed with a heterologous nucleic acid so as to express a bioactive molecule, or heterologous protein or to overexpress an endogenous protein. As an example, a progenitor cell or a fibrochondrocyte or

fibrochondrocyte-like cell can be genetically modified to expresses a fluorescent protein marker. Exemplary markers include GFP, EGFP, BFP, CFP, YFP, and RFP. In another example, a progenitor cell or a fibrochondrocyte or

fibrochondrocyte-like cell can be genetically modified to express an

angiogenesisrelated factor, such as activin A, adrenomedullin, aFGF, ALKl , ALK5, ANF, angiogenin, angiopoietin- 1 , angiopoietin-2, angiopoietin-3, angiopoietin-4, angiostatin, angiotropin, angiotensin-2, AtT20-ECGF, betacellulin, bFGF, B61 , bFGF inducing activity, cadherins, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen, collagen receptors αί, 6ί [and α26ί, connexins, Cox-2, ECDGF (endothelial cell-derived growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, endothelins, endostatin, endothelial cell growth inhibitor, endothelial cell-viability maintaining factor, endothelial

differentiation shpingolipid G-protein coupled receptor- 1 (EDGl ), eph ns, Epo, HGF, TNF-alpha, TGF-beta, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin and fibronectin receptor a56l, Factor X, HB-EGF, HBNF, HGF, HUAF, heart derived inhibitor of vascular cell proliferation, IFN- gamma, IL1 , IGF-2 IFN-gamma, integrin receptors, K- FGF, LIF, leiomyoma-derived growth factor, MCP-1 , macrophage-derived growth factor, monocyte-derived growth factor, MD-ECI, MECIF, MMP 2, MMP3, MMP9, urokiase plasminogen activator, neuropilin (NRP1 , NRP2), neurothelin, nitric oxide donors, nitric oxide synthases (NOSs), notch, occludins, zona occludins, oncostatin M, PDGF, PDGF-B, PDGF receptors, PDGFR-β, PD-ECGF, PAI-2, PD-ECGF, PF4, PI GF, PKR1 , PKR2, PPAR- gamma, PPAR-gamma ligands, phosphodiesterase, prolactin, prostacyclin, protein S, smooth muscle cell-derived growth factor, smooth muscle cell- derived migration factor, sphingosine- 1 -phosphate-1 (Si PI), Syk, SLP76, tachykinins, Tie 1, Tie2, TGF-β, and TGF-β receptors, TIMPs, TNF-ct, TNF-6, transferrin, thrombospondin, urokinase, VEGF -A, VEGF-B, VEGF-C, VEGF- D, VEGF-E, VEGF, VEGF.sub.164, VEGI, EG-VEGF, VEGF receptors, PF4, 16 kDa fragment of prolactin, prostaglandins El and E2, steroids, heparin, 1 - butyryl glycerol (monobutyrin), and/or nicotinic amide. As another example, a progenitor cell or a fibrochondrocyte or fibrochondrocyte-like cell can be transfected with genetic sequences that are capable of reducing or eliminating an immune response in a host {e.g., expression of cell surface antigens such as class I and class II histocompatibility antigens can be suppressed). This can allow the transplanted cells to have reduced chance of rejection by the host. The invention further discloses methods to induce fibrochondrogenic

differentiation of progenitor cells by further sequential or concurrent treatment of growth factors, such as connective tissue growth factor (CTGF) and transforming growth factor β (TGF 6) on substrates, such as monolayer or 3D pellet culture of hMSCs. As shown herein, treatment of hMSCs with FGF-2 and Wnta3 more readily induces differentiation towards chondrogenesis than application of these compounds individually or combinations of other components, showing a synergistic effects. Further, the treatment also resulted in an enhancement of the proliferation rate (see Example 1).

A progenitor cell, more preferably a MSC, and even more preferably a BMSC can be contacted with FGF-2 and Wnt3a sequentially or simultaneously so as to stimulate proliferation and chondrogenic differentiation. Forexample, progenitor cells can be contacted with FGF2 and Wnt3a. As another example, progenitor cells can be contacted with FGF2 followed by Wnt3a, or, cells can be contacted with Wnt3a followed by FGF2. As another example, cells can be contacted concurrently with FGF2 and Wnt3a.

MSCs can be cultured by a variety of means known to the art. Cells can be incubated with FGF2 or Wnt3a under conditions allowing proliferation and differentiation. Methods of culturing progenitor cells are generally known in the art and such methods can be adapted so as to provide optimal conditions for differentiation of progenitor cells contacted with FGF2 or Wnt3a (see e.g., Vunjak-Novakovic and Freshney (2006) Culture of Cells for Tissue

Engineering, Wiley-Liss, ISBN 0471629359).

FGF2 is available from a variety of commercial sources (e.g., AbD Serotec). FGF2 can be present at a concentration of about 100, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 700, about 750, about 800, about 850, about 900, about 950, about 1000 pg/ml or at a concentration of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 ng/mL. For example, FGF2 can be present at a concentration of about 1 ng/mL (see e.g., Example 1 ).

Wnt3a is preferably produced in cell culture, like in a system using insect cells or using mammalian cells (Willert, K. et al, Nature 2003; 423:448-452) or the system as described in US 7, 153,832, which herewith is incorporated by reference. From these the protein then can be isolated. Wnt3a can be present in a concentration of about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 325, about 350, about 375, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950 or about 1000 ng/ml.

