Methods and compositions for generating chondrocyte lineage cells and/or cartilage like tissue

Abstract

A method for generating chondrocytes and/or cartilage, optionally articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or hypertrophic chondrocyte like cells and/or cartilage like tissue, the method comprising: a. culturing a primitive streak-like mesoderm population, optionally a CD56+, PDGFRalpha+ KDR- primitive streak-like mesoderm population, with a paraxial mesoderm specifying cocktail comprising: i. a FGF agonist; ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and iii. optionally one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta; b. generating a chondrocyte precursor population comprising: i. culturing the paraxial mesoderm population expressing CD73, CD105 and/or PDGFR-beta at a high cell density optionally in serum free or serum containing media; ii. culturing the high cell density CD73+, CD105+ and/or PDGFRbeta+ paraxial mesoderm population with a TGFbeta3 agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and c. either i. culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with the TGFbeta3 agonist for an extended period of time to produce an articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue; or ii. culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte like cells and/or cartilage like tissue.

1 . A method for generating chondrocytes and/or cartilage, optionally articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or hypertrophic chondrocyte like cells and/or cartilage like tissue, the method comprising:
 a. culturing a primitive streak-like mesoderm population, optionally a CD56+ and PDGFRalpha+ primitive streak-like mesoderm population, with a paraxial mesoderm specifying cocktail comprising: 
 i. a FGF agonist; 
 ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and 
 iii. optionally one or more of a TGFbeta inhibitor, optionally SB431542; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; 
 to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta; 
   b. generating a chondrocyte precursor population comprising: 
 i. culturing the paraxial mesoderm population expressing CD73, CD105 and/or PDGFR-beta at a high cell density optionally in serum free or serum containing media; 
 ii. culturing the high cell density CD73+, CD105+ and/or PDGFRbeta+ paraxial mesoderm population with a TGFbeta agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and 
   c. either 
 i. culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with the TGFbeta agonist for an extended period of time to produce an articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue; or 
 ii. culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte like cells and/or cartilage like tissue. 
   
     
     
         2 . The method of  claim 1  for generating articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue, wherein step c) comprises culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with a TGFbeta agonist for an extended period of time to produce articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue, optionally greater than 3 weeks. 
     
     
         3 . The method of  claim 1  for generating hypertrophic chondrocyte like cells and/or cartilage like tissue 
 wherein step c) comprises culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce hypertrophic chondrocyte like cells and/or cartilage like tissue. 
 
     
     
         4 . The method of any one of  claims 1 to 3  wherein the paraxial mesoderm population is comprised in embryoid bodies, monolayer culture and/or a combination thereof. 
     
     
         5 . The method of  claim 1  for generating articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue, the method comprising:
 a. culturing a starting population of pluripotent stem cells with a primitive streak inducing cocktail to induce a primitive streak-like mesoderm population expressing CD56 and PDGFR-alpha; 
 b. culturing a primitive streak-like mesoderm population with a paraxial mesoderm specifying cocktail comprising: 
 i. a FGF agonist; 
 ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and 
 iii. one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; 
 to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and PDGFR-beta; 
 
 c. generating a chondrocyte precursor population comprising:
 i. culturing the paraxial mesoderm population expressing CD73, CD105 and/or PDGFR-beta at a high cell density optionally in serum free or serum containing media; 
 ii. culturing the high cell density CD73+, CD105+ and PDGFRbeta+ paraxial mesoderm population with a TGFbeta agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and 
 
 d. culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with a TGFbeta agonist for an extended period of time to produce an articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue. 
 
