Methods for Producing Cartilage and Bone

Abstract

This application relates to methods for inducing differentiation of stem cells into cartilage precursors and chondrocytes in vitro, the methods also relate to the production of in vitro engineered cartilage and bone and related biomaterials, and methods for drug screening and modeling of bone- and cartilage-related diseases and disorders.

Description

[Technical field]

[0001] This application relates to methods for inducing differentiation of stem cells into chondrocyte precursors and chondrocytes in vitro, as well as to the production of in vitro engineered cartilage and bone and related biomaterials, and methods for drug screening and modeling of bone- and cartilage-related diseases and disorders. [Background technology]

[0002] The major connective tissues of the body, such as skin, tendons, ligaments, cartilage, and bone, provide the framework of important structures and information required for development. The extensive extracellular matrix (ECM) of connective tissues is a complex interacting network of proteins, glycoproteins, and proteoglycans that provides a dynamic and essential three-dimensional environment that supports cell maintenance, growth, and differentiation. In addition to providing a highly organized framework, the ECM mediates the shuttling of signals to and from cells involved in important biological processes such as cell differentiation and migration during development and repair processes.

[0003] Chondrocytes at the developing articular joint surface form a unique population of articular chondrocytes that produce permanent articular cartilage. These articular chondrocytes synthesize a unique cartilage extracellular matrix with special properties required to provide resilience to repeated mechanical loading occurring during diarthrodial joint function. These permanent articular chondrocytes produce the characteristic extracellular components of cartilage, such as the major structural collagen II and aggrecan. They have a specific repertoire of gene expression and stoichiometry of cartilage extracellular matrix components that support their important role in joint homeostasis and function. These important biological properties are necessary to be able to provide biomaterials with tissue-specific ECMs that are phenotypically accurate and suitable for use in tissue repair / regeneration or disease modeling.

[0004] Embryonic chondrocytes may follow a developmental pathway that forms the transitional chondrocytes of the cartilage growth plate. Chondrocytes that enter this pathway generate the highly organized growth plate structures that are the key drivers of longitudinal bone growth by sequential maturation, hypertrophy, and migration to bone.

[0005] Despite advances in orthopedic surgery, finding effective treatments for musculoskeletal trauma, cartilage and meniscal trauma, genetic disorders of cartilage and bone development and homeostasis, and other joint-related diseases and disorders, such as osteoarthritis, remains a significant clinical challenge. Furthermore, the complex organization of cartilage and endochondral bone formation offers a fruitful perspective for human genetic disorders (osteochondrodysplasias) with abnormal cartilage development and bone growth. The current disease classification of genetic skeletal disorders includes more than 450 disorders, reflecting their diverse manifestations. Recent advances in genomic technology have revealed many underlying genetic mutations, but understanding of the mechanisms of cellular diseases has lagged and few targeted therapies are available. In addition to genetic skeletal diseases, acquired cartilage disorders such as osteoarthritis are a major clinical challenge. The central pathological feature of osteoarthritis is the progressive destruction of articular cartilage. The ability of articular cartilage to recover is poor, and it is cartilage erosion that ultimately represents the terminal disease. Approaches to cartilage regeneration include cartilage repair with autologous or allogeneic neocartilage constructs. Although this is a rapidly developing field, there is an unmet need, as optimal conditions for robust production of cartilage for therapeutic applications have yet to be determined. Human iPSC-derived chondrocytes, whose developmental stage and extracellular matrix composition can be precisely controlled, provide an important tool to produce neocartilage (containing cells) for cartilage regeneration or decellularized cartilage as a biomaterial scaffold. Controlled in vitro methods will make it possible to provide enhanced biomaterials with more desirable biological and biomechanical properties. Furthermore, in vitro model systems of diseases, in which the processes of endochondral cartilage maturation and bone formation are faithfully reproduced, are needed to provide a better understanding of the molecular pathology of the disease and to allow the identification of new therapeutic options.