Compositions including FGF2 and Wnt3a can be formulated or encapsulated for controlled release, for stimulation of progenitor cells in situ. For example, compositions including FGF2 and Wnt3a can be encapsulated in microspheres, such as PLGA microspheres. Differing PLGA ratios can be used to provide sequential release of FGF2 and Wnt3a. For example, a composition including FGF2 can be encapsulated in 50:50 PLGA microspheres. As another example, a composition including Wnt3a can be encapsulated in 75:25 PLGA

microspheres. Encapsulated compositions including FGF2 and/or Wnt3a can be embedded in a biocompatible matrix A (e.g., a 3D fibrin gel) loaded with mesenchymal stem cells and cultured in vitro

As another example, a composition including Wnt3a can be incorporated in liposomes (Morrell, N.T. et al, PLoS One 2008, 3: e2930).

Mesenchymal stem cells can be cultured by a variety of means known to the skilled person. For example, cells can be plated (e.g., about 100,000 cells per well) for 2D culture. As another example, progenitor cells can be centrifuged (e.g., about 0.2 million cells to form a 3D pellet. Monolayer (2D) or 3D cell pellets can be cultured in a growth medium. Monolayer (2D) or 3D cell pellets can be treated with FGF2 and Wnt3a sequentially or concurrently. An induction medium can be provided in conjunction with FGF2 and Wnt3a (e.g., as a component of encapsulated composition or provided separately).

Methods described herein can increase the number of formed

fibrochondrocyte or fibrochondrocyte-like cells as compared to conventional methods. For example, culture methods described herein can increase differentiation towards fibrochondrocyte or fibrochondrocyte-like cells from progenitor cells. For example, culture methods described herein can increase proliferation of mesenchymal stem cells, fibrochondrocyte or fibrochondrocyte- like cells. In some embodiments, a progenitor cell or a fibrochondrocyte or fibrochondrocyte-like cell can be co-cultured with one or more additional cell types. Such additional cell types can include (but are not limited to) cardiac cells, skin cells, liver cells, heart cells, kidney cells, pancreatic cells, lung cells, bladder cells, stomach cells, intestinal cells, cells of the urogenital tract, breast cells, skeletal muscle cells, skin cells, bone cells, cartilage cells, keratinocytes, hepatocytes, gastro-intestinal cells, epithelial cells, endothelial cells, mammary cells, skeletal muscle cells, smooth muscle cells, parenchymal cells, osteoclasts, or chondrocytes. In such a way more complex tissue constructs or organs may be produced in such co-culture conditions. This applies both to in vitro co-culturing and to implantation of cells as described herein.

Various embodiments described herein employ a scaffold or matrix material. For example, a composition including FGF2 and Wnt3a and possible

mesenchymal stem cells can be included in or on a scaffold.

A scaffold can be fabricated with any matrix material recognized as useful by the skilled artisan. A matrix material can be a biocompatible material that generally forms a porous, microcellular scaffold, which provides a physical support for cells. Such matrix materials can allow cell attachment and migration; can deliver and retain cells and biochemical factors; can enable diffusion of cell nutrients and expressed products; or exert certain mechanical and biological influences to modify the behavior of the cell phase. The matrix material generally forms a porous, microcellular scaffold of a biocompatible material that provides a physical support and an adhesive substrate for recruitment and growth of cells during in vitro or in vivo culturing.

Suitable scaffold and matrix materials are discussed in, for example, Ma and Elisseeff, ed. (2005) Scaffolding In Tissue Engineering, CRC, ISBN

1574445219; Saltzman (2004) Tissue Engineering: Engineering Principles for the Design of Replacement Organs and Tissues, Oxford ISBN 019514130X. For example, matrix materials can be, at least in part, solid xenogenic {e.g., hydroxy apatite) (Kuboki et al. 1995 Connect Tissue Res 32, 219-226; Murata et al. 1998 Int J Oral Maxillofac Surg 27, 391 -396), solid alloplastic (polyethylene polymers) materials (Saito and Takaoka 2003 Biomaterials 24 2287-93; Isobe et al. 1999 J Oral Maxillofac Surg 57, 695-8), or gels of autogenous (Sweeney et al. 1995 . J Neurosurg 83, 710-715), allogenic (Bax et al. 1999 Calcif Tissue Int 65, 83-89; Viljanen et al. 1997 Int J Oral Maxillofac Surg 26, 389-393), or alloplastic origin (Santos et al. 1998 . J Biomed Mater Res 41 , 87-94), and combinations of the above (Alpaslan et al. 1996 Br J of Oral Maxillofac Surg 34, 414- 418).

The matrix comprising the scaffold can have an adequate porosity and an adequate pore size so as to facilitate cell recruitment and diffusion throughout the whole structure of both cells and nutrients. The matrix can be

biodegradable providing for absorption of the matrix by the surrounding tissues, which can eliminate the necessity of a surgical removal. The rate at which degradation occurs can coincide as much as possible with the rate of tissue or organ formation. Thus, while cells are fabricating their own natural structure around themselves, the matrix is able to provide structural integrity and eventually break down, leaving the neotissue, newly formed tissue or organ which can assume the mechanical load. The matrix can be an injectable matrix in some configurations. The matrix can be delivered to a tissue using minimally invasive endoscopic procedures.