     
     
         6 . The method of  claim 1  for generating hypertrophic chondrocyte like cells and/or cartilage like tissue, the method comprising:
 a. culturing a starting population of pluripotent stem cells with a primitive streak inducing cocktail to induce a primitive streak-like mesoderm population expressing CD56 and PDGFR-alpha; 
 b. culturing a primitive streak-like mesoderm population with a paraxial mesoderm specifying cocktail comprising: 
 i. a FGF agonist; 
 ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and 
 iii. optionally one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; 
 to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and PDGFR-beta; 
 
 c. generating a chondrocyte precursor population comprising: 
 i. culturing the paraxial mesoderm population expressing CD73, CD105 and/or PDGFR-beta at a high cell density optionally in serum free or serum containing media; 
 ii. culturing the high cell density CD73+, CD105+ and PDGFRbeta+ paraxial mesoderm population with a TGFbeta agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and 
 
 d. culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte like cells and/or cartilage like tissue. 
 
     
     
         7 . The method of  claim 1  for generating chondrocytes, the method comprising:
 a. culturing a starting population of pluripotent stem cells with a primitive streak inducing cocktail to induce a primitive streak-like mesoderm population expressing CD56 and PDGFR-alpha; 
 b. culturing a primitive streak-like mesoderm population with a paraxial mesoderm specifying cocktail comprising: 
 i. a FGF agonist; 
 ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and 
 iii. one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; 
 to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and PDGFR-beta; 
 
 c. generating a chondrocyte precursor population comprising: 
 i. culturing the paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta at a high cell density, optionally in serum free or serum containing media; 
 ii. culturing the high cell density CD73+, CD105+ and PDGFRbeta+ paraxial mesoderm population with a TGFbeta agonist in serum free media to produce a high cell density Sox9+, collagen 2+ chondrocyte precursor population; and 
 
 d. either 
 i. culturing the high cell density Sox9+, collagen 2+ chondrocyte precursor population with a TGFbeta agonist for an extended period of time to produce an articular like non-hypertrophic chondrocyte cell and/or cartilage like tissue; or 
 ii. culturing the high cell density Sox9+ collagen2+ chondrocyte precursor population with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte like cell and/or cartilage like tissue. 
 
 
     
     
         8 . The method of any one of  claims 5 to 7 , wherein the starting population is a human embryonic stem cell population (hESC) or an induced pluripotent stem cell population (iPSC), optionally primary hESC and/or iPSC. 
     
     
         9 . The method of  claim 8  wherein the hESC population is selected from a HES2 H1, H9, or any human iPS cell line. 
     
     
         10 . The method of any one of  claims 5 to 9 , wherein the starting population is aggregated into embryoid bodies. 
     
     
         11 . The method of any one of  claims 5 to 10 , wherein the starting population is contacted with the primitive streak inducing cocktail for about 1 to about 5 days. 
     
     
         12 . The method of any one of  claims 5 to 11 , wherein the primitive streak inducing cocktail comprises an activin agonist, optionally activin A or nodal, a BMP4 agonist, optionally BMP4, BMP2, BMP6, BMP7 and/or, BMP10, and a FGF agonist, optionally bFGF, FGF2, FGF4, FGF9 and/or optionally FGF 19, 21, 3, 5, 6, 8a, 16-18, 20 and/or 23. 
     
     
         13 . The method of any one of  claims 5 to 12 , wherein the primitive streak inducing cocktail further comprises a wnt agonist, optionally selected from Wnt3a and a GSK3b inhibitor such as such as CHIR-99021 (Stemolecule™ CHIR99021 Stemgent), 6-Bromolndirubin-3′-Oxime (BIO) (Cayman Chemical (cat:13123)), or Stemolecule™ BIO from Stemgent (cat:04003). 
     
     
         14 . The method of any one of  claims 1 to 13 , wherein the paraxial mesoderm is specified in a monolayer culture. 
     
     
         15 . The method of any one of  claims 1 to 14 , wherein the BMP inhibitor is a type 1 BMP receptor inhibitor, BMP ligands and/or soluble BMP receptors, optionally selected from dorsomorphin (DM), noggin, Chordin, LDN-193189, soluble BMPR1a, soluble BMPR1b. 
     
