WO2018114832A1 - Methods and media for preparing cells for healing bone and joint disorders - Google Patents

Abstract

The invention relates to in vitro methods of inducing hypertrophy in chondrogenic cells comprising the step of incubating aggregates of chondrogenic cells in a medium comprising BMP4 or BMP2, a Wnt agonist and a thyroid hormone. The invention further relates to the medical use of cells obtained by this method to repair bone and joint disorders.

Description

METHODS AND MEDIA FOR PREPARING CELLS FOR HEALING BONE AND JOINT DISORDERS

FIELD OF THE INVENTION

The application provides novel cell based methods for making cellular compositions that allow healing of bone, cartilage and joint disorders. The application further provides pharmaceutical compositions comprising said cellular compositions and method of treatments using said cellular compositions. The application further relates to said cellular compositions made by said methods and to their use in the treatment of bone disorders, cartilage disorders and joint disorders. The current invention further relates to method of treatments of bone and joint disorders.

BACKGROUND OF THE INVENTION

Bone fracture healing is a highly orchestrated process that involves many steps. Immediately after fracture, inflammatory cells infiltrate in the hematoma and start to secrete pro-inflammatory cytokines such as interleukin-1 beta (IL-Ιβ) and interleukin-6 to recruit macrophages for debris clean up and tissue removal. Local mesenchymal progenitors proliferate and start to differentiate towards chondrocytes. These cells secrete a cartilaginous matrix and form the soft callus within the fracture site. Chondrocytes gradually mature and become hypertrophic. Matrix vesicles are secreted from (pre)hypertrophic chondrocytes. On the surface of these vesicles, alkaline phosphatase initiates accumulation of phosphate ions, whereas annexins bind calcium. The accumulation of both ions causes precipitation towards hydroxyapatite. These crystals continue to grow and will initially cause the vesicles to burst. The released crystals continue to grow and will bind to collagens to induce callus tissue mineralization. Secretion of soluble signals such as vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs), by the hypertrophic cells, stimulates migration of osteogenic cells and blood vessels. Coupled with this, chondroclasts invade into the soft callus and start to resorb the mineralized cartilaginous matrix. The balanced activities of matrix resorption and osteoblast mediated bone tissue formation leads to a replacement of the soft callus by the bony hard callus. Further remodelling of this tissue leads to restoration of both morphological appearance as well as mechanical properties of the original bone. Successful bone healing is dependent on a pleiotropic set of tissue intermediates yet, it is generally accepted that soft callus formation is one of the most important steps during the healing process. The soft callus is the first tissue intermediate that allows bone bridging, provides the initial mechanical stabilization, is responsible for bone tissue revascularization and recruits osteoprogenitors. It is therefore not surprising that reparative strategies are undergoing a paradigm shift whereby in vitro cartilage like tissue intermediates are assembled to serve as a trigger and template for bone formation. The different processes present in bone fracture repair appear quite similar to those observed during embryonic endochondral bone development and potentially explain why bone tissue is one of the only tissues that heals without scar formation upon damage.

Increased understanding in the cellular and molecular processes of bone fracture healing has led to significant advances in regenerative bone biology.

Craft et al. (2015) Nat Biotechnol. 33, 638-645 and WO2014161075 provide methods to produce chondrocytes from human pluripotent stem cells and indicate that BMP-4 treatments shows signs of mineralization and hypertrophy.

Yamashita et al. (2015) Stem Cell Reports. 4, 404-418 describe the generation of hyaline cartilaginous tissue from human iPSCs.

Scotti et al. (2013) Proc Natl Acad Sci USA. 110, 3997-4002 disclose that engineered hypertrophic cartilage requires IL-Ιβ to be efficiently remodelled into bone and bone marrow upon subcutaneous implantation.

However, no conclusive therapies have been established for the healing of critical size defects. Currently applied approaches include bone transport and transplantation of autologous viable bone grafts into the defect site. Despite bone formation, complications such as refracture of the newly formed bone segment, failure of distraction osteogenesis, deformity of the newly formed bone column and non-union at the docking site have been reported with bone transport. While bone graft transplantation techniques are limited by the availability of these grafts. Alternative strategies are currently being developed, however many of these suffer from unpredictable outcomes or require the addition of supraphysiological doses of growth factors e.g. bone morphogenetic protein-2 (BMP2), thus increasing the risk of potential oncogenic cell transformation. The yearly accumulation of (unsuccessfully treated) patients suffering from pseudoarthrosis urges the development of a more robust and effective therapy for healing critical size defects. Accordingly, there remains a need for safer and better methods to treat bone, cartilage and joint disorders. SUMMARY OF THE INVENTION

The present invention provides a novel approach for healing critical size osteochondral and especially bone defects through the use of human pluripotent stem cells (hPSCs). Upon mesodermal induction and subsequent chondrogenic differentiation, human pluripotent stem cells condense and form glycosaminoglycan rich nodules. Subsequent suspension culture leads to gradual maturation of these nodules into safranin-o and collagen type II rich mature cartilaginous aggregates. Following ectopic implantation, the aggregates remain cartilaginous and display hyaline cartilage like tissue characteristics. Upon hypertrophic stimulation, the cartilage nodules mature and mineralize, however no bone formation can be detected following ectopic and orthotopic implantation. Interestingly, when treated with IL-Ιβ, the aggregates progressively become more mineralized which allows bone bridging within 4 weeks of implantation. Furthermore, newly formed bone tissue can be detected. These results demonstrate that soft callus like tissue, if "primed and conditioned" appropriately, can be generated from human pluripotent stem cells and that these are capable of inducing in vivo bone formation. Our strategy emphasizes the promise of pluripotent stem cells for the creation of functional skeletal tissue intermediates that are capable of healing critical size long bone defects.

The present invention is based on the unexpected finding that certain treatment of cells, including specific culturing in serum-free conditions, results in cell-based compositions with improved in vivo properties and capacities. These treatments or specific culture conditions or combinations not being suggested by the prior art, and these cellular compositions show unexpected biological properties, in particular have significant capacities in the treatment of especially bone disorders, as well as osteochondral joint disorders. The improved biological properties relate to an improved in vivo effect, compared to untreated or not pre-conditioned cells in cellular compositions or any other cellular composition that is currently known. The methods, uses and compounds of the present invention are summarised in the following statements:

1. An in vitro method of inducing hypertrophy in chondrogenic cells comprising the step of incubating said chondrogenic cells in a medium comprising :

- BMP-4 (Bone Morphogenetic Protein 4) or BMP-2 (Bone Morphogenetic Protein 4),

- a Wnt agonist, and

-a thyroid hormone. 2. The method according to statement 1, wherein the chondrogenic cells are aggregates of chondrogenic cells.

3. The method according to statement 1 or 2, wherein the aggregates have a size of between 25 to 1000 cells/aggregate.

4. The method according to any one of statements 1 to 3, wherein the chrondrogenic cells Sox positive cells, or wherein the chondrogenic cells are safranin O and col II positive.

5. The method according to any one of statements 1 to 4, comprising the step of incubating said chondrogenic cells in a medium comprising BMP4 (Bone Morphogenetic Protein 4), a Wnt agonist and a thyroid hormone.

6. The method according to any one of statements 1 to 5, wherein the Wnt agonist is a GSK-3 inhibitor.

7. The method according to any one of statements 1 to 6, wherein the Wnt agonist is BIO (6-bromoindirubin-3'-oxime).

8. The method according any one of statements 1 to 7, wherein the thyroid hormone is T3 (3,3,5-Triiodo-L-thyronine).

9. The method according any one of statements 1 to 8, wherein the medium comprises BMP4 and BIO and T3.

10. The method according to any one of statements 1 to 9, wherein the incubation of said chondrogenic cells in said medium is performed for between 3 to 4 weeks.

11. The method according to any one of statements 1 to 10, wherein the incubation is followed by an further incubation in a medium comprising BMP-4 or BMP-2, a Wnt agonist (typically canonical Wnt agonist) and a thyroid hormone and a compound selected from the group consisting of ILIA, IL1 β, IL-6 and TNF.

12. The method according to statement 11, wherein the further incubation is performed in a medium comprising BMP-4, a Wnt agonist (typically canonical Wnt agonist) and a thyroid hormone and IL1 β.

13. The method according to statement 11 or 12, wherein the further incubation is performed for about 10 days.

14. The method according to any one of statements 1 to 13, wherein the chondrogenic cells are obtained by differentiation of pluripotent stem cells, such as iPSC.

15. Use of a combination of BMP-4 or BMP-2, a Wnt agonist (typically canonical Wnt agonist) and a thyroid hormone for inducing hypertrophy in chondrogenic cells. 16. The use according to statement 15, using a combination of BMP4, BIO and T3.

17. A cell culture medium for inducing hypertrophy in chondrogenic cells comprising BMP-4 or BMP-2, a Wnt agonist (typically canonical Wnt agonist) and a thyroid hormone.

18. The cell culture medium according to statement 17, comprising BMP-4, a Wnt agonist (typically canonical Wnt agonist) and a thyroid hormone.

19. The cell culture medium according to statement 17 or 18, which comprises about 0, 1 μΜ to about 10 μΜ thyroid hormone, more preferably about 1 μΜ thyroid hormone, and about 0,01 μΜ to about 1 μΜ canonical Wnt agonist, more preferably about 0,1 μΜ canonical Wnt agonist.

20. The cell culture medium according to statement 20, wherein the thyroid hormone is T3 and the canonical Wnt agonist is BIO.

21. The cell culture medium according to any one of statements 17 to 20, further comprising IL-1B.

22. The serum free cell culture medium according to any one of statements 17 to 21, comprising one or more basal cell culture media, insulin, transferrin, selenium, a-ketoglutarate, ceruloplasmin, cholesterol, phosphatidyl ethanolamine, a-tocopherol, reduced glutathione, taurine and ascorbic acid.

23. The cell culture medium according to any one of statements 17 to 22, further comprising ascorbic acid, dexamethasone and β-glycerophosphate.

24. The cell culture medium according to any one of statements 17 to 23, which is a serum free medium comprising PDGF.

25. The cell culture medium according to any one of statements 17 to 24, comprising PDGF at a concentration between 5ng/ml to 500 ng/ml, typically between about bout 50 ng/ml to 500 ng/ml, and BMP4 at a concentration between lOng/ml and 1000 ng/ml, typically at a concentration of about 100 ng/ml.

26. Cells obtained by the method of any one of statements 1 to 14, for use in treating bone or joint defects.

27. The cells according to statement 26, for use in treating a disorder selected from the group consisting of a bone fracture, a non-healing bone defect, an osteochondral defect, a damaged joint surface, a subchondral defect or a metabolic bone disease.

28. The cells according to statement 26, in admixture with a biocompatible carrier, for use in treating a disorder according to statement 26 or 27. 29. A method of treating a bone defect, comprising the step of administering cells obtained by the method of any one of statements 1 to 14, to the bone or joint defect.

30. The method according to statement 29, wherein the cells administered together with a biocompatible carrier.

Other embodiments, objects, features and advantages will be set forth in the detailed description of the embodiments that follows. The summary above is to be considered as a brief and general overview of some of the embodiments disclosed herein, is provided solely for the benefit and convenience of the reader, and is not intended to limit in any manner the scope encompassed by the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Collagen type II expression analysis. At day 56, human pluripotent stem cell derived aggregates expressed similar levels of collagen type II as compared to human articular chondrocytes.

Figure 2. Chondrogenic differentiation of human pluripotent stem cells. Following mesoderm induction, a decrease in pluripotency markers Nanog, Oct3/4 and Sox2 was detected, while the primitive streak markers Brachyury, MIXL1 and KDR transiently upregulated after 36 hours of differentiation. Upon chondrogenic differentiation, an increase in Col2Al and ACAN was detected. Although Sox9 was upregulated during mesoderm induction, its expression remained steady during differentiation (A). Glycosaminoglycan rich nodules were detected on day 14 (B). These nodules progressively matured into safranin-o (C) and collagen type II (D) positive aggregates. Statistical significance is represented as follow: *p<0.05, **p<0.01, ***p<0.001, and ****p < 0.0001.

Figure 3. Hypertrophic differentiation of cartilaginous aggregates. Gene expression levels of Sox9, Runx2, Col2Al and CollOAl were evaluated. Upon BBT3 (BMP4 + BIO +T3) stimulation, a trend towards decrease in Sox9 and Col2Al was observed, while Col lOAl was upregulated. The CollOAl to Col2Al ratio indicate that BBT3 (BMP4 + BIO +T3) treated samples underwent hypertrophic differentiation. Statistical significance is represented as follow: *p<0.05.

Figure 4. IL-Ιβ treatment of BBT3 (BMP4 + BIO +T3) treated samples. Upon IL- 1β treatment a trend towards decrease of chondromodulin was detected while IL- 1β upregulated VEGF and MMP13.

In the figures, the values in the Y-axis show expression values compared to the house keeping gene ACTB using the 2-ACT method. DETAILED DESCRIPTION OF THE INVENTION AND DEFINITIONS

The present invention will be described with respect to particular embodiments but the invention is not limited thereto only by the claims. Any reference signs in the claims shall not be construed as limiting the scope thereof.

Disclosed in this application are :

Methods for producing a cellular composition with in vivo bone forming potential, comprising the steps of:

a) culturing iPSC in a serum free medium;

a) a mesoderm induction phase, comprising culturing the cells of step (a) in a serum free medium comprising a mesoderm specifying cocktail; and

b) a chondrocyte induction phase; comprising culturing the cells of step (b) in a serum free medium, comprising a chondrocyte specifying cocktail, the cocktail comprising PDGF.

These methods can further comprise a step (d) wherein the cells at the end of step (c) are further cultured in a serum free medium comprising a hypertrophy specifying cocktail, the cocktail further comprising PDGF and BMP4.

These methods can further comprise a step (e) wherein the cells at the end of step (d) are further cultured in a serum free medium comprising at least one factor selected from the group IL-1, IL-6, and TNF.

Typically the factor in step (e) is IL-Ιβ.

The mesoderm induction phase (b) in these methods can comprises two steps: bl) wherein the cells of step (a) are further cultured in a serum free medium comprising a cocktail which further comprises a Wnt agonist (such as CHIR99021) and hbFGF; and

b2) wherein the cells at the end of step (bl) are further cultured in a serum free medium comprising a cocktail which further comprises Retinoic acid and hbFGF . Herein in step (bl) the Wnt agonist can be used in a final concentration in the serum free medium of about 0,8 μΜ to about 80 μΜ and hbFGF can be used in a final concentration in the serum free medium of about 2ng/ml to about 200ng/ml; and in step (b2) Retinoic acid can be used in a final concentration in the serum free medium of about 0, 1 μΜ to about 10 μΜ, and hbFGF can be used in a final concentration in the serum free medium of about 0,8ng/ml to about 80ng/ml. More specifically in step (bl) the Wnt agonist can be used in a final concentration in the serum free medium of about 8 μΜ and hbFGF can be used in a final concentration in the serum free medium of about 20ng/ml; and in step (b2) Retinoic acid can be used in a final concentration in the serum free medium of about 1 μΜ, and hbFGF can be used in a final concentration in the serum free medium of about 8ng/ml.

More specifically, PDGF is used in step (c) in a final concentration in the serum free medium of about 5ng/ml to about 500 ng/ml, typically in a final concentration in the serum free medium of about 50ng/ml.

The medium used in step (c) can further comprise the factors TGFpi, GDF5, BMP2, or ascorbic acid, and in addition can further comprise the factor hbFGF in a first period and wherein the cells are further cultured in the serum free medium without hbFGF for a second period.

TGFpi, GDF5 and BMP-2 can be used each in a final concentration in the serum free medium of about lng/ml to about 100 ng/ml, and ascorbic acid can be is used in a final concentration in the serum free medium of about 5 μg/ml to about 500 μg/ml; typically TGFpi, GDF5 and BMP-2 are all used in a final concentration in the serum free medium of about lOng/ml, and ascorbic acid is used in a final concentration in the serum free medium of about 50 μg/ml.

hbFGF can be used in a final concentration in the serum free medium of about lng/ml to about 100 ng/ml, typically of about lOng/ml.

PDGF used in step (d) can be used in a final concentration in the serum free medium of about 5ng/ml to about 500 ng/ml, and BMP4 is used in step (d) in a final concentration in the serum free medium of about lOng/ml to about 1000 ng/ml.

In specific versions of the methods PDGF is used in step (d) in a final concentration in the serum free medium of about 50ng/ml, and BMP4 is used in step (d) in a final concentration in the serum free medium of about lOOng/ml.

The cocktail in step (d) generally comprises a thyroid such as T3 (3,3,5-Triiodo-L- thyronine), a canonical Wnt agonist such as Bio (6-bromoindirubin-3'-oxime), and further ascorbic acid, dexamethasone and β-glycerophosphate.

The final concentration in the serum free medium of the factors in step (d) is typically about 1 μΜ T3 (3,3,5-Triiodo-L-thyronine), about 0, 1 μΜ canonical Wnt agonist, about 50 μg/ml ascorbic acid, about ΙΟΟηΜ dexamethasone is used in a final concentration in the serum free medium of about 5 μg/ml to about 500 μg/ml and about 7mM β-glycerophosphate.

In these methods IL-1, IL-6, and TNF in step (e) is generally used in a final concentration in the serum free medium of about 1 ng/ml to about 100 ng/ml, typically at a final concentration in the serum free medium of about 10 ng/ml. The serum free medium in step (e) may further comprise the factors ascorbic acid, dexamethasone and β-glycerophosphate, whereby generally the final concentration of the factors (e) in the serum medium is about 5 μς/ιτιΙ to about 500 μς/ιτιΙ Ascorbic acid, about InM to about ΙΟΟηΜ dexamethasone, and about 0,7mM to about 70mM β-glycerophosphate, typically the concentration of the factors (e) in the serum medium is about 50 μς/ιτιΙ Ascorbic acid, about ΙΟΟηΜ dexamethasone, and about 7mM β-glycerophosphate.

In these methods step (b) is generally performed for at least 24 hours and step (c) is generally performed for at least 2 weeks. Alternatively (b) is performed for about 2 days to about 1 week and step (c) is performed for about 2 weeks to about 10 week. Alternatively, step (bl) is performed for about 2 to about 3 days, and step (b2) is performed for about 2 to about 3 days. Alternatively, in step (c) the first period is about 1 to about 3 weeks and the second period is about 3 to about 9 weeks. Alternatively in step (c) the first period is about 2 weeks and the second period is about 6 weeks. Step (d) may be performed for about 1 week to about 6 weeks, such as about 3 weeks.

Step (e) may be performed for about 1 week to about 2 weeks, such as about 10 days.

In the above method methods a serum free medium may be used . Such medium may comprise two basal cell culture media in a ratio of about 1 : 1 (v/v) insulin, transferrin, selenium, a-ketoglutarate, ceruloplasmin, cholesterol, phosphatidyl ethanolamine, a-tocopherol acid succinate, reduced glutathione, Taurine, and L- ascorbic acid 2-sulphate.

The cellular composition obtainable by the method above have in vivo bone and/or cartilage forming potential. This composition may further comprise a biocompatible carrier, comprising for example collagen, calcium phosphate, carboxymethyl cellulose, hydrogel or combinations thereof.

The above cellular composition can be formulated into a pharmaceutical composition using pharmaceutically acceptable carrier, excipient or solution, and used as a medicament for use in the treatment of a bone or joint disorders, such as a bone fracture, a non-healing bone defect, an osteochondral defect or damaged joint surface, or a metabolic bone disease.

Any eukaryotic cells can be used in the initial step of culturing the stem cells, provided they are pluripotent stem cells, for example embryonic stem cells or reprogrammed somatic cells (iPSC) or partially reprogrammed somatic cells. Also pluripotent cells that are induced to mesoderm or are mesoderm derived can be used according to the methods of the present invention, starting right after the mesoderm induction phase, such as for Mesenchymal Stem Cells (MSCs). The entire method, including pre- or de-differentiation of such cells together with the proliferation and differentiation methods as described in detail in this invention, are contemplated in the present invention.

The invention is also directed to the cellular compositions produced by the methods of the present invention and to the use of the cellular compositions in the treatment of cartilage, joint or bone disorders, in particular bone fractures, more particularly non-union fractures (bone fractures that do not heal naturally).

The invention is also directed to pharmaceutical compositions comprising the cellular compositions produced by the methods of the present invention and to the use of pharmaceutical compositions in the treatment of joint or bone disorders, in particular bone fractures, more particularly non-union fractures (bone fractures that do not heal naturally). In certain embodiments of the present invention, the pharmaceutical compositions comprise further a biocompatible carrier and/or a pharmaceutically acceptable carrier, excipient or solution.

The present inventions relates to methods wherein chondrogenic cells are transformed into hypertrophic cells using a medium comprising a mixture of BMP- 4, an agonist of the canonical Wnt pathway and a thyroid hormone. Chondrogenic cells may be obtained via different methods. The present description describes methods wherein the chondrogenic cells are obtained from stem cells which are differentiated into the mesodermal lineage and further into chondrogenic cells. The invention is however equally applicable to chondrogenic cells obtained via other methods.

Disclosed are cellular composition comprising cells, wherein the cells are stem cells, more preferably mesenchymal cells, such as periosteum derived cells. Preferably the cells are of mammalian in particular human origin.

Herein disclosed are methods for producing a cellular composition, comprising the culturing of cells, wherein said starting cells are stem cells, more specifically iPSC In a preferred embodiment, said cells are of mammalian in particular human origin.

Herein disclosed are methods for producing a cellular composition, more specifically a cellular composition as further described herein. In general, methods for producing a cellular composition with in vivo bone and/or cartilage forming potential are disclosed, the methods comprise the steps of:

a) culturing iPSC in a medium, more preferably serum free medium;

b) a mesoderm induction phase, comprising culturing the cells of step (a) in a medium, more preferably serum free medium comprising a mesoderm specifying cocktail; and

c) a chondrocyte induction phase comprising culturing the cells of step (b) in a medium, more preferably serum free medium, comprising a chondrocyte specifying cocktail, said cocktail comprising PDGF in the serum free condition. More specially said initial cells are iPSC or human IPSC. In another embodiment, said initial cells are any type of cells or that can be differentiated towards the mesodermal lineage, such as mesenchymal cells, or periosteum derived cells, more specifically said cells are mammalian cells and even more specific, said cells are of human origin.

The methods may further comprise a step (d) wherein the cells at the end of step (c) are further cultured in a medium, more preferably serum free medium comprising a hypertrophy specifying cocktail, said cocktail further comprising PDGF and BMP4.

The methods may further comprises a step (e) wherein the cells at the end of step (d) are further cultured in a medium, more preferably serum free medium comprising at least one factor selected from the group IL-1, including ILIA and

IL1 β, IL-6, and TNF. Typically the factor is IL-Ιβ.

The mesoderm induction phase (b) may comprise two steps:

bl) wherein the cells of step (a) are further cultured in a medium, more preferably serum free medium comprising a cocktail which further comprises hbFGF and a

Wnt agonist, typically CHIR99021 ; and

b2) wherein the cells at the end of step (bl) are further cultured in a medium, more preferably serum free medium comprising a different cocktail compared to the cocktail in step (bl), said cocktail now comprises Retinoic acid and hbFGF. The Wnt agonist in step bl is typically a GSK3 inhibitor such as BIO (6- BromoIndirubin-3'-Oxime) (commercially available form e.g. Axon Medchem). The BIO Wnt agonist [CAS 667463-62-9]] has the chemical structure as depicted below in formula I :

The Wnt agonist can be used in a final concentration in the medium, more preferably serum free medium of about 0,8 μΜ to about 80 μΜ, more preferably of about 8 μΜ and said hbFGF is used in a final concentration in the medium, more preferably serum free medium of about 2ng/ml to about 200ng/ml, more preferably of about 20 ng/ml, and in step (b2) said Retinoic acid is used in a final concentration in the medium, more preferably serum free medium of about 0,1 μΜ to about 10 μΜ, more preferably of about 1 μΜ, and said hbFGF is used in a final concentration in the medium, more preferably serum free medium of about 0,8ng/ml to about 80ng/ml, more preferably of about 8 ng/ml.

PDGF is used in step (c) can be in a final concentration in the medium, more preferably serum free medium of about 5ng/ml to about 500 ng/ml, more preferably of about 50 ng/ml.

The cocktail in step (c) can further comprise the factors TGFpi, GDF5, BMP2, and ascorbic acid. Typically TGFpi, GDF5 and BMP-2 are all used in a final concentration in the medium, more preferably serum free medium of about lng/ml to about 100 ng/ml, more preferably of about 10 ng/ml, and said ascorbic acid is used in a final concentration in the medium, more preferably serum free medium of about 5 μg/ml to about 500 μg/ml, more preferably of about 50 μg/ml. More specifically the cocktail in step (c) further comprises the factor hbFGF in a first period and wherein the cells are further cultured in said medium, more preferably serum free medium without hbFGF for a second period, and said hbFGF is used in a final concentration in the medium, more preferably serum free medium of about lng/ml to about 100 ng/ml in said first period, even more preferably said concentration of hbFGF is about lOng/ml.

In step (d) said PDGF may be used in a final concentration in the medium, more preferably serum free medium of about 5ng/ml to about 500 ng/ml, more preferably of about 50 ng/ml, and said BMP4 is used in a final concentration in the medium, more preferably serum free medium of about lOng/ml to about 1000 ng/ml, more preferably of about 100 ng/ml.

In step (d) the cocktail in step (d) may further comprise the factors 3,3,5-Triiodo- L-thyronine (T3), a canonical Wnt agonist, more preferably BIO (6- BromoIndirubin-3'-Oxime), ascorbic acid, dexamethasone and β- glycerophosphate. The final concentration in the medium, more preferably serum free medium of said factors in step (d) can be about 0,1 μΜ to about 10 μΜ T3 (3,3,5-triiodo-l-thyronine), more preferably about 1 μΜ T3 (3,3,5-triiodo-l- thyronine), about 0,01 μΜ to about 1 μΜ canonical Wnt agonist, more preferably about 0, 1 μΜ canonical Wnt agonist, about 5 to about 500 μg/ml said ascorbic acid, more preferably about 50 μg/ml said ascorbic acid, about ΙΟηΜ to about ΙΟΟΟηΜ dexamethasone, more preferably about ΙΟΟηΜ dexamethasone,, and about 0,7mM to about 70 mM β-glycerophosphate, more preferably about 7mM - β-glycerophosphate.

The present invention is illustrated with embodiments wherein BMP-4, BIO and T3 are used.

Apart from BMP-2, which is the preferred embodiment, also the use of BMP4 is envisaged, since both proteins bind to the same receptor and are phylogenetically related. Equally, mutants and truncated forms of BPM-2 and of BMP-4, which retain binding and activation of its receptor are suitable in the methods of the present invention. Specific embodiments of the methods of the invention relate to the use of both BMP-2 and BMP4. Apart from BIO other Wnt agonists are envisaged in the present invention such as WAY-316606 (SFRP Inhibitor), (hetero)arylpyrimidines, IQ1 (PP2A activator) QS11 (ARFGAP1 activator), SB-216763 (GSK3 inhibitor), LY2090314 (GSK3 inhibitor), DCA (beta-catenin activator) and 2-amino-4-[3,4-(methylene- dioxy)benzyl-amino]-6-(3-methoxyphenyl) pyrimidine.