The scaffold can comprise a matrix material having different phases of viscosity. For example, a matrix can have a substantially liquid phase or a substantially gelled phase. The transition between phases can be stimulated by a variety of factors including, but limited to, light, chemical, magnetic, electrical, and mechanical stimulus. For example, the matrix can be a thermosensitive matrix with a substantially liquid phase at about room temperature and a substantially gelled phase at about body temperature. The liquid phase of the matrix can have a lower viscosity that provides for optimal distribution of growth factors or other additives and injectability, while the solid phase of the matrix can have an elevated viscosity that provides for matrix retention at or within the target tissue.

The scaffold can comprise a matrix material formed of synthetic polymers. Such synthetic polymers include, but are not limited to, polyurethanes, polyorthoesters, polyvinyl alcohol, polyamides, polycarbonates, polyvinyl pyrrolidone, marine adhesive proteins, cyanoacrylates, analogs, mixtures, combinations and derivatives of the above. Alternatively, the matrix can be formed of naturally occurring biopolymers. Such naturally occurring

biopolymers include, but are not limited to, fibrin, fibrinogen, fibronectin, collagen, and other suitable biopolymers. Also, the matrix can be formed from a mixture of naturally occurring biopolymers and synthetic polymers.

The scaffold can include one or more matrix materials including, but not limited to, a collagen gel, a polyvinyl alcohol sponge, a poly(D,L-lactide-co- glycolide) fiber matrix, a polyglactin fiber, a calcium alginate gel, a polyglycolic acid mesh, polyester (e.g., poly-(L-lactic acid) or a polyanhydride), a

polysaccharide (e.g. alginate), polyphosphazene, polyacrylate, or a polyethylene oxide-polypropylene glycol block copolymer. Matrices can be produced from proteins (e.g. extracellular matrix proteins such as fibrin, collagen, and fibronectin), polymers (e.g., polyvinylpyrrolidone), or hyaluronic acid.

Synthetic polymers can also be used, including bioerodible polymers (e.g., poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates), degradable polyurethanes, non-erodible polymers (e.g., polyacrylates, ethylene -vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof), non-erodible

polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon(R), or nylon.

The scaffold can further comprise any other bioactive molecule, for example an antibiotic or an additional chemotactic growth factor or another osteogenic, dentinogenic, amelogenic, or cementogenic growth factor. In some

embodiments, the scaffold is strengthened, through the addition of, e.g., human serum albumin (HSA), hydroxyethyl starch, dextran, or combinations thereof. Suitable concentrations of these compounds for use in the

compositions of the application are known to those of skill in the art, or can be readily ascertained without undue experimentation.

The concentration of a compound or a composition in the scaffold will vary with the nature of the compound or composition, its physiological role, and desired therapeutic or diagnostic effect. A therapeutically effective amount is generally a sufficient concentration of therapeutic agent to display the desired effect without undue toxicity. For example, the matrix can include a

composition comprising FGF2 and/or Wnt3a at any of the above described concentrations. The compound(s) can be incorporated into the scaffold or matrix material by any known method. In some embodiments, the compound is imbedded in a gel, e.g., a collagen gel incorporated into the pores of the scaffold or matrix material or applied as a coating over a portion, a substantial portion, substantially all of, or all of the scaffold or matrix material.

Alternatively, chemical modification methods can be used to covalently link a compound or a composition to a matrix material. The surface functional groups of the matrix can be coupled with reactive functional groups of a compound or a composition to form covalent bonds using coupling agents well known in the art such as aldehyde compounds, carbodiimides, and the like. Additionally, a spacer molecule can be used to gap the surface reactive groups and the reactive groups of the biomolecules to allow more flexibility of such molecules on the surface of the matrix. Other similar methods of attaching biomolecules to the interior or exterior of a matrix will be known to one of skill in the art. Several methods can be used for fabrication of porous scaffolds, including particulate leaching, gas foaming, electrospinning, freeze drying, foaming of ceramic from slurry, and the formation of polymeric sponge. Other methods can be used for fabrication of porous scaffolds include computer aided design (CAD) and synthesizing the scaffold with a bioplotter (e.g., solid freeform fabrication) (e.g., Bioplotter (TM), EnvisionTec, Germany).

Biologic drugs that can be added to compositions of the invention include immunomodulators and other biological response modifiers. A biological response modifier generally encompasses a biomolecule (e.g., peptide, peptide fragment, polysaccharide, lipid, antibody) that is involved in modifying a biological response, such as the immune response or tissue or organ growth and repair, in a manner that enhances a particular desired therapeutic effect, for example, the cytolysis of bacterial cells or the growth of tissue- or organ- specific cells or vascularization. Biologic drugs can also be incorporated directly into the matrix component. Those of skill in the art will know, or can readily ascertain, other substances which can act as suitable non-biologic and biologic drugs.

Compositions described herein can also be modified to incorporate a diagnostic agent, such as a radiopaque agent. The presence of such agents can allow the physician to monitor the progression of wound healing occurring internally. Such compounds include barium sulfate as well as various organic compounds containing iodine. Examples of these latter compounds include iocetamic acid, iodipamide, iodoxamate meglumine, iopanoic acid, as well as diatrizoate derivatives, such as diatrizoate sodium. Other contrast agents that can be utilized in the compositions can be readily ascertained by those of skill in the art and can include, for example, the use of radiolabeled fatty acids or analogs thereof.

The concentration of an agent in the composition will vary with the nature of the compound, its physiological role, and desired therapeutic or diagnostic effect. A therapeutically effective amount is generally a sufficient concentration of therapeutic agent to display the desired effect without undue toxicity. A diagnostically effective amount is generally a concentration of diagnostic agent which is effective in allowing the monitoring of the

integration of the tissue graft, while minimizing potential toxicity. In any event, the desired concentration in a particular instance for a particular compound is readily ascertainable by one of skill in the art.

In the present invention, the role of Wnt3a is to simulate the Wnt pathway. The Wnt/beta-catenin signalling pathway regulates a variety of cellular processes during the development of vertebrates and invertebrates, including cell proliferation and differentiation, cell fate, and organogenesis. In addition, the pathway controls tissue homeostasis and regeneration in response to damage in Zebra fish, Xenopus, planarians, and even adult mammals. Wnt signaling is initiated by interaction of Wnt proteins with a variety of receptors, including members of the Frizzled (Fz) family of transmembrane receptors and members of the low-density-lipoprotein receptor-related protein (LRP) family (e.g., LRP5/LRP6). The extracellular Wnt signal stimulates intracellular signal transduction cascades including the canonical pathway, which regulates gene expression in the nucleus (see Logan CY and Nusse, R. Annu. Rev. Cell Dev. Biol., 20:781 -810, 2004) and several non-canonical pathways (reviewed by Kohn, AD and Moon, RT, Cell Calcium, 38: 439-446, 2005). Briefly, Wnt signaling via the canonical pathway leads to stabilization and nuclear localization of beta-catenin, which assembles with members of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to form complexes that generally activate transcription. In the absence of Wnt signalling, beta-catenin is instead targeted for degradation by the beta- catenin destruction complex, and TCF/LEFs form complexes that generally repress transcription. In the absence of Wnt signaling, kinases such as glycogen synthase kinase-3 (GSK3) and casein kinase 1 (CK1) phosphorylate beta-catenin, which as a consequence is ubiquinated and targeted for destruction by the proteasome. Activation of the Wnt pathway thus results in diminished phosphorylation of beta-catenin, thereby leading to its

stabilization. Several endogenous proteins have been identified as inhibitors of Wnt signaling, including Dickkopf (Dkk), breakpoint cluster region protein (Bcr), proteins comprising a WIF (Wnt inhibitory factor) domain etc.

The Wnt proteins form a large family of isoforms. Among these, Wnt3a has been shown to enhance self-renewal and to maintain the undifferentiated state of embryonic stem cells (ESCs) and haematopoietic stem cells (HSCs) through the accumulation of [beta]-catenin in the cell nucleus.

Wnt signaling has been implicated in the expansion of certain stem cells. For example, US2008/0213892 describes the use of Wnt proteins for the expansion of neural progenitor cells. Wnt proteins also play a role in the gut, where in the crypt of the colon the loss of transcription factor TCF4 can lead to depletion of stem cells.

DEFINITIONS

Wnt/ *-catenin signalling pathway. Detailed reviews of this pathway are described in Logan and Nusse (2004), Annu. Rev. Cell Dev. Biol. 20, 781-810 and Wodarz and Nusse (1998), Annu. Rev. Cell Dev. Biol. 14, 59-88. The latter document also describes a number of assays for monitoring Wnt signalling.

Wnt pathway activity. This term, used synonymously with "Wnt signaling" refers to the series of biochemical events that ensues following binding of a ligand (eg., a Wnt protein) to a receptor for a Wnt family member, ultimately leading to changes in gene transcription and, if in vivo, often leading to a characteristic biological effect in an organism. Wnt. The term "Wnt" or "Wnt protein" refers to a polypeptide having a naturally occurring amino acid sequence of a Wnt protein or a fragment, variant, or derivative thereof that at least in part retains the ability of the naturally occurring protein to bind to Wnt receptor(s) and activate Wnt signaling. In addition to naturally-occurring allelic variants of the Wnt sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms "Wnt", "Wnt protein" and the like. Wnt proteins are a family of secreted proteins important for a wide array of developmental and physiological processes (Mikels, AJ and Nusse, R., Oncogene, 25: 7461-7468, 2006). Wnts are related to one another in sequence and strongly conserved in structure and function across multiple species. Thus a Wnt protein displaying activity in one species may be used in other species to activate the Wnt pathway in such species and may be expected to display similar activity. Wnt family members include Wntl , Wnt2, Wnt2b (also called Wntl3), Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt9a, WntlOb, Wntl 1, Wntl6, WntlOa, Wntl Ob, Wntl 1 , Wntl4, Wntl 5, or Wntl. Sequences of Wnt genes and proteins are known in the art. One of skill in the art can readily find the Gene ID, accession numbers, and sequence information for Wnt family members and other genes and proteins of interest herein in publicly available databases.

Wnt/p*-catenin signalling pathway activation. This term means to cause or facilitate a qualitative or quantitative increase of Wnt pathway activity. An "activator of the Wnt/6-catenin signalling pathway" will cause or facilitate a qualitative or quantitative increase in Wnt pathway activity. The "activator of the Wnt/6-catenin signalling pathway" may be an agonist of the Wnt/6-catenin signalling pathway.