     
         16 . The method of any one of  claims 1 to 15 , wherein the primitive streak-like mesoderm population is contacted with the BMP inhibitor for about 1, 2, 3 or 4 days to inhibit cardiomyocyte specification. 
     
     
         17 . The method of any one of  claims 1 to 16 , wherein the FGF agonist for specifying the primitive streak-like mesoderm population and/or the paraxial mesoderm population is selected from FGF2, bFGF, FGF4 and FGF9. 
     
     
         18 . The method of any one of  claims 1 to 17 , wherein the primitive streak-like mesoderm population is contacted with the FGF agonist for at least 5 days, 6 days, 7 days, 8 days, 9 days, 10, days, 11 days or more to increase the proportion of cells expressing CD73 and/or CD105 by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% compared to FGF agonist untreated cells. 
     
     
         19 . The method of any one of  claims 1 to 18 , wherein the paraxial mesoderm population also expresses transcription factors Meox1 and Nkx3.2 and is negative for Nkx2.5. 
     
     
         20 . The method of any one of  claims 1 to 19 , wherein the paraxial mesoderm population is plated in a micromass culture, pellet culture or a filter culture or in any high cell density format. 
     
     
         21 . The method of  claim 20 , wherein the paraxial mesoderm population is plated at a cell density between 10 million cells/ml and 50 million cells/ml, optionally at least 10 million cells per 1 ml, 20 million cells/ml, 30 million cells/ml, 40 million cells/ml or 50 million cells/ml in a micromass culture; or between 500,000 and 2 million, optionally about 500,000, about 750,000, about 1 million, about 1.25 million, about 1.5 million, about 1.75 million, about 2 million in a membrane filter culture. 
     
     
         22 . The method of any one of  claims 1 to 21 , wherein the populations are cultured in serum free basal media comprising insulin, transferrin and optionally selenium (ITS). 
     
     
         23 . The method of  claim 22 , wherein the basal media comprises DMEM with insulin, transferrin, selenium supplement, proline, and dexamethasone. 
     
     
         24 . The method of any one of  claims 1 to 23 , wherein a paraxial mesoderm population is cultured at high cell density for about 0 to about 4 days, optionally 0, 1, 2, 3, or 4 days before addition of TGFb3 agonist. 
     
     
         25 . The method of any one of  claims 1 to 24 , wherein the CD73+, CD105+ and/or PDGFRbeta+ paraxial mesoderm population is cultured with the TGFbeta3 agonist in serum free media for at least 3 days, or for about 3 days to about 14 days, optionally at least a week, to produce a Sox9+, collagen 2+ chondrocyte precursor population. 
     
     
         26 . The method of any one of  claims 1 ,  2 ,  4 to 24 , wherein the extended period of time the Sox9+, collagen 2+ chondrocyte precursor population is cultured with a TGFbeta agonist is at least 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more to produce an articular cartilage like tissue. 
     
     
         27 . The method of  claim 26 , wherein the chondrocyte precursor population is cultured with the TGFb agonist until lubricin and/or cartilage intermediate layer protein 2 (CILP2) is expressed. 
     
     
         28 . The method of any one of  claims 1 to 27 , wherein the paraxial mesoderm population and/or the Sox9+, collagen 2+ chondrocyte precursor population is cultured with a TGFb agonist selected from TGFb3 TGFb1, and/or TGFb2. 
     
     
         29 . The method of any one of  claims 1 ,  3 to 27 , wherein the G hypertrophic chondrocyte like cells and/or cartilage like tissue, is cultured with the BMP4 agonist to produce a collagen 10+ and/or Runx2+ hypertrophic chondrocyte like cells and/or cartilage like tissue. 
     
     
         30 . The method of any one of  claims 1 ,  3 to 27  and  29 , wherein the extended period of time the high cell density Sox9+ collagen2+ chondrocyte precursor population is cultured with the BMP4 agonist is at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or more to generate a cartilage tissue that expresses collagen 2 or a hypertrophic chondrocyte population that expresses collagen 10. 
     