Apart from T3 other natural or synthetic thyroid hormone are envisaged such as T4, levothyroxine and liothyronine.

The factor in step (e) can be used in a final concentration in the medium, more preferably serum free medium of about 1 ng/ml to about 100 ng/ml, more preferably about 10 ng/ml.

The medium, more preferably serum free medium in step (e) may further comprise the factors ascorbic acid, dexamethasone and β-glycerophosphate. The final concentration of said factors (e) in the serum medium is typically about 5 μg/ml to about 500 μg/ml ascorbic acid, more preferably about 50 μg/ml, about ΙΟηΜ to about ΙΟΟΟηΜ dexamethasone, more preferably 100 nM dexamethasone, and about 0,7mM to about 70mM β-glycerophosphate, more preferably about 7 mM β- glycerophosphate. The mesoderm induction step (b) may be performed for at least 24 hours, or for about 2 days to about one week. Step (bl) may be performed for about 2 to about 3 days, and step (b2) may be performed for about 2 to about 3 days.

The chondrocyte induction step (c) may be performed for at least 2 weeks, or may be performed for about 2 weeks to about 10 weeks. The said first period in step (c) may be about 1 to about 3 weeks, preferably about 2 weeks, and the second period may be about 3 to about 9 weeks, more preferably about 6 weeks.

Step (d) may be performed for about 1 week to about 6 weeks, more preferably for about 3 weeks.

Step (e) is performed for about 1 week to about 2 weeks, more preferably for about 10 days.

The culturing step (c) may be a culturing step in aggregates, meaning that said cells are further cultured under conditions that form aggregates, meaning that they can form spontaneously or by manipulation (such as the method described in this invention, in the examples) aggregates of 1 to 5000 cells/aggregate, 25 to 1000 cells/aggregate, 50 to 500 cells/aggregate, 20 to 250 cells/aggregate, these aggregates have a cell density of about 50, 100, or 250 cells/aggregate. Other methods of generating aggregates are well known to the skilled person. An example of such an aggregation method is described in Moreira Teixeira et al. (2012) Eur Cell Mater. 523, 387-399.

A suitable medium used in the present invention is a serum free medium comprising : two basal cell culture media in a ratio of about 1 : 1 (v/v), such as Ham's F12 and DMEM, insulin, preferably at a concentration of about 6,25 μg/ml, transferrin, preferably at a concentration of about 6,25 μg/ml, selenium, preferably at a concentration of about 6,25 μg/ml, a-ketoglutarate, preferably at a concentration of about 10^~4 M, ceruloplasmin, preferably at a concentration of about 0,25U/ml, cholesterol, preferably at a concentration of about 5 μg/ml, phosphatidyl ethanolamine, preferably at a concentration of about 2 μg/ml, a- tocopherol acid succinate, preferably at a concentration of about 9 x 10^~4 M, reduced glutathione, preferably at a concentration of about 10 μg/ml, Taurine, preferably at a concentration of about 1,25 μg/ml, and L-ascorbic acid 2-sulphate, preferably at a concentration of about 50 μg/ml.

Typically said serum free medium is a mixture of two basal cell culture media in a ratio of about 1 : 1 (v/v), such as Ham's F12 and DMEM, which further contains insulin, preferably at a concentration of about 6,25 μg/ml, transferrin, preferably at a concentration of about 6,25 μg/ml, selenium, preferably at a concentration of about 6,25 μg/ml, a-ketoglutarate, preferably at a concentration of about 10^~4 M, ceruloplasmin, preferably at a concentration of about 0,25U/ml, cholesterol, preferably at a concentration of about 5 μς/ηηΙ, phosphatidyl ethanolamine, preferably at a concentration of about 2 μς/ηηΙ, α-tocopherol acid succinate, preferably at a concentration of about 9 x 10^"4 M, reduced glutathione, preferably at a concentration of about 10 μς/ηηΙ, taurine, preferably at a concentration of about 1,25 μς/ηηΙ, and L-ascorbic acid 2-sulphate, preferably at a concentration of about 50 μς/ιτιΙ.

The serum free medium can be a mixture of Ham's F12 and DMEM in a ratio of about 1 : 1 (v/v), which further contains the following compounds: insulin, preferably at a concentration of about 6,25 μς/ηηΙ, transferrin, preferably at a concentration of about 6,25 μς/ηηΙ, selenium, preferably at a concentration of about 6,25 μς/ηηΙ, α-ketoglutarate, preferably at a concentration of about 10^~4 M, ceruloplasmin, preferably at a concentration of about 0,25U/ml, cholesterol, preferably at a concentration of about 5 μς/ηηΙ, phosphatidyl ethanolamine, preferably at a concentration of about 2 μς/ηηΙ, α-tocopherol acid succinate, preferably at a concentration of about 9 x 10^~4 M, reduced glutathione, preferably at a concentration of about 10 μς/ηηΙ, taurine, preferably at a concentration of about 1,25 μς/ηηΙ, and L-ascorbic acid 2-sulphate, preferably at a concentration of about 50 μς/ιτιΙ.

The serum free medium as described hereinabove may be used in all steps of the describe methods. In specific versions of these methods the serum free medium as described hereinabove is used in all steps of the methods of this invention, except for the mesoderm induction step (b) wherein the serum free medium used in that specific step (b) is APEL Stemdiff medium and/or Essential 6 medium or the like.

Disclosed are cellular compositions with in vivo bone and/or cartilage forming potential produced by any of the methods herein described . The cellular compositions may further comprising a biocompatible carrier, comprising for example collagen, calcium phosphate, carboxymethyl cellulose, hydrogel or combinations thereof.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising : the cellular composition according to the present invention and a pharmaceutically acceptable carrier, excipient or solution.

One embodiment of the present invention relates the cellular composition of the present invention for use as a medicine. Another embodiment of the present invention relates to the pharmaceutical composition, comprising the cellular composition of the present invention, for use as a medicine.

More specific embodiments of the present invention relate to the cellular composition or the pharmaceutical composition of the present invention for use as a medicine for the treatment of a subject or animal having a bone disorder, a cartilage disorder or a joint disorder. In a more specific embodiment thereof, said bone, cartilage or joint disorder is a bone fracture, a non-healing bone defect, an osteochondral defect or damaged joint surface, or a metabolic bone disease. Typically said bone disorder is a non-healing bone defect. A specific embodiment of the present invention relates to the cellular composition or the pharmaceutical composition of the present invention for use as a medicine for the treatment of a subject or animal having a non-healing bone defect, more specifically said subject is a mammal and even more specifically said mammal is a human patient. For such bone defects, the preferred cellular composition of the present invention is the one that is generated by the method of the present invention, comprising all 5 steps (a) to (e). For other defects, more related to cartilage disorders, the preferred cellular composition of the present invention is the one that is generated by the method of the present invention, comprising the first 3 steps (a) to (c). Another aspect of the present invention relates to method of treatment of a bone, cartilage or joint disorder in an animal, comprising the administration to said animal of the cellular composition or the pharmaceutical composition of the present invention.

In specific embodiments of the present invention said animal is a mammal. In more specific embodiments of the present invention said animal is a human patient. In another embodiment the animal is a horse.

Alternatively, the present invention concerns the use of the cellular composition produced according to any one of the methods of this invention or a pharmaceutical composition according to the present invention for use in medicine, more particularly for use in the treatment of a subject with a bone disorder. A more particular embodiment thereof relates to the treatment of a subject with a non-healing bone defect, more particularly said subject is a human patient.

In certain preferred embodiments, the subject, patient or animal is a human, more particularly a human with a bone defect, such as a non-healing bone defect.

One embodiment of the present invention concerns a method of treatment comprising administering a therapeutically effective amount of the cells or the cellular composition produced according to any one of the methods of this invention, to a subject with a cartilage, joint or bone disorder, said bone disorder includes a bone fracture. A specific embodiment of the present invention relates to said method of treatment or the use of the cellular composition to treat a subject, preferably a human, with a bone disorder such as a non-healing bone defect.

In general, stem cells useful in this invention can be maintained and expanded in standard basal cell culture media that are available to and well-known in the art. Such media include, but are not limited to, Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 medium®, Eagle's Minimum Essential Medium®, F-12K medium®, Iscove's Modified Dulbecco's Medium® and RPMI-1640 medium®. Many media are also available as low-glucose formulations, with or without sodium pyruvate. The stem cells are typically cultured in a mixture of 2 of said basal cell culture media (about 1 : 1, v/v). An example of said two basal media are Ham's F12 and DMEM, but the use of other combinations of said basal cell culture media are also contemplated in the present invention.

Stem cells may be cultured in said basal cell culture media in serum-free conditions. The serum free cell culture conditions can comprise the addition of the following compounds: insulin, transferrin, selenium, a-ketoglutarate, ceruloplasmin, cholesterol, phosphatidyl ethanolamine, a-tocopherol acid succinate, reduced glutathione, taurine, and L-ascorbic acid 2-sulphate.

More specifically, said compounds are in about the following concentrations in said basal culture media : insulin : 6,25 μg/ml, transferrin : 6,25 μg/ml, selenium : 6,25 μg/ml, alpha-ketoglutarate: 10^"4 M, ceruloplasmin : 0,25U/ml, cholesterol : 5 g/ml, phosphatidyl ethanolamine: 2 μg/ml, a-tocopherol acid succinate: 9 x 10^"4 M, reduced glutathione: 10 μg/ml, taurine 1,25 μg/ml, and L-ascorbic acid 2- sulphate: 50 μg/ml.