Any means for activation of the Wnt/6-catenin signalling pathway may be used in the present invention. Such may include, for example, increasing the expression and/or activity of one or more endogenous genes encoding any member of the Wnt/6-catenin signalling pathway at the transcriptional, translational or post-translational level, such as increasing the persistence messenger RNAs or proteins. Thus, any of the components of the Wnt/6- catenin signalling pathway may be modulated in order to activate the pathway provided that the desired result is obtained. Methods for determining whether the Wnt/6-catenin signalling pathway is activated are described herein.

In some embodiments, Wnt/6-catenin signalling pathway may be activated by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or 100% or more as compared to the pathway in the absence of an activator.

The Wnt/6-catenin signalling pathway may be activated for more than 3 hours, more than 6 hours, more than 12 hours or more than 24 hours. In some embodiments, the Wnt/6-catenin signalling pathway is activated for 2 days or more, such as 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more or 10 days or more. It will be evident that the Wnt/6-catenin signalling pathway may be activated for as long as necessary, depending on the application, for example, 2 weeks, 3 weeks, 4 weeks, etc, as required.

Assays for Wnt/p*-catenin signalling pathway activation

Activation of the Wnt/6-catenin signalling pathway in a cell may be assessed in a number of ways, as known in the art. In general, such an assay will seek to detect the modulation of the target component, or a component downstream of the component which is the target of the activation.

One assay for determining the activation of the Wnt/6-catenin signalling pathway comprises detecting the activity of GSK-3.beta. One assay for determining the activation of the Wnt/6-catenin signalling pathway comprises the detection of a reduced activity of GSK-3.beta. Such an assay may be particularly suitable where GSK-3.beta activity is targeted for inhibition as a means to activate the Wnt signalling pathway.

Alternatively, or in addition, an assay for activation of the Wnt/6-catenin signalling pathway may comprise detecting the amounts of 6-catenin. This may be achieved by making extracts of cells using means known in the art, and detecting the amount of 6-catenin protein by antibody Western blots. Particularly useful assays include those which detect active β-catenin, or non- phosphorylated forms of β-catenin, using antibodies specific for such forms, for example. A monoclonal antibody capable of detecting specifically the active non-phosphorylated form of β-catenin is described in van Noort et al., (2002) J Biol. Chem. 2002 277(20): 17901-5. The assay for measuring activation involves detecting the accumulation of 6-catenin.

Activation of the pathway may further be detected by measuring the expression of Axin2, using Western blots with anti-Axin2 antibodies or by measuring the phosphorylation of Dishevelled, or the LRP tail (Tamai 2004 Mol. Cell. (2004) 13(1): 149-56).

Further, the activation of the pathway may be detected through use of appropriate reporter plasmids, which are transfected into cells of interest. Expression of the reporter may be sensitive to the activation of Wnt/6-catenin signalling, as a result of, for example, the promoter for the reporter comprising a response element. One reporter which may be used in such an assay is a TOP Flash reporter, as described in Molenaar et al, (1996) Cell 86(3):391-9. TOP-Flash comprises a TCF Reporter Plasmid with two sets of three copies of the TCF binding site upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame. A control plasmid is FOP-Flash containing mutated and non-active TCF-binding sites.

Activation of the Wnt/p*-catenin signalling pathway As illustrated above, the methods of the invention comprise activating the Wnt/6-catenin signalling pathway in a cell by applying, amongst other, the factor Wnta3. However, in stead of, or next to Wnta3, a variety of agents may be used to increase Wnt pathway activity. The Wnt/6-catenin signalling pathway may be activated by any of the receptors for Wnt/6-catenin signalling. For example, any of the Frizzled receptors may be activated to activate the pathway. Examples of Frizzled receptors well known in the art. Receptor activation may be achieved in a number of ways, for example, by up regulating the expression of the receptor(s) by transfecting a suitable expression vector expressing the receptor into a cell. Furthermore, receptor activation may be achieved by introduction of a constitutively active Frizzled receptor to the cell, for example by transfection into the cell as an expression vector encoding the constitutively active receptor. Receptors for Wnt, such as Frizzled receptors, may also be activated by binding of Wnt ligand. Thus, the Wnt signalling pathway may be activated by increasing the activity or expression of Wnt ligand, or by decreasing the activity or expression of antagonists of Wnt or Frizzled. Wnt ligands are known in the art, and include, but are not limited to WNT1, WNT2, WNT2B/13, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT 7 A, WNT7B, WNT8A, WNT8B, WNT9A (previously WNT 14), WNT9B (previously WNT 15), WNT10A, WNT10B, WNT11 and WNT 16. Exemplary Wnt ligands include Wntl, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a,

Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntll and Wntl6.

The Wnt protein may be isolated from naturally occurring sources (e.g., mammalian cells that naturally produce the protein), produced in eukaryotic or prokaryotic cells using recombinant expression technology, or chemically synthesized. Soluble, biologically active Wnt proteins may be prepared in purified form using methods known in the art. See, e.g., U.S. Pat. Pub. No. 20040248803 and Willert, K., et al., Nature, 423: 448-52, 2003. In certain embodiments the soluble, biologically active Wnt protein is Wnt3a. In certain embodiments the Wnt protein is co- or post-translationally modified as occurs when the Wnt protein is produced in a host cell that naturally expresses the Wnt protein. In other embodiments the Wnt protein is not co- or post- translationally modified as in nature. In certain embodiments the soluble, biologically active Wnt protein is modified with a lipid moiety such as palmitate. The lipid moiety may be attached to a conserved cysteine. For example, in certain embodiments the Wnt protein is palmitoylated on a conserved cysteine as known in the art. In certain embodiments the Wnt protein is glycosylated as occurs when the Wnt protein is produced in a mammalian host cell that naturally expresses the Wnt protein. In other embodiments the Wnt protein is not glycosylated as found in nature.

Recombinant mouse Wnt3a is commercially available (e.g., from Millipore cat. no. GF 145 or R& D Systems cat. no. 1324- WN- 002). In addition, the Norrin ligand (Xu et al., (2004) Cell 116(6):883-95), which binds to Frizzled with high affinity, may be used to activate the Wnt signalling pathway. The R-spondin protein (Kazanskaya et al (2004) Dev Cell. 7(4):525-34 and Kim et al., (2005) Science 309(5738): 1256-9) also binds to the Frizzled receptors and may similarly be used, alone or in combination with other Wnt pathway agonists. The Wnt signalling pathway may be activated by exposing the cell to a medium containing the Wnt ligand.

The pathway may be activated by increasing the expression of Wnt ligands. For example, activation of the Wnt signalling pathway by Wnt over-expression is described in detail in WO 2004/0014209.

The pathway may also be activated by down-regulation of any antagonist or negative regulator or component of that pathway, for example, glycogen synthase kinase (GSK) 3 beta or alpha, whether by inhibiting enzymatic activity or lowering protein concentration. Blocking negative regulators of Wnt signaling, such as Axin and APC through use for example of RNAi will also activate the Wnt pathway.

In some embodiments, the kinase activity that is inhibited is GSK-3 beta kinase activity. GSK-3 beta activity may be inhibited by inhibiting the enzymatic activity of GSK-3 beta, for example by use of chemical inhibitors or antagonists, which may be competitive or non -competitive. Such inhibitors may include kinase inhibitors. GSK-3 beta activity may also be down- regulated by down-regulating the expression of GSK-3 beta protein, such as by use of antisense RNA, or RNAi, or siRNA or by inhibiting the conversion of inactive forms of GSK-3 beta to active forms, or by increasing the rate of degradation of GSK-3 beta. The methods and compositions described here may also employ loss of function and dominant negative mutations in GSK-3 beta, described for example in Hedgepeth et al. (1997) Dev Biol. 185(1):82-91.

Wnt signalling may be activated via up-regulation, for example, over- expression, of FRAT1, a negative regulator of GSK-3 (see Crowder and

Freeman (2000), J. Biol. Chem. 275, 34266-34271 and Culbert, et al., (2001) FEBS Lett. 507, 288-294.

A number of chemical inhibitors of GSK-3 beta activity are known in the art, as described in for example US6,441,053. Methods for identifying inhibitors of GSK-3.beta activity are also set out in that document. In one embodiment, the inhibitor of GSK-3 beta activity is the aminopyrimidine CHIR99021, which is the most selective inhibitor of GSK-3 beta reported to date (Ring D. B. et al., Diabetes, 52:588-595 (2003) and Hall R. K. et al., J Biol Chem, 275:30169- 30175 (2000)). Other non-limiting examples of inhibitor of GSK-3 beta activity are alsterpaullone, kenpaullone, SB214763, and SB415286. The chemical inhibitors of GSK-3 beta may comprise indirubins, for example, Tyrian purple indirubins, as described in detail in Meijer et al., (2003) Chemistry & Biology, Vol. 10, 1255-1266. The GSK-3.beta. inhibitor may comprise 6- bromoindirubin-3'-oxime (BIO). Other inhibitors may include 1- Azakenpaullone, FRATtide, GSK-3b Inhibitor VII, GSK-3b Inhibitor XI, GSK- 3b Inhibitor I, GSK-3b Inhibitor II, GSK-3b Inhibitor III, GSK-3 Inhibitor IX, GSK-3 Inhibitor X, GSK-3 Inhibitor XIII, GSK-3 Inhibitor XIV, GSK-3b

Inhibitor VI, GSK-3b Inhibitor XII, TWS119, GSK-3b Inhibitor VIII, GSK-3b Peptide Inhibitor, GSK-3b Peptide Inhibitor, Indirubin-3'-monoxime, which are widely available, for example from Calbiochem. Many potent and selective small molecule inhibitors of GSK3 have been identified. Exemplary inhibitors include (2'Z,3'E)-6-Bromoindirubin-3'-oxime; N-(4-Methoxybenzyl)- N'-(5-nitro-l ,3-thiazol-2-yl)urea; 3-(2.4- DichlorophenylV4-( 1 -methyl- 1 H-indol-3-vlV 1 H-pyrrole-2.5-dione; 3-[(3- Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-lH-pyrrol-2,5-dione; 4- Benzyl-2-methyl-l ,2,4-thiadiazolidine-3,5-dione; Lithium chloride; sodium valproate; GSK3 inhibitor II; casein kinase 1 (CK1) inhibitor - such as D4476, IC261 , and CKI-7; and cyclin dependent kinase (CDK) inhibitors. Activation of the Wnt/6-catenin signalling pathway may also be achieved by activation of beta-catenin which leads to an increase in beta-catenin activity in the cell, such as accumulation of active (non-phosphorylated) beta-catenin in the cell. The activator may be an RNAi agent.