[0006] Through detailed studies, the inventors have developed a pluripotent stem cell differentiation protocol to recapitulate paraxial mesoderm development in vitro to produce sclerotomes induced to differentiate into articular chondrocytes, chondrocyte precursors, and then chondrocytes, or chondrocytes that can mature into hypertrophic chondrocytes, transition to osteoblasts, and produce mineralized extracellular matrix. The protocol described herein recapitulates the key steps of growth plate development and endochondral ossification in vitro, thereby providing a phenotypically accurate biomaterial with a tissue-specific ECM. The methods and biomaterials described herein provide a model system for studying the mechanisms of diseases, disorders, and conditions that affect cartilage and endochondral bone formation, as well as for screening drugs in diseases, disorders, and conditions that affect cartilage and endochondral bone formation. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] https: / / string-db.org [Non-Patent Document 2] Lilianty J, Bateman JF, Lamande SR. Stem Cell Res. 2021 Aug 25; 56:102515 [Non-Patent Document 3] Howden S, et al. Stem Cell Res. 2019 Jul;38:101453 [Non-Patent Document 4] The Merck Manual of Diagnosis and Therapy, 20th edition, Merck Sharp & Dohme Corp., 2018 [Non-Patent Document 5] Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 2008 [Non-Patent Document 6] Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995. [Non-Patent Document 7] Immunology by Wemer Luttmann, Elsevier, 2006 [Non-Patent Document 8] Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2016 [Non-Patent Document 9] Lewin's Genes XI, Jones & Bartlett Publishers, 2014 [Non-Patent Document 10] Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) [Non-Patent Document 11] Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) [Non-Patent Document 12] Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 [Non-Patent Document 13] Laboratory Methods in Enzymology: RNA, Jon Lorsch (ed.), Elsevier, 2013 [Non-Patent Document 14] Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 [Non-Patent Document 15] Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005 [Non-Patent Document 16] Current Protocols in Immunology (CPI), John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, arren Strobe (eds.), John Wiley and Sons, Inc., 2003 [Non-Patent Document 17] Immunological Methods, Ivan Lefkovits, Benvenuto Pemis (eds.), Elsevier Science, 2014 [Non-Patent Document 18] https: / / www.gsea-msigdb.org / gsea / msigdb / Summary of the Invention [Means for solving the problem]

[0008] According to a first aspect, the present invention provides a method of generating chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, the method comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte progenitor cells; and b) culturing the three-dimensional aggregates of chondrocyte precursor cells generated in step a) in the absence of an activator of the FGF pathway to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells expressing COL2A1 and ACAN. Including, The method includes transferring the three-dimensional aggregates of sclerotome cells or the three-dimensional aggregates of chondrocyte precursor cells into orbital culture.

[0009] In an embodiment of the first aspect, the method further comprises culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like progenitor cells with triiodothyronine (T3) to produce a population of hypertrophic chondrocytes or hypertrophic chondrocyte-like cells expressing COL10A1, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof.

[0010] In another embodiment of the first aspect, the method further comprises culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with TGF-beta3, preferably for a period of at least 2 weeks to about 4 weeks, to produce a population of articular chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof. In some embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with TGF-beta3 to produce a population of articular chondrocytes or articular chondrocyte-like cells expressing PRG4, articular cartilage-like tissue, articular cartilage, or a combination thereof.

[0011] In a second aspect, the present invention provides a method for producing articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, the method comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte precursor cells; b) culturing the three-dimensional aggregates of chondrocyte precursor cells with an activator of the FGF pathway and an agonist of TGF-beta to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells expressing COL2A1 and ACAN; c) culturing the three-dimensional aggregates of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta for an extended period of time to produce articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof. Including, The method includes transferring the three-dimensional aggregates of sclerotome cells, the three-dimensional aggregates of chondrocyte precursor cells, or the three-dimensional aggregates of chondrocytes or chondrocyte-like cells into orbital culture.