A more preferred serum free medium, useful for the culturing of the stem cells of the present invention is "CDM", which consist of: Ham's F12 and DMEM (1 : 1) with the addition of: insulin : 6,25 μg/ml, transferrin : 6,25 μg/ml, selenium : 6,25 μg/ml, a-ketoglutarate: 10^~4 M, ceruloplasmin : 0,25U/ml, cholesterol : 5 μg/ml, phosphatidyl ethanolamine: 2 μg/ml, α-tocopherol acid succinate: 9 x 10^~4 M, reduced glutathione: 10 μg/ml, taurine 1,25 μg/ml, and L-ascorbic acid 2- sulphate: 50 μg/ml, wherein the concentrations of the compounds are the final concentrations in said medium. It is known to the skilled person that the concentrations of the compounds in the medium, more particularly in said serum free media can be varied, maximum by 1 log scale, preferably by less than 50% or more preferably less than 20% or less than 10%. By way of example the a-ketoglutarate concentration in that medium can be between 10^~5 M and 10 ³ M, preferably between 1,5 x 10^~4 M and 0,5 x 10^{" 4} M, or more preferably between 1,2 x 10^~4 M and 0,8 x 10^~4 M or between 1, 1 x lO ⁴ M and 0,9 x 10^"4 M. As specified herein said basal medium can be any basal medium as contemplated by the description of the present invention, including other mixes of 2 basal media as compared to Ham's F12 and DMEM (1 : 1, v/v).

Definitions

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of" when referring to recited components, elements or method steps also include embodiments which "consist of" said recited components, elements or method steps.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. As an example, in case the term about is used in combination with a certain amount of days, it includes said specific amount of days plus or minus 1 day, e.g. about 6 days include any amount of days between 5 and 7. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term "animal", "patient" or "subject" is used herein to describe an animal, especially including a domesticated mammal and preferably a human, to whom a treatment or procedure is performed. For treatment of those conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a domesticated/agricultural animal or human patient of either gender.

As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures. Beneficial or desired clinical results include, but are not limited to, prevention of an undesired clinical state or disorder, reducing the incidence of a disorder, alleviation of symptoms associated with a disorder, diminishment of extent of a disorder, stabilized (i.e., not worsening) state of a disorder, delay or slowing of progression of a disorder, amelioration or palliation of the state of a disorder, remission (whether partial or total), whether detectable or undetectable, or combinations thereof. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the terms "therapeutic treatment" or "therapy" and the like, refer to treatments wherein the object is to bring a subjects body or an element thereof from an undesired physiological change or disorder to a desired state, such as a less severe or unpleasant state (e.g., amelioration or palliation), or back to its normal, healthy state (e.g., restoring the health, the physical integrity and the physical well-being of a subject), to keep it at said undesired physiological change or disorder (e.g., stabilization, or not worsening), or to prevent or slow down progression to a more severe or worse state compared to said undesired physiological change or disorder.

As used herein the terms "prevention", "preventive treatment" or "prophylactic treatment" and the like encompass preventing the onset of a disease or disorder, including reducing the severity of a disease or disorder or symptoms associated therewith prior to affliction with said disease or disorder. "Preventing" also encompasses preventing the recurrence or relapse-prevention of a disease or disorder for instance after a period of improvement.

A 'therapeutic amount' or 'therapeutically effective amount' as used herein refers to the amount of an active compound or pharmaceutical agent (e.g., a cell-based product) effective to treat a disease or disorder in a subject, i.e., to obtain a desired local or systemic effect. The term thus refers to the quantity of the cells, the compound or the agent that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. Such amount will typically depend on the specific cell type, the compound or the agent and the severity of the disease, but can be decided by the skilled person, possibly through routine experimentation. The term "prophylactically effective amount" refers to an amount of cells, an active compound or pharmaceutical agent (e.g., a cell-based product) that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.

As used herein and unless otherwise stated, the term " mesenchymal cells " means any cell type derived from tissues originating from the mesoderm or neural crest during embryonic development or have the phenotype as described in Dominici et al. (2006) Cytotherapy 8, 315-317).

As used herein and unless otherwise stated, the term " periosteum derived cells " means any cell type that is isolated from the periosteum well known to a person skilled in the art.

"Stem cell" means a cell that can undergo self-renewal (i.e., progeny with the same differentiation potential) and also produce progeny cells that are more restricted in differentiation potential. Within the context of the invention, a stem cell would also encompass a more differentiated cell that has dedifferentiated, for example, by nuclear transfer, by fusions with a more primitive stem cell, by introduction of specific transcription factors, or by culture under specific conditions. See, for example, Wilmut et al. (1997) Nature 385, 810-813; Ying et al. (2002) Nature 416, 545-548 ; Guan et al. (2006) Nature 440, 1199-1203; Takahashi et al. (2006) Cell 126, 663-676; Okita et al. (2007) Nature 448, 313- 317 and Takahashi et al. (2007) Cell 131, 861-872.

"Induced pluripotent stem cells (IPSC or IPS cells)" is a designation that pertains to somatic cells that have been reprogrammed, for example, by introducing exogenous genes that confer on the somatic cell a less differentiated phenotype. These cells can then be induced to differentiate into less differentiated progeny. IPS cells have been derived using modifications of an approach originally discovered in 2006 (Yamanaka, S. et al. (2007) Cell Stem Cell 1, 39-49). For example, in one instance, to create IPS cells, scientists started with skin cells that were then modified by a standard laboratory technique using retroviruses to insert genes into the cellular DNA. In one instance, the inserted genes were Oct4, Sox2, Lif4, and c-myc, known to act together as natural regulators to keep cells in an embryonic stem cell-like state. These cells have been described in the literature. See, for example, Wernig et al. (2008) PNAS 105, 5856-5861; Jaenisch et al., (2008) Cell 132, 567-582; Hanna et al. (2008) Cell 133, 250-264 (2008); and Brambrink et al. (2008) Cell Stem Cell 2, 151-159.

IPS cells have many characteristic features of embryonic stem cells. For example, they have the ability to create chimeras with germ line transmission and tetraploid complementation and they can also form teratomas containing various cell types from the three embryonic germ layers. On the other hand, they may not be identical as some reports demonstrate. See, for example, Chin et al. (2009) Cell Stem Cell 5, 111-123 showing that induced pluripotent stem cells and embryonic stem cells can be distinguished by gene expression signatures.

As used herein and unless otherwise stated, the term "bone disorders " or "bone diseases" means any medical condition that affects the bone, examples of such bone disorders include but are not limited to bone diseases such as osteoporosis, Paget's disease, congenital pseudoarthrosis, osteoarthritis, osteosarcoma, diabetes, osteopetrosis, brittle bone disease, McCune-Albright Syndrome and Neurofibramatosis and also include bone injuries such as bone fractures, delayed union fractures and non-healing bone disorders and bone injuries resulting from trauma, infections and prosthesis revision.

Bone disorder also relates, complications which occur during bone formation such as refracture of the newly formed bone segment, failure of distraction osteogenesis, deformity of the newly formed bone column and non-union at the docking site have been reported with bone transport.

As used herein and unless otherwise stated, the term " non-healing bone defect" or "non-healing bone disorder" or "nonunion bone defects" means permanent failing of healing of a structural defect of the bone leading to loss of integrity. Large non-healing bone defects can also be caused by other situations such as traumatic bone defects, as a result of infection, irradiation, prosthetic revision or in compromised patients such as diabetes, ostepoporosis and vascular disease and smokers. Examples of such non-union bone defects include but are not limited to atrophic, hypertrophic fractures and large bone defects as known to a person skilled in the art.

As used herein, the terms "cartilage disorder" or "cartilage disease" or "joint disorder" or "joint disease" refer to developed or genetic inherited disorders of cartilage and/or joints such as spondylo-, ankylo- and osteoarthritis. In the context of the present invention joint disorder/disease relates to damage of the subchondral, non-cartilage part of the joint. As used herein, the terms "aggregates" or "microaggregates" refer to cells that condense together, spontaneously or by manipulation (such as described in the example section and detailed description in Materials and Methods), to aggregates of 1 to 5000 cells/aggregate. Individual aggregates can then be combined, 1 to > 1*10¹⁰ to custom size, depending of defect size. Typical amounts of cells per aggregate are about 50, about 100, about 150, about 200 or about 250 cells per aggregate. Other methods of generating aggregates or microaggregates are well known to the skilled person. An example of such an (micro)aggregation method is described in Moreira Teixeira et al. (2012) Eur. Cells Materials 23, 387 - 399. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the invention. Any methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention, but the preferred methods and products are described herein.

Chondrogenic refers to the capacity of cells to form hyaline cartilage when implanted in vivo. The expression of sox9 is a widely accepted marker to classify cells as chondrogenic cells. Other markers which are used to identify chondrogenic cell are e.g. staining with Saffranin O, expression of collagen II, ACAN.

Bone is an important tissue that has many functions, it protects the underlying soft organs, allows locomotion by providing attachment points for tendons and muscles, regulates critical endocrine processes and it allows haematopoiesis through the bone marrow. However, upon fracture many of these functions are lost, leading to a decrease in patient's life quality. Fortunately, bone is a unique tissue that has a remarkable healing capacity. Bone fracture healing can take place by direct bone healing but most frequently it largely recapitulates embryonic endochondral bone formation processes in which a cartilaginous template, called fracture callus, precedes bone formation. Yet, when healing processes are hampered, fracture healing can be delayed or even abrogated and thus resulting in permanent failure of healing also known as non-union. In this invention we aimed - amongst others - to develop a new healing strategy for bone defects through the use of human pluripotent stem cell derived cartilaginous aggregates. Large non healing bone defects can also be caused by other situations such as traumatic bone defects, as a result of infection, tumour resection and irradiation, prosthetic revision or in compromised patients such as diabetes, osteoporosis and vascular disease; and smokers. Upon chondrogenic differentiation, hPSC derived mesodermal precursors condensed and formed glycosaminoglycan rich clusters. These clusters continued to mature and developed into Safranin-0 positive and collagen type II rich tissue intermediates. TGF-βΙ, BMP2 and GDF5 were added to basal chondrogenic media to stimulate matrix deposition. These growth factors were previously shown to be capable of inducing chondrogenic differentiation of pluripotent and mesenchymal stem cells (Yamashita et al.(2015) Stem Cell Reports 4, 404-418). However combinations of these growth factors were mainly used in the context of inducing stable articular cartilage like chondrocytes. Our experiments corroborated these findings as non-hypertrophically stimulated aggregates were not remodelled into bone tissue and remained cartilaginous. These observations can potentially be attributed to the use of GDF5 in our growth factor cocktail. Lineage tracing revealed that GDF5 expressing cells are precursors of future articular chondrocytes (ACs). These chondrocytes are known to express high levels of Sox9 and are capable of giving rise to ectopic cartilage upon intramuscular injection. High levels of Sox9 can explain the lack of tissue remodelling within the hPSC derived aggregates. Indeed, it was previously shown that Sox9 is a negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Furthermore, Sox9 is capable of inducing the expression of chondromodulin-1, a potent VEGF inhibitor that prevents migration of osteogenic and vascular cells. The lack of vascular migration might delay or even block the entry of chondroclasts and thus directly contributes to the stability of the implanted tissue.

Endochondral bone formation based strategies are becoming increasingly popular for treating bone fractures, as this approach mimics or even stimulates endogenous repair mechanisms. Recently, Bahney and colleagues investigated the bone inductive capacities of soft callus tissue in critical size tibial defects (Bahney et al. (2014) J. Bone Miner. Res. 29, 1269-1282). The authors harvested the cartilaginous callus from unstable fractures and used it as graft in recipient mice. Although successful bone healing was achieved, this strategy would have limited applications in human settings as it would require additional surgical manipulations and inherently does not differ from bone grafting procedures. Nevertheless, Bahney and colleagues provide us with proof of principle that transplantation of soft-callus like tissues into critical sized defects can lead to successful bone bridging and healing.

To derive soft callus like tissue, cartilaginous aggregates were further stimulated with various cytokines that are involved in the synthesis and maturation of the soft callus. BMP and Wnt proteins are known to be expressed within the fracture callus and aid in promoting chondrocyte proliferation and hypertrophy. Although an increase in hypertrophic markers was detected upon BMP4 treatment, no bone formation was seen in ectopically and orthotopically implanted aggregates. The lack of bone formation in BMP4 treated samples may be attributed to the bipotent role of BMPs in promoting chondrogenesis. Although BMP dependent upregulation of Runx2, the master transcription factor regulating hypertrophy, has been reported, BMPs were also shown to induce Sox9, which stabilizes the articular phenotype in chondrocytes. The lack of bone remodelling in BMP4 treated aggregates can thus potentially be explained by the dominance of Sox9 over Runx2's transcriptional activities. Wnt proteins are known to be tightly regulated within cartilaginous tissues. Gain of function of canonical Wnt signalling (also known as Wnt/p-catenin pathway)in chondrocytes leads to progressive maturation towards hypertrophy whereas loss of function results in tissue damage and chondrocyte death. The absence of bone remodelling in BIO (6-bromoindirubin- 3'-oxime) treated samples is therefore likely to be the result of endogenously secreted Wnt antagonists such as frizzled-related and dickkopf-related proteins, which are present in healthy articular cartilage. Recently, it has been shown that the expression of both proteins is induced upon GDF5 treatment. These results further support that the inclusion of GDF5 during chondrogenic differentiation favours the derivation of stable articular like chondrocytes from progenitor cells. However, Craft and colleagues reported that GDF5 derived cartilaginous constructs were not articular-like and failed to remain cartilaginous upon implantation. These discrepancies can potentially be attributed to the differences in cartilage tissue derivation. Whereas Craft and colleagues required the addition of BMP-inhibitors during mesoderm specification our approach did not and thus might be indicative for a different mesodermal origin and subsequent cellular behaviour.

The identification of thyroid hormone inducing chondrocyte hypertrophy stems from clinical studies whereby an excess or deficiency in thyroid hormone resulted in skeletal dysplasias. Thyroid hormone was found to be able to directly repress Sox9 while promoting chondrocyte hypertrophy. However upon T3 (3,3,5-Triiodo- L-thyronine) treatment, the hPSC derived cartilaginous aggregates failed to induce in vivo bone formation. This can potentially be attributed to the heterogeneity of chondrocyte maturation levels within the aggregate. Indeed, non-hypertrophic chondrocytes are known to express Sox9, which induces the expression of parathyroid hormone related protein (PTHrP). This protein is known to be present in healthy articular chondrocytes and is able to suppress chondrocyte hypertrophy. The lack of cartilage tissue remodelling can thus be explained by the presence of paracrine PTHrP signalling, which negates the recruitment of chondroclasts and osteoprogenitors.

Combination of BMP, Wnt and TH signalling induced in vitro cartilage mineralization. The cumulative effect of these cytokines was further confirmed using in silico modelling (data not shown). However despite the presence of hypertrophic chondrocytes and mineralized cartilage, no bone formation was observed in the 8-weeks implantation window. One could conclude that the lack of cartilage remodelling is simply the result of insufficient in vivo incubation time. Indeed, researchers have reported that a total in vivo period of at least 12 weeks to over a year is necessary before cartilage remodelling and subsequent bone formation can be seen in pluripotent stem cell derived tissues. Even if bone formation would take place after prolonged in vivo incubation, this approach would not be clinically attractive for treating large bone defects, since prolonged absence of bone bridging increases the risk of pseudoarthrosis and graft failure. During fracture repair, IL-Ιβ is expressed in two waves. Shortly after fracture proinflammatory cells secrete IL-Ιβ to stimulate progenitor cell proliferation and macrophage recruitment. The expression of IL-Ιβ gradually decreases but becomes re-expressed during soft callus remodelling. We hypothesized that by treating the hypertrophic cartilaginous aggregates with IL-Ιβ, a faster bone bridging would be seen and that this will lead to successful bone healing through endochondral ossification. Indeed upon orthotopic implantation, IL-Ιβ treated aggregates progressively mineralized and induced bone bridging after 4 weeks. Cartilage remodelling and bone formation was observed.

In summary, the present invention demonstrates that soft callus like tissue could be generated from human pluripotent stem cells and that these were capable of inducing in vivo bone formation and fracture healing in critical size defects. The progressive development of (induced) pluripotent stem cell banks will ultimately allow the fabrication of soft callus like tissue intermediates off-the-shelf that are capable of mediating bone fracture repair in non-union situations. EXAMPLES

Example 1. hPSCs are able to undergo chondrogenic differentiation in vitro

Chondrogenic differentiation of human pluripotent stem cells was carried out by using a three-step protocol. Following mesoderm induction, the pluripotency markers Nanog, Oct3/4 and Sox2 gradually decreased. Nanog was decreased by an 8.34- and 7.21-fold when day 0 cells were compared to day 3 and day 14. Similarly, Oct3/4 decreased by a 2.51- and 29.95-fold. Sox2 was found to be decreased by a 1.22- (day 0 compared to day 3) and 2.12-fold (day 0 compared to day 14) upon differentiation. During primitive streak induction, a transient increase of Brachyury (463.57-fold; day 0 compared to day 1.5) was detected. Although no significant differences were detected for the mesodermal markers MIXL1 and KDR, a trend towards transient increase was observed. Upon chondrogenic stimulation, the expression of Col2Al progressively increased by a 109.67-, 35-, 5.75-fold when day 0, 1.5 and 3 cells were compared to day 14. Col2Al expression levels continued to increase until day 56, similar expression levels were detected when compared to human articular chondrocytes (Fig. 1) A trend towards increase in aggrecan (ACAN) was detected although no significant differences have been observed. Interestingly, Sox9 was found to be upregulated at day 1.5 by a 3.51-fold (when compared to day 0) and might indicate that chondrogenic cells were specified early on during primitive streak development. Sox9 expression remained consistent during subsequent chondrogenic differentiation (Fig. 2).

During chondrogenic differentiation, cells condensed into glycosaminoglycan rich nodules. Upon detachment and suspension culture the nodules progressively matured into safranin-o and collagen type II positive aggregates (data not shown).

Example 2. Cartilaginous aggregates are able to undergo hypertrophic differentiation in vitro

Chondrogenic aggregates were stimulated towards hypertrophy by using BMP4, BIO (6-bromoindirubin-3'-oxime) and T3 (3,3,5-Triiodo-L-thyronine). The effect of each component was evaluated separately and used in combination (BMP4, BIO, T3, BBT3) (Fig 3). BBT3 is the combination of BMP4 (bone morphogenic protein 4), BIO (6-bromoindirubin-3'-oxime), T3 (3,3,5-Triiodo-L-thyronine).

Upon hypertrophic differentiation, a trend towards decrease in Sox9 and Col2Al was detected in BBT3 (BMP4 + BIO +T3)treated samples. While Runx2 was mainly detected in T3 (3,3,5-triiodo-l-thyronine) treated samples, the CollOAl - Col2Al ratio suggest that samples treated with BBT3 (BMP4 + BIO +T3), progressively underwent hypertrophic differentiation. Interestingly, although T3 (3,3,5-Triiodo- L-thyronine) has mainly been used for inducing hypertrophic differentiation, the high increase in Col2Al would suggest that T3 (3,3,5-Triiodo-L-thyronine) promotes both chondrogenic and hypertrophic differentiation. Although no increase in Col lOAl expression was detected.

Interestingly, mineral deposits were detected in BBT3 (BMP4 + BIO +T3) treated samples. Upon SEM-EDAX analysis, it was confirmed that these deposits were enriched in calcium and phosphate and are thus indicative for hypertrophic differentiation and early mineralization (data not shown).

Example 3. Cartilaginous aggregates remain cartilaginous following ectopic implantation.

Stability and safety of the in vitro derived cartilaginous aggregates were evaluated through ectopic implantation in nude mice for 8 weeks. Upon implant harvesting, histological sections were processed and tissue formation was evaluated. All implants contained safranin-o cartilaginous tissues, indicative of proteoglycan deposition, while no teratoma formation was observed. However, despite in vitro hypertrophic differentiation, no cartilage tissue remodelling or bone formation was detected. Samples that were treated with BBT3 (BMP4 + BIO +T3) showed progressive signs of matrix mineralization and are thus indicative for a slow but steady progression towards hypertrophy (data not shown).

Example 4. IL-Ιβ priming leads to successful bone bridging and induces healing in critical size long bone defects

The stability of the in vitro derived cartilaginous aggregates was further challenged through orthotopic implantation in critical size tibial defects of nude mice. Neo- tissue formation was continuously monitored through in vivo μΟΤ. Upon implantation, none of the stimulated aggregates were able to induce de novo bone formation. These results are indicative for the articular cartilage-like (stable) phenotype of the in vitro derived aggregates (data not shown).

The BBT3 (BMP4 + BIO +T3) aggregates were treated for an additional 10 days of BBT3 (BMP4 + BIO +T3) supplemented with IL-Ιβ prior to implantation. Upon stimulation, a trend towards decrease in chondromodulin (VEGF inhibitor), increase in VEGF and matrix metalloproteinase-13 (MMP13) was detected (Fig. 4). The increase of VEGF and MMP13, suggests that these aggregates acquired a soft- callus like phenotype. As a result, we evaluated the endochondral bone formation potential of these aggregates, by implanting these in critical size long bone defects. Remarkably, progressive mineralization was detected and bone bridging was achieved after 4 weeks. The aggregates were slowly remodelling into bone tissue and allowed successful repair of critical size bone fractures (data not shown).

Example 5. Serum free protocol leads to successful bone bridging and induces healing in critical size long bone defects

The described serum free protocol also allows in vitro derivation of cartilage aggregates. These aggregates expressed sulphated glycosaminoglycans (as assessed by toluidine blue and safranin o staining). Furthermore, gene expression analysis revealed that in vitro derived cartilage aggregates expressed similar levels of collagen type II when compared to native cartilaginous tissues.

Following ectopic implantation similar results were observed with the serum free protocol as compared to the protocol with serum.

Example 6. Materials and methods

Cell culture

Human pluripotent stem cell lines H9, 604B1, CY2 and NCRM 1 were used for differentiation into chondrogenic cells. Briefly, hPSCs were maintained on mitomycin-c (Sigma) treated SNL feeder cells in human embryonic stem cell medium consisting of Dulbecco's modified eagle medium (DMEM) : Ham's F12 nutrient mix (F12) (Sigma) supplemented with 20% knockout serum replacement, 2mM Glutamax, 1% sodium pyruvate (SP), 1% non-essential amino acids (NEAA), 0.1 mM 2-mercaptoethanol (2ME), 50 U and 50 mg/ml penicillin/streptomycin (Pen/Strep) and 10 ng/ml human basic fibroblast growth factor (hbFGF). Cells were grown in a humidified incubator at 37°C and 5% CO2. Medium was replenished daily and hPSCs were passaged once a week on fresh feeder cells, a) Chondrogenic differentiation : with serum

Prior to differentiation, hPSCs were transferred and maintained in feeder-free conditions using Essential 8 and Matrigel (Becton Dickinson) coated well plates. Mesoderm induction was carried out by using a two-step protocol : cells were treated with a primitive streak induction medium that consists of Stemdiff APEL medium (APEL, Stem Cell Technologies) supplemented with 8 μΜ canonical wingless related integration protein (Wnt) agonist CHIR99021 (GSK3 inhibitor, CAS number 252917-06-9 commercially available from e.g. Axon Medchem), 20 ng/ml hbFGF, 50 U and 50 mg/ml Pen/Strep for 36 hours followed by another 36 hours of APEL supplemented with 1 μΜ retinoic acid (RA), 8 ng/ml hbFGF, 50 U and 50 mg/ml Pen/Strep. The resultant mesodermal precursors were subsequently chondrogenically differentiated until day 14 using chondrogenic medium (CM) consisting of DMEM supplemented with 1% Foetal Bovine Serum (FBS, Hyclone), 1% L-Glutamine, 1% NEAA, 1% SP, 1% insulin-transferrin-selenite X (ITS-X), 50 μg/ml ascorbic acid (AA, Sigma), 0.1 mM 2ME, 10 ng/ml hbFGF, 10 ng/ml TGF- βΐ, 10 ng/ml BMP2, 10 ng/ml GDF5 (Prospec), 50 U and 50 mg/ml Pen/Strep. Subsequently, cartilage like nodules were detached and cultured in suspension by using CM without hbFGF for 6 weeks.

Cartilaginous aggregates were stimulated to enter hypertrophy using basal hypertrophic differentiation medium (HM) : DMEM supplemented with 1% FBS, 1% SP, 1% ITS-X, 50 g/ml AA, 10 nM dexamethasone (Sigma), 7 mM β- glycerophosphate (Sigma), 50 U and 50 mg/ml Pen/Strep. HM was further supplemented with 100 ng/ml BMP4, 0.1 μΜ BIO (6-bromoindirubin-3'-oxime) (canonical Wnt agonist), 1 μΜ 3,3,5-Triiodo-L-thyronine (T3) (Sigma), for 3-4 weeks, depending on the differentiation condition followed by IL-1 priming for 10 days withlO ng/ml IL-Ιβ, in HM with or without 100 ng/ml BMP4, 0.1 μΜ BIO (6- bromoindirubin-3'-oxime) (, 1 μΜ 3,3,5-Triiodo-L-thyronine (T3) (Sigma), more preferably without. All components with the exception of the growth factors and cytokines (Peprotech) were purchased from Invitrogen, unless otherwise stated. 2b) Chondrogenic and osteogenic differentiation can also be performed serum free according to the following serum free protocol

Mesoderm induction

APEL of E6 medium + 8μΜ CHIR99021 + 20ng/ml hbFGF for 24-36 hours APEL of E6 medium + 1 μΜ Retinoic acid + 8ng/ml hbFGF for 24-36 hours

Chondrocyte induction

CDM + lOng/ml TGFpi + lOng/ml GDF5 + lOng/ml BMP2 + 10 ng/ml hbFGF + 50ng/ml PDGFBB + 50 μg/ml ascorbic acid for 14 days

CDM + 10ng/ml TGFpi + lOng/ml GDF5 + lOng/ml BMP2 + 50ng/ml PDGFBB + 50 μg/ml ascorbic acid for 6 weeks

Hypertrophy induction

CDM + lOOng/ml BMP4 + 1 μΜ T3 + 0.1 μΜ BIO (6-bromoindirubin-3'-oxime) + 50ng/ml PDGFBB + 50 μg/ml ascorbic acid + 100 nM dexamethasone + 7mM β- glycerophosphate for 3 weeks IL1 Priming:

CDM + 10 ng/ml IL-1B + 50 μς/ιτιΙ ascorbic acid + 100 nM dexamethasone + 7mM β-glycerophosphate for 10 days. In general, in the methods of the present invention, culturing and differentiation methods can be used in serum containing conditions. However, serum-free conditions are preferred and all culture steps are similar to the conditions with serum, including the addition of all cocktails and all factors, such as hbFGF, BMP4, TGFpi, GDF5, ascorbic acid, dexamethasone, β-glycerophosphate, with the exception that in the serum free condition, one additional factor is added, i.e. PDGF, including PDGFBB, AB, and/or AA. In the serum free conditions of the present invention, said PDGF is used in a concentration of about 5ng/ml PDGDF to about 500ng/ml PDGDF, more preferably about 50ng/ml PDGDF. Extracellular matrix analysis

Extracellular matrix deposition was analysed after 14 days of differentiation. Briefly, alcian blue staining was performed by fixing the hPSCs with ice-cold methanol for 1 hour at 4°C. Afterwards, 1 ml 0.1 % alcian blue in 0.1 M HCI (Fluka) staining solution was added to each well and incubated for 1 hour at room temperature. Subsequently, well plates were washed with water until clear of any dye. Well plates were visualized and images were acquired by using an inverted microscope (IX83-P22F, Olympus).

Gene expression analysis

RNA extraction and cDNA synthesis was carried out at each time point using the RNeasy mini kit (Qiagen) and PrimeScriptTM reagent kit (Takara), respectively. Gene expression analysis was carried out using a SYBR® Select Master Mix (Invitrogen) or Taqman Fast Universal Mastermix (Invitrogen) based qPCR reaction on the StepOnePlus system (Applied Biosystems®). Quantification of gene expression was calculated using the 2-ACT method. All experiments were carried according to each manufacturer's protocol. All primer sequences are detailed in table 1. Table 1. Primer sequences for SYBR gene expression analysis. The above indicated rimer se uences were utilized for ene ex ression anal sis

In vivo tissue formation

To investigate the stability and bone inductive capacity of the cartilaginous aggregates, the aggregates were either ectopically implanted at the cervical region or orthotopically implanted in critical size long bone (tibiae) defects of NMRI nu/nu mice. Bone healing was continuously monitored in orthotopically treated mice through in vivo x-ray microtomography analysis (μΟΤ; Skyscan 1076 system [Bruker microCT, Kontich, Belgium] at 9 μιτι, 50 kV and 100 μΑ equipped with a 1 mm aluminium filter) at 1, 2, 4, 6 and 8 weeks post-implantation. All mice were sacrificed at 8 weeks and implants were collected and histologically processed. The performed animal experimental procedures were approved by the local ethical committee for animal research (KU Leuven). The animals were housed according to the guidelines provided by the Animalium Leuven (KU Leuven).

Histological processing and analysis

Explants were fixed in 2% paraformaldehyde (PFA) overnight at 4°C before decalcification using EDTA/PBS (pH 7.5) for 3 weeks. Subsequently, explants were paraffin embedded and 5 μιτι histological sections cut.

All sections were deparaffinised in HistoclearTM (Laborimpex, Brussels, Belgium) followed by methanol, and rinsed with water before performing histological stainings. Sections were stained with safranin-0 (SAF-O), haematoxylin and eosin (HE) as previously described. Immunostaining for collagen type II (Col2) was carried out by blocking endogenous peroxidase with 3% hydrogen peroxidase for 2 x 15 minutes, followed by sequential degradation of the cartilaginous matrix using 2 mg/ml hyaluronidase (Sigma) for 40 minutes, 200 mU/ml chondroitinase (Sigma) and 2.5 mU/ml heparinase II (Sigma) for 1 hour at 37°C. Antigen retrieval was carried out by using a 0.02% pepsin (Sigma) in 0.02 M HCI solution for 11 minutes at 37°C. Sections were then blocked with 20% goat serum, before being incubated with a rabbit anti-human Col2 antibody (1 : 200, Millipore) at 4°C overnight. Subsequently, sections were rinsed in PBS before blocking in 20% goat serum for 1 hour. Sections were afterwards incubated with a horseradish peroxidase conjugated goat anti-rabbit antibody (1 : 100, Jackson) for 1 hour before being stained by using the DAB + substrate chromogen kit (Dako). Nuclei were counterstained with haematoxylin and sections were dehydrated in graded ethanol before mounting. Sections were visualized and images were acquired by using an inverted microscope (IX83-P22F, Olympus).

Scanning electron microscopic analysis

Chondrocyte deposited minerals were analysed by using a scanning electron microscope coupled with energy dispersive x-ray analysis (SEM-EDAX, XL30 FEG Philips). Briefly, rehydrated paraffin tissue sections were chemically dried by using hexamethyldisilazane for 3 minutes followed by gold sputtering before SEM analysis at 10 kV. Calcium, phosphorus and oxygen elements were detected by EDAX to indicate hydrochloride formation. Statistical analysis

All experiments were carried out in triplicate to assess statistical significance, with the exception of in vivo studies (n=4). Data represented in each graph are depicted as mean ± standard deviation. Statistical significance was calculated in Excel (Microsoft) using the unpaired two-tailed t-test. All graphs were generated using Prism software (Graphpad). Statistical significance is represented on each graph as follow: *p<0.05, **p<0.01, ***p<0.001, and ****p < 0.0001.

Claims

1. An in vitro method of inducing hypertrophy in chondrogenic cells comprising the step of incubating said chondrogenic cells in a medium comprising :

- BMP-4 (Bone Morphogenetic Protein 4) or BMP-2 (Bone Morphogenetic Protein 4),

- a Wnt agonist, and

-a thyroid hormone.

2. The method according to claim 1, wherein the chondrogenic cells are aggregates of chondrogenic cells.

3. The method according to claim 1 or 2, wherein the aggregates have a size of between 25 to 1000 cells/aggregate.

4. The method according to any one of claims 1 to 3, wherein the chrondro- genic cells Sox positive cells, or wherein the chondrogenic cells are safranin O and col II positive.

5. The method according to any one of claims 1 to 4, comprising the step of incubating said chondrogenic cells in a medium comprising BMP4 (Bone Morphogenetic Protein 4), a Wnt agonist and a thyroid hormone.

6. The method according to any one of claims 1 to 5, wherein the Wnt agonist is a GSK-3 inhibitor.

7. The method according to any one of claims 1 to 6, wherein the Wnt agonist is BIO (6-bromoindirubin-3'-oxime).

8. The method according any one of claims 1 to 7, wherein the thyroid hormone is T3 (3,3,5-Triiodo-L-thyronine).

9. The method according any one of claims 1 to 8, wherein the medium comprises BMP4 and BIO and T3.

The method according to any one of claims 1 to 9, wherein the incubation of said chondrogenic cells in said medium is performed for between 3 to 4 weeks.

The method according to any one of claims 1 to 10, wherein the incubation is followed by an further incubation in a medium comprising BMP-4 or BMP- 2, a Wnt agonist and a thyroid hormone and a compound selected from the group consisting of ILIA, IL1 β, IL-6 and TNF.

The method according to claim 11, wherein the further incubation is performed in a medium comprising BMP-4, a Wnt agonist and a thyroid hormone and IL1 β.

The method according to claim 11 or 12, wherein the further incubation is performed for about 10 days.

The method according to any one of claims 1 to 13, wherein the chondrogenic cells are obtained by differentiation of pluripotent stem cells, such as iPSC.

Use of a combination of BMP-4 or BMP-2, a Wnt agonist and a thyroid hormone for inducing hypertrophy in chondrogenic cells.

The use according to claim 15, using a combination of BMP4, BIO and T3.

A cell culture medium for inducing hypertrophy in chondrogenic cells comprising BMP-4 or BMP-2, a Wnt agonist and a thyroid hormone.

18. The cell culture medium according to claim 17, comprising BMP-4, a Wnt agonist and a thyroid hormone.

19. The cell culture medium according to claim 17 or 18, which comprises about 0, 1 μΜ to about 10 μΜ thyroid hormone, more preferably about 1 μΜ thyroid hormone, and about 0,01 μΜ to about 1 μΜ canonical Wnt agonist, more preferably about 0,1 μΜ canonical Wnt agonist.

The cell culture medium according to claim 20, wherein the thyroid hormone is T3 and the canonical Wnt agonist is BIO.

The cell culture medium according to any one of claims 17 to 20, further comprising IL-1B.

The serum free cell culture medium according to any one of claims 17 to 21, comprising one or more basal cell culture media, insulin, transferrin, selenium, a-ketoglutarate, ceruloplasmin, cholesterol, phosphatidyl ethanolamine, a-tocopherol, reduced glutathione, taurine and ascorbic acid.

The cell culture medium according to any one of claims 17 to 22, further comprising ascorbic acid, dexamethasone and β-glycerophosphate.

The cell culture medium according to any one of claims 17 to 23, which is a serum free medium comprising PDGF.

The cell culture medium according to any one of claims 17 to 24, comprising PDGF at a concentration between 5ng/ml to 500 ng/ml, typically between about bout 50 ng/ml to 500 ng/ml, and BMP4 at a concentration between lOng/ml and 1000 ng/ml, typically at a concentration of about 100 ng/ml.

Cells obtained by the method of any one of claims 1 to 14, for use in treating bone or joint defects.

The cells according to claim 26, for use in treating a disorder selected from the group consisting of a bone fracture, a non-healing bone defect, an osteochondral defect, a damaged joint surface, a subchondral defect or a metabolic bone disease.

28. The cells according to claim 26, in admixture with a biocompatible carrier, for use in treating a disorder according to claim 26 or 27. A method of treating a bone defect, comprising the step of administering cells obtained by the method of any one of claims 1 to 14, to the bone or joint defect. 30. The method according to claim 29, wherein the cells administered together with a biocompatible carrier.