Exposure to fibroblast growth factor (FGF) activates Akt and thus inhibits GSK-3 beta, as described in Hashimoto et al., (2002), J. Biol. Chem. 277, 2985- 32991. FGF may therefore also acts as an activator of Wnt signalling.

In one embodiment, the activator of the Wnt/ -catenin signalling pathway is Wnt3a, preferably in combination with FGF2.

In the same sense as described above for Wnt3a also for FGF2 various alternatives are available. First of all, FGF2 maybe replaced by other FGF family members, such as FGF1, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGFll (FHFl), FGF 12 (FHF2), FGF 13 (FHF3), FGF 14 (FHF4), FGF 15, FGF 16, FGF 17, FGF18, FGF19, FGF20, FGF21, FGF22, or FGF23. Also, small segments of the native FGF2 protein may mimick the function of the intact protein (Ramaiah A. et al., Acta Derma Venereol (Stockholm), 1989, 69, 323-327).

It has further been described that peptide agonists, so called hexafins, that correspond to the 66-67 loop of the FGF2 molecule, can be used as an agonist for its function (Li, S. et al., Dev Neurobiol. 2009, 69(13):837-54). Further peptide agonists of FGF2 are mentioned in_US 2009/069233.

Example

Materials and Methods Purified Wnt3a and Wnt -inhibitors

Wnt3a was purified from cell culture medium conditioned by Drosophila S2 cells modified with a mouse Wnt3a expression vector, using affinity and gel filtration chromatography as described [1]. The purified protein was obtained as a solution of approximately 50 g/ml in PBS + 1% of 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS). Final concentration used during MSC culture was 250 ng/ml. Fz8CRD-IGg fusion protein (soluble Wnt-receptor) was produced as previously described [2] and used at final concentration of 2 g/ml. IWP2 (Wnt secretion-inhibitor) [3] was purchased from Stemgent. A stock solution of 2 mM IWP2 in dimethyl sulfoxide (DMSO) was prepared and used at a final concentration of 2 μΜ. This small molecule specifically inhibits the maturation of Wnt proteins by blocking the acyltransferase enzyme porcupine. Cell source and isolation of BMSC (pre -expansion)

Human bone marrow-derived mesenchymal stem cells (hBMSC) were obtained from femoral biopsies of 3 donors (age 50-78 years) undergoing total hip replacement, after undersigned informed consent and in accordance with the local ethical committee (MEC-2004-142). Cells from bone marrow aspirate were seeded at the density of 45-55 x 106 nucleated cells/cm2 in alpha-MEM (Gibco), supplemented with 10% FCS, 1 ng/ml FGF2 (AbD Serotec), 25 g/ml ascorbic acid-2 -phosphate (Sigma-Aldrich), 1.5 g/ml fungizone and 50 g/ml gentamicin. hBMSC were isolated by their ability to adhere to the plastic flasks and, after 24 hours, nonadherent cells were washed out. Cells were allowed to proliferate in standard conditions (5% C02 at 37°C) and media were renewed every 48 hrs.

Expansion phase

When hBMSC neared confluence, they were trypsinized using 0.05% trypsin and replated at a density of 2300 cells/cm2 in seven different expansion media: (1) alpha-MEM + 10%FCS + 25 g/ml ascorbic acid-2 -phosphate (FCS); (2) FCS + lng/ml FGF2 (FGF); (3) FCS+Wnt3a (FCS+Wnt); (4) FCS+FGF+Wnt3a (FGF+Wnt); (5) FCS+FGF+Wnt3a+IWP2 (FGF+Wnt+IWP2); (6)

FCS+FGF+IWP2 (FGF+IWP2); (7) FCS+FGF+Fz8CRD (FGF+FZ). At this stage, media were renewed every 24 hrs and after one passage cells were harvested, counted and used to assay the chondrogenic potential.

In the conditions without Wnt3a or IWP2, equal volumes of PBS + 1% CHAPS solution or DMSO were added to verify that the observed effects were due to the active compound (Wnt3a or IWP2).

Chondrogenic differentiation

After expansion, hBMSC were harvested and centrifuged at 1200 rpm for 8 minutes to obtain pellets of 0.2 x 106 cells. Chondrogenic differentiation was induced by culturing the cells for 35 days in chondrogenic medium, consisting of DMEM-high glucose Glutamax+ (Gibco), 1: 100 Insuline Transferring Selenic acid (ITS+; B&D Bioscience), 40 pg/ml L-proline (Sigma-Aldrich), lmM sodium pyruvate (Gibco), ΙΟΟηΜ dexamethasone (Sigma-Aldrich),

Transforming Growth Factor-6l (TGF-61; R&D Systems), 1.5 pg/ml fungizone and 50 pg/ml gentamicin. Medium was renewed twice a week.

Evaluation of chondrogenic differentiation: transcript analysis

After 21 and 35 day in chondrogenic medium, 3 pellet/condition/donor were used for gene expression analysis. Pellets were manually homogenized in RNA-BeeTM (TEL-TEST) and RNA was extracted by adding 20% chloroform. RNAs were purified with RNeasyMicro Kits (Qiagen) and 1 pg of total RNA was reverse-transcribed into complementary DNA (cDNA) using RevertAid First Strand cDNA synthesis Kit (MBI Fermentas). Polymerase chain reactions were performed with TaqMan Universal PCR MasterMix (Applied Biosystems) using an ABI PRISM 7000 apparatus. Expression level of aggrecan (AGC), total collagen type-II (COL2) and collagen type-2b (COL2b) were studied as markers for chondrogenic differentiation. Transcript levels of matrix metalloproteinase-13 (MMP- 13), collagen type-X (COL 10) and alkaline phosphatise (ALP) were detected to assay the (undesired) hypertrophic differentiation. Primers/probe sequences and amplification settings were previously reported [4-7]. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that was stably expressed across sample conditions. Relative expression was calculated according to the 2~ Ct formula. Evaluation of chondrogenic differentiation: (immuno)histochemistry

For (immuno)histochemistry pellets were fixed in 4% formalin after 35 days in chondrogenic medium. Once paraffin-embedded, at least two sections (6 pm) of each sample were stained with Thionine to detect glycosaminoglycans (GAGs) and with immunohistochemistry to detect collagen type-II (COL2). Antigen retrieval was performed with 0.1% pronase (Sigma-Aldrich) in PBS for 30 minutes followed by 10 mg/mL hyaluronidase 1 (Sigma-Aldrich) in PBS for 30 minutes. Then, sections were incubated 2 hrs with primary antibody for COL2 (Π/Π6Β3; Developmental Studies Hybridoma Bank, University of Iowa).

Alkaline phosphatase-labeled secondary antibody was used in combination with Neu Fuchsine substrate (Chroma) resulting in a red staining. An isotype IgGl monoclonal antibody was used as negative control.

Results Cells cultured with Wnt3a, with or without FGF2, had smaller size and polygonal shape. Similarly, FGF2 or Wnt3a enhanced the proliferation rate compared to FCS-only while the combination FGF2 and Wnt3a synergistically promoted cell growth.

Combination of FGF2 and Wnt3a displayed the best chondrogenic potential (Collagen-2 and Glycosaminoglycans), while FCS+Wnt3a or FCS-only failed to promote chondro gene sis. Interestingly, condition FCS+Wnt3a displayed also the lowest osteogenic and adipogenic capacity, respectively detected by

VonKossa or Oil-Red staining. No hypertrophic differentiation was observed in cells treated with FGF2 + Wnt3a.

Our data show that during expansion Wnt3a and FGF signals synergistically promote proliferation and chondrogenic potential of BMSC.

References

1. Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448-52.

2. Hsieh JC, Rattner A, Smallwood PM, Nathans J. Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein. Proc Natl Acad Sci U S A 1999;96:3546-51.

3. Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat Chem Biol 2009;5: 100-7.

4. Clockaerts S, Bastiaansen-Jenniskens YM, Feijt C, Verhaar JA, Somville J, De Clerck LS, et al.

Peroxisome proliferator activated receptor alpha activation decreases inflammatory and destructive responses in osteoarthritic cartilage. Osteoarthritis Cartilage 2011 ; 19:895-902.

5. McAlinden A, Johnstone B, Kollar J, Kazmi N, Hering TM. Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation. Matrix Biol 2008;27:254-66.

6. van der Windt AE, Jahr H, Farrell E, Verhaar JA, Weinans H, van Osch GJ. Calcineurin inhibitors promote chondrogenic marker expression of dedifferentiated human adult chondrocytes via stimulation of endogenous TGFbetal production. Tissue Eng Part A 2010;16: 1-10.

7. Farrell E, van der Jagt OP, Koevoet W, Kops N, van Manen CJ, Hellingman CA, et al.

Chondrogenic priming of human bone marrow stromal cells: a better route to bone repair? Tissue Eng Part C Methods 2009;15:285-95.

Claims

Claims
A method for fast expansion of progenitor cells comprising culturing said cells in the presence of fibroblast growth factor 2 (FGF2) and Wnt3a.
The method according to claim 1, wherein the progenitor cell is a mesenchymal stem cell, preferably a bone-marrow-derived
mesenchymal stem cell.
The method according to claim 1, wherein the cells are cultured in vitro.
The method according to claim 1, wherein the cells are implanted in vivo.
A culture medium comprising components for culturing progenitor cells, further comprising FGF2 and Wnt3a.
The culture medium according to claim 5, wherein the FGF2 is present in a concentration of about 0.1 to about 100 ng/ml, preferably about 0.1 to about 10 ng/ml, more preferably about 0.5 to about 2 ng/ml, most preferably at about 1 ng/ml.
The culture medium according to claim 5, wherein the Wnt3a is present in a concentration of about 1 to about 1000 ng/ml, preferably about 10 to about 500 ng/ml, more preferably about 100 to about 500 ng/ml, most preferably about 250 ng/ml. The culture medium of claim 5, where said culture medium is expansion culture medium.
A matrix for culturing and/or production of chondrocytes comprising FGF2 and Wnt3a.
The matrix of claim 9 which has been seeded with mesenchymal stem cells.
The method of claim 1, wherein the cells are cultured in a matrix as defined in claim 9.
PCT/NL2013/050340 2012-05-03 2013-05-03 Culturing of mesenchymal stem cells WO2013165252A1 (en)

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