[0012] In a third aspect, the present invention provides a method for producing a population of osteoblasts or bone-like organoids, the method comprising: a) deriving hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, cartilage-like tissue, cartilage according to the method of the first aspect, and b) culturing the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, cartilage-like tissue, or cartilage in orbital culture with an osteogenic culture medium to generate osteoblasts or bone-like organoids expressing COL1A1 and COL1A2; Includes.

[0013] In a fourth aspect, the present invention provides chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof produced according to the method of the first aspect.

[0014] In a fifth aspect, the present invention provides articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof produced according to the method of the second aspect.

[0015] In a sixth aspect, the present invention provides an osteoblast or bone-like organoid produced according to the method of the third aspect.

[0016] In a seventh aspect, the present invention provides a decellularized scaffold prepared by decellularizing chondrocytes or chondrocyte-like cells, cartilage-like tissue, or cartilage of the fourth aspect, or a combination thereof, or articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof of the fifth aspect, or three-dimensional aggregates of osteoblasts or bone-like organoids of the sixth aspect to produce a decellularized scaffold.

[0017] In a seventh aspect, the present invention provides a composition comprising a homogenous population of iPSC-derived chondrocytes, the cells expressing collagen 2 (COL2A1) and ACAN in a ratio ranging from 20:1 to 5:1, preferably at a ratio of about 10:1, and substantially no expression of COL10A1.

[0018] In an eighth aspect, the invention provides a composition comprising a homogenous population of iPSC-derived hypertrophic chondrocytes, said cells expressing collagen 2 (COL2A1) and COL10A1 in a ratio ranging from 1:1 to about 2.5:1.

[0019] In a ninth aspect, the present invention provides a method for treating a cartilage or osteochondral defect in a subject in need thereof comprising the steps of a) deriving chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof according to the first aspect, articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof according to the second aspect, or osteoblasts or bone-like organoids according to the third aspect, and b) administering to the subject the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, or the osteoblasts or bone-like organoids.

[0020] In a tenth aspect, the present invention provides a method for testing a candidate chondrogenic or osteogenic modulating substance, the method comprising: a) carrying out a method according to any one of the first, second or third aspects, wherein the test substance is included in any one or more of the incubation steps of the method; b) evaluating the effect of the test substance on the proliferation, maintenance, and / or differentiation of chondrocytes or osteoblasts compared to a control population generated in the absence of the test substance; and c) identifying the test substance as a candidate chondrogenic or osteogenic regulator if it increases or decreases proliferation compared to a control and / or affects the maintenance or differentiation of chondrocytes or osteoblasts. Includes.

[0021] In an eleventh aspect, the present invention provides the use of chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, of the first aspect, articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, of the second aspect, or osteoblasts or bone-like organoids according to the third aspect, for disease modelling or testing of therapeutics for a bone or cartilage disease or disorder.