     
         31 . The method of any one of  claims 1 to 30 , wherein CD73+ CD105+ cells and/or CD73+ PDGFR-beta+ cells are isolated, optionally by flow cytometry, from the paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta prior to high cell density culture. 
     
     
         32 . The method of any one of  claims 1 to 31 , wherein the chondrocyte precursors are cultured in TGFbeta agonist or BMP4 agonist for cartilage tissue formation. 
     
     
         33 . The method of  claim 1  for generating chondrocyte like cells comprising:
 a. culturing chondrocyte precursor cells at a high cell density, optionally in serum free or serum containing media; 
 b. culturing the high cell density chondrocyte precursor cells with a TGFbeta3 agonist in serum free media; and 
 c. either 
 i. culturing the chondrocyte precursor cell with a TGFbeta agonist for an extended period of time to produce an articular cartilage like chondrocyte population; or 
 ii. culturing the chondrocyte precursor cell with a BMP4 agonist for an extended period of time to produce a hypertrophic chondrocyte population of cells and/or cartilage like tissue. 
 
 
     
     
         34 . The method of  claim 33 , wherein the chondrocyte precursor cells are primary fetal chondrocytes or passaged fetal chondrocytes. 
     
     
         35 . The method of  claim 33 , wherein the chondrocyte precursor cells are primary cells obtained from a subject with a cartilage or bone condition or disease. 
     
     
         36 . The method of any one of  claims 1 to 35 , wherein the steps are performed in vitro. 
     
     
         37 . The method of any one of  claims 1 to 36 , wherein the generated cells and/or tissues are administered to a subject. 
     
     
         38 . An isolated population of articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or hypertrophic chondrocyte like cells and/or cartilage like tissue, generated according to the method of any one of  claims 1 to 37 . 
     
     
         39 . A composition comprising the population of articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or hypertrophic chondrocyte like cells and/or cartilage like tissue, and optionally a diluent or a carrier, optionally PEG, hydrogel, bone scaffolding, bone substitute scaffolding or matrigel. 
     
     
         40 . The composition of  claim 39 , wherein the composition is a cell slurry. 
     
     
         41 . The isolated cell population of  claim 38  or the composition of  claims 39  or  40  further comprising endothelial cells or fibroblasts. 
     
     
         42 . A cartilage or bone tissue product comprising the cells or composition of any one of  claims 1 to 41 , and a scaffold. 
     
     
         43 . The bone tissue product of  claim 42 , wherein the scaffold is a bone substitute. 
     
     
         44 . A method for ameliorating symptoms and/or treating a subject in need thereof comprising administering the population of cells and/or tissues of any one of  claims 38 to 41  and/or inserting the product of  claim 42  or  43 . 
     
     
         45 . Use of the population of cells and/or tissues or composition of any one of  claims 38 to 41  and/or product of  claim 42  or  43  for ameliorating symptoms and/or treating a subject in need thereof. 
     
     
         46 . The method or use of any one of  claims 44  or  45 , wherein the population of cells is induced from autologous cells. 
     
     
         47 . The method or use of any one of  claims 44 to 46 , wherein the population of cells is enriched for articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue. 
     
     
         48 . The method or use of any one of  claims 44 to 47  wherein the subject has a joint condition such as osteoarthritis, osteochondritis dissecans, polychondritis, and other chondropathies, or joint injuries affecting the cartilage. 
     
     
         49 . The method or use of any one of  claims 44 to 45  wherein the population of cells is enriched for hypertrophic chondrocyte like cells and/or cartilage like tissue. 
     
     
         50 . The method or use of  claim 49 , wherein the enriched hypertrophic chondrocyte like cells and/or cartilage like tissue, is adhered to a scaffold. 
     
     
         51 . The method or use of  claims 44 to 46  and  50 , wherein the subject has a bone condition such as a bone fracture, bone break or is in need of a bone replacement for example due to a malignancy, trauma or has achondroplasia, osteogenesis imperfect, osteoporosis or other osteopathies. 
     
     
         52 . A method of generating a paraxial mesoderm population of cells comprising:
 a. culturing a starting population of pluripotent stem cells with a primitive streak inducing cocktail to induce a primitive streak-like mesoderm population expressing CD56 and PDGFR-alpha;   b. culturing a primitive streak-like mesoderm population with a paraxial mesoderm specifying cocktail comprising: 
 i. a FGF agonist; 
 ii. a BMP inhibitor; optionally Noggin, LDN-193189, Dorsomorphin; and 
 iii. one or more of a TGFbeta inhibitor, optionally SB431524; and a Wnt inhibitor, optionally DKK1, IWP2, or XAV939; 
 to specify a paraxial mesoderm population expressing cell surface CD73, CD105 and/or PDGFR-beta. 
   
     
     
         53 . The method of  claim 52 , wherein the method further comprises enriching CD73, CD105 and/or PDGFRbeta expressing cells. 
     
     
         54 . A method of isolating paraxial chondrogenic mesoderm population of cells comprising:
 a. contacting a population of cells comprising paraxial chondrogenic mesoderm cells with a cocktail comprising a CD73 specific binding agent, CD105 specific binding agent, and a PDGFRbeta specific binding agent; and   b. enriching for CD73+, CD105+ and PDGFRbeta+ cells.   
     
     
         55 . The method of  claim 53  or  54  wherein the cells are enriched using flow cytometry. 
     
     
         56 . An isolated paraxial chondrogenic mesoderm population of cells prepared according to the method of  claim 52 to 55 . 
     
     
         57 . A method of testing candidate chondrogenic modulating substance, the method comprising:
 a. contacting a test substance with a chondrocyte precursor lineage cell population, the test substance contacted with the chondrocyte precursor lineage cell population at any step in the method of any one of  claims 1 to 37 ,  52  or  53 ;   b. assessing the effect of the test substance on chondrocyte proliferation, maintenance and/or differentiation compared to a control population generated in the absence of test substance; and   c. identifying the test substance as a candidate chondrogenic modulating substance if the test substance increases or decreases proliferation, and/or affects chondrocyte maintenance or differentiation compared to the control.   
     
     
         58 . The method of  claim 57 , wherein the candidate chondrogenic modulating substance is a factor isolated from a subject with diseased cartilage or bone. 
     
     
         59 . The method of  claim 58 , wherein the factor is isolated from a fat pad in a joint, optionally a knee joint, of a subject with arthritis and/or obese or from healthy subjects as controls. 
     
     
         60 . The method of  claim 57 , wherein the test substance is added with the BMP4 agonist and the test substance is assessed for its ability to inhibit hypertrophy compared to controls treated in the absence of the test substance. 
     
     
         61 . The method of  claim 60 , wherein hypertrophy is assessed using flow cytometry, optionally by assessing forward and side scatter. 
     
     
         62 . A method of isolating articular chondrocytes comprising:
 a. contacting a mixed population of cells comprising chondrocytes with an antibody that binds CD73 under conditions that allow for the formation of an antibody:CD73 cell complex; and   b. isolating the antibody:CD73 cell complex.   
     
     
         63 . The method of  claim 62 , wherein the mixed population of cells comprises non chondrocyte like cells, non-articular chondrocyte like cells and hypertrophic chondrocyte like cells. 
     
     
         64 . The method of  claim 62  or  63 , wherein the antibody is coupled to a tag such as a bead such as a sepharose bead or magnetic bead. 
     
     
         65 . A method of assessing a candidate articular chondrocyte proliferation inducer comprising:
 a. obtaining articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue generated according to the method of any one of  claims 1 ,  2 ,  4 to 37 ;   b. culturing the articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue with a test substance;   c. measuring the articular like non-hypertrophic chondrocyte like cell proliferation; and   d. detecting an increase in proliferation compared to articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue cultured in the absence of the test substance indicating that the test substance is a candidate articular chondrocyte proliferation inducer.   
     
     
         66 . A method of assessing a candidate hypertrophic chondrocyte proliferation inducer comprising:
 a. obtaining hypertrophic chondrocyte like cells and/or cartilage like tissue, generated according to the method of any one of  claims 1 ,  3 to 37 ;   b. culturing the hypertrophic chondrocyte like cells and/or cartilage like tissue, with a test substance;   c. measuring hypertrophic chondrocyte cell proliferation; and   d. detecting an increase in proliferation compared to hypertrophic chondrocyte like cells and/or cartilage like tissue, cultured in the absence of the test substance indicating that the test substance is a candidate hypertrophic chondrocyte proliferation inducer.   
     
     
         67 . The method of  claims 65 , wherein a CD73 articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue is isolated prior to culture with the test substance, optionally isolated by flow cytometry. 
     
     
         68 . The method of any one of  claims 65 to 67 , wherein the articular like non-hypertrophic chondrocyte cell and/or hypertrophic chondrocyte like cell, cell comprises a reporter gene functionally coupled to an articular chondrocyte specific promoter (i.e. articular chondrocyte reporter system), optionally a lubricin promoter element and/or a reporter gene functionally coupled to a hypertrophic chondrocyte specific promoter, optionally of collagen 10 promoter element (i.e. hypertrophic chondrocyte reporter system); and a compound that induces articular chondrocyte differentiation are identified by measuring the articular chondrocyte reporter system activity and a compound that induces hypertrophic chondrocyte differentiation are identified by measuring hypertrophic chondrocyte reporter system activity. 
     
     
         69 . The method of any one of  claims 65 to 68  wherein an increase in proliferation is measured using one or more of the following methods: a 3H Thymidine incorporation assay; a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay; a propidium iodine assay. 
     
     
         70 . A method of assessing AC cell and/or GPC cell protective activity and/or toxicity of a test compound, comprising:
 a. generating articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or GPC like cells and/or growth plate cartilage according to the method of any one of  claims 1 to 37 ;   b. culturing the articular like non-hypertrophic chondrocyte cells and/or cartilage like tissue and/or GPC cells and/or cartilage like tissue with the test substance;   c. measuring cell/tissue toxicity or chondrocyte protective activity of the test substance; and   d. detecting an increase in cell toxicity compared to articular like non-hypertrophic chondrocyte cells and/or GPC cells and/or tissue cultured in the absence of the test substance indicating that the test substance is toxic to articular chondrocyte and/or GPC cells or detecting an increase in protective activity (e.g. a decrease in cell toxicity) compared to articular like non-hypertrophic chondrocyte cells and/or GPC cells and/or tissue cultured in the absence of the test substance indicating the test substance is protective .   
     
     
         71 . The method of  claim 70 , wherein cell toxicity is measured using one of the following assays: a Trypan blue dye assay; a luciferase assay; a tetrazolim salt conversion assay such as a MTT assay and a WST-1 assay. 
     
     
         72 . The method of any one of  claims 65 to 70  wherein the proliferation, cell toxicity and/or protective activity is assessed using one or more of the following analyses or assays:
 histological analysis, biochemical assays such as those that quantify the production of glycosaminoglycans and proteoglycans, gene expression analyses, gain/loss of a fluorescent reporter such as lubricin or collagen 10 by microscopy or flow cytometry, gain or loss of CD73 cell surface receptor expression, assays for cell death, flow cytometry for cell size which can indicate chondrocyte hypertrophy. 
 
     
     
         73 . The method of any one of  claims 65-72  wherein the AC like chondrocytes and/or cartilage like tissue or the hypertrophic like chondrocytes and/or cartilage like tissue is/are contacted with a disease mediator optionally prior to culture with the test substance. 
     
     
         74 . The method of  claim 73  wherein the disease mediator is a cytokine, optionally IL-1beta, or a joint fat pad component.