[0022] The numbering description of the present invention is as follows: 1. A method for producing chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte progenitor cells; and b) culturing the three-dimensional aggregates of chondrocyte precursor cells generated in step a) in the absence of an activator of the FGF pathway to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells expressing COL2A1 and ACAN. Including, A method comprising the step of transferring a three-dimensional aggregate of sclerotome cells or a three-dimensional aggregate of chondrocyte precursor cells into orbital culture. 2. The sclerotome cells are i) culturing a population of human multipotent progenitor cells with a composition comprising an activator of the TGF-beta pathway, an activator of the Wnt pathway, an activator of the FGF pathway, and an inhibitor of P13K for a period of about 24 hours; ii) culturing the cells of step i) with a composition comprising an inhibitor of the TGF-beta pathway, an activator of the Wnt pathway, an activator of the FGF pathway, and an inhibitor of the BMP pathway for a period of about 24 hours; iii) culturing the cells of step ii) with a composition comprising an inhibitor of the Wnt pathway, an inhibitor of the BMP pathway, and an inhibitor of the MEK / ERK pathway for a period of about 24 hours; iv) contacting the cells of step iii) with a composition comprising an inhibitor of the Wnt pathway and an activator of the Hedgehog pathway for a period of about 72 hours to generate sclerotome cells. 2. The method of claim 1, wherein the compound is obtained by a process comprising the steps of: 3. The method of claim 1 or 2, wherein the sclerotome cells express any one of PAX1, SOX9, FOXC2, PAX9, NKX3.2 / BAPX1, and TWIST1. 4. The method of any one of aspects 1 to 4, wherein the sclerotome cells are derived from a human embryonic stem cell population (hESC) or a human induced pluripotent stem cell population (iPSC). 5. The method of claim 4, wherein the iPSCs are derived from a feeder-free cell culture. 6. The method of any one of aspects 1 to 5, wherein step a) comprises transferring the aggregates of sclerotome cells to orbital culture at any time after the initiation of step a). 7. The method of any one of aspects 1 to 6, wherein the three-dimensional aggregate of chondrocytes expresses one or more additional genes selected from the group consisting of COL11A1, COL11A2, COL9A1, COL9A2, COL9A3, MATN1, and MATN3. 8. The method of any one of aspects 1 to 7, wherein the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof express collagen 2 (COL2A1) and ACAN in a ratio ranging from 20:1 to 5:1, preferably at a ratio of about 10:1, and do not substantially express COL10A1. 9. The method of any one of aspects 1 to 8, wherein the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof do not express collagen COL1A1 or COL1A2 at a level greater than about 0.2% of the level of COL2A1. 10. The method of any one of aspects 1 to 9, further comprising culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with triiodothyronine (T3) to produce a population of hypertrophic chondrocytes or hypertrophic chondrocyte-like cells expressing COL10A1, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof. 11. The method of any one of aspects 1 to 10, comprising transferring the aggregates of sclerotome cells to orbital culture at any time from day 7 of step a) onwards. 12. The method of aspect 10 or 11, wherein the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof, express COL2A1 and COL10A1. 13. The method of claim 12, wherein the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof, express COL2A1 and COL10A1 in a ratio ranging from about 1:1 to about 2.5:1. 14. The method according to any one of aspects 1 to 9, further comprising culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with TGF-beta3, preferably for a period of at least 2 weeks to about 7 weeks, to produce a population of PRG4-expressing articular chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof. 15. A method for producing articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte precursor cells; b) culturing the three-dimensional aggregates of chondrocyte precursor cells with an activator of the FGF pathway and an agonist of TGF-beta to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells expressing COL2A1 and ACAN; c) culturing the three-dimensional aggregates of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta for an extended period of time to produce articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof. Including, A method comprising the step of transferring a three-dimensional aggregate of sclerotome cells, a three-dimensional aggregate of chondrocyte precursor cells, or a three-dimensional aggregate of chondrocytes or chondrocyte-like cells into orbital culture. 16. The method of claim 14 or 15, wherein the articular chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof express one or more further genes selected from the group consisting of COL2A1, ACAN, PRG4, and optionally ASPN, CILP, and CILP2. 17. The method of any one of aspects 1 to 16, wherein the culturing in step a) is for a period of at least 7 days. 18. The method according to any one of aspects 1 to 16, wherein the culturing in step a) is for a period of 7 to 21 days. 19. The method according to any one of aspects 1 to 16, wherein the culturing in step a) is for a period of about 10 to 17 days. 20. The method of any one of aspects 1 to 16, wherein the culturing in step a) is for a period of about 14 days. 21. The method of claim 15 or 16, wherein the culturing in step a) is for a period of about 7 days. 22. The method of any one of aspects 1 to 21, wherein the culturing in step b) is for a period of at least 7 days. 23. The method of any one of aspects 1 to 21, wherein the culturing in step b) is for a period of 7 to 21 days. 24. The method of any one of aspects 1 to 21, wherein the culturing in step b) is for a period of about 10 to 17 days. 25. The method of any one of aspects 1 to 21, wherein the culturing in step b) is for a period of about 14 days. 26. The method of any one of aspects 1 to 21, wherein the culturing in step b) is for a period of about 7 days. 27. The method according to any one of Forms 15 or 16, or Forms 17 to 25 when subordinate to Form 15 or 16, wherein the culturing in step c) is for a period of about 2 to about 5 weeks. 28. The method of any one of the preceding aspects, wherein the FGF pathway activator is selected from the group consisting of FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20, and FGF23. 29. The method of claim 28, wherein the FGF pathway activator is FGF2. 30. The method of any one of forms 2 to 29, wherein the activator of the TGF-beta pathway is selected from the group consisting of activin A, TGF-beta 1, TGF-beta 2, TGF-beta 3, IDE1 / 2 (IDE1 (1-[2-[(2-carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (heptanedioic acid-1-(2-cyclopentylidenehydrazide)), and Nodal. 31. The method of claim 30, wherein the activator of the TGF-beta pathway is activin A. 32. Activators of the Wnt pathway include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, Wnt-16b, RSPO coagonists, lithium chloride, TDZD8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), and BI 32. The method of any one of forms 2 to 31, wherein the compound is selected from the group consisting of O-acetoxime ((2'Z,3'E)-6-bromoindirubin-3'-acetoxime), A1070722 (1-(7-methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea), HLY78 (4-ethyl-5,6-dihydro-5-methyl-[1,3]dioxolo[4,5-j]phenanthridine), CID11210285 hydrochloride (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride), WAY-316606, (hetero)arylpyrimidine, IQ1, QS11, SB-216763, and DCA. 33. The method of claim 32, wherein the Wnt pathway activator is CHIR99021. 34. Inhibitors of the PI3K pathway include AS 252424 (5-[[5-(4-fluoro-2-hydroxyphenyl)-2-furanyl]methylene]-2,4-thiazolidinedione), AS 605240 (5-(6-quinoxalinylmethylene)-2,4-thiazolidine-2,4-dione), AZD 6482 ((-)-2-[[(1R)-1-[7-methyl-2-(4-morpholinyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl]ethyl]amino]benzoic acid), BAG 956 (α,α,-dimethyl-4-[2-methyl-8-[2-(3-pyridinyl)ethynyl]-1H-imidazo[4,5-c]quinolin-1-yl]-benzeneacetonitrile), CZC 24832 (5-(2-amino-8-fluoro[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(1,1-dimethylethyl)-3-pyridinesulfonamide), GSK 1059615 (5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione), KU 0060648 (4-ethyl-N-[4-[2-(4-morpholinyl)-4-oxo-4H-1-benzopyran-8-yl]-1-dibenzothienyl]-1-piperazineacetamide), LY 294002 hydrochloride (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride), 3-methyladenine (3-methyl-3H-purin-6-amine), PF 04691502 (2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), PF 05212384 (N-[4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea), PI 103 hydrochloride (3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride), PI 828 (2-(4-morpholinyl)-8-(4-aminophenyl)-4H-1-benzopyran-4-one), PP 121 (1-cyclopentyl-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,34. The method of any one of forms 2 to 33, wherein the β-aminobutyric acid ester is selected from the group consisting of β-aminobutyric acid ester, ... 35. The method of claim 34, wherein the inhibitor of the PI3K pathway is PIK90. 36. Inhibitors of the TGF-beta pathway include A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), and LY 788388 (4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide). 364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), RepSox (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), SB-505124 (2-[4-(1,3-benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine), SB 525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), SD208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine), ITD1 (4-[1,1'-biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester), DAN / Fc, antibodies against TGF-beta and TGF-beta receptor, TGF-beta inhibitory nucleic acids. 37. The method of claim 36, wherein the inhibitor of the TGF-beta pathway is A-83-01. 38. The method of any one of forms 2 to 37, wherein the inhibitor of the BMP pathway is selected from the group consisting of chordin, soluble BMPR1a, soluble BMPR1b, noggin, LDN-193189, and dorsomorphin. 39. The method of claim 38, wherein the inhibitor of the BMP pathway is LDN-193189. 40. Inhibitors of the Wnt pathway include C59 (4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1, IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide), Ant1.4Br, and Ant2. 1.4CI, niclosamide, apicularen, bafilomycin, XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR-1 (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide), NSC668036 (N-[(1,1-dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3-methylbutanoyl-L-alanine-(1S)-1-carboxy-2-methylpropyl ester hydrate), 2,4-diaminobutyric acid, 1,2 ... 40. The method of any one of forms 2 to 39, wherein the antibody is selected from the group consisting of no-quinazoline, quercetin, ICG-001 ((6S,9aS)-hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide), PKF115-584, BML-284 (2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, antibodies against Wnt and Wnt receptors, and Wnt inhibitory nucleic acids. 41. The method of claim 40, wherein the inhibitor of the Wnt pathway is C59. 42. Inhibitors of the MEK / ERK pathway include AP 24534 (3-(2-imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]-benzamide), PD173074 (N-[2-[[4-(diethylamino)butyl]amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea), and FIIN 1 hydrochloride (N-(3-((3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(4-(diethylamino)butylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)methyl)phenyl)acrylamide), PD 161570 (N-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea), SU 5402 (2-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-4-methyl-1H-pyrrole-3-propanoic acid), SU 6668 (5-[1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid), PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide), BIX 02189 ((3Z)-3-[[[3-[(dimethylamino)methyl]phenyl]amino]phenylmethylene]-2,3-dihydro-N,N-dimethyl-2-oxo-1H-indole-6-carboxamide), FR 180204 (5-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine), Pluripotin (N-[3-[7-[(1,3-dimethyl-1H-pyrazol-5-yl)amino]-1,4-dihydro-1-methyl-2-oxopyrimide[4,5-d]pyrimidin-3(2H)-yl]-4-methylphenyl]-3-(trifluoromethyl)benzamide), TCS ERK 11e (4-[2-[(2-chloro-4-fluorophenyl)amino]-5-methyl-4-pyrimidinyl]-N-[(1S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1H-pyrrole-2-carboxamide), TMCB (2-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)acetic acid), XMD 8-92 (2-[[2-ethoxy-4-(4-hydroxy-1-piperidinyl)phenyl]amino]-5,11-dihydro-5,11-dimethyl-6H-pyrimido[4,5-b][1,4]benzodiazepin-6-one), SU5402, AZD4547, BGJ398, AL 8697, AMG 548, CMPD-1, DBM 1285 dihydrochloride, EO 1428, JX 401, ML 3403, RWJ 67657, SB 202190, SB-203580, SB 239063, SB 706504, Scio-469, SKF 86002 dihydrochloride, SX 011, TA 42. The method of any one of forms 2 to 41, wherein the compound is selected from the group consisting of TA 01 (4-(2-(2,6-difluorophenyl)-4-(fluorophenyl)-1H-imidazol-5-yl)pyridine), TA 02 (4-(2-(2-fluorophenyl)-4-(fluorophenyl)-1H-imidazol-5-yl)pyridine), TAK 715, VX-702, and VX-745. 43. The method of claim 42, wherein the inhibitor of the MEK / ERK pathway is PD0325901. 44. The method of any one of forms 2 to 43, wherein the activator of the Hedgehog pathway is selected from the group consisting of Hedgehog family ligands (Hh, Shh, Ihh, Dhh, etc.) and fragments thereof, benzothiophene smoothened agonists, SAG (Hh-Ag1.3), SAG21k (3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-((1r,4r)-4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide), Hh-Ag1.1, Hh-Ag1.5, and palmorfamine. 45. The method of claim 44, wherein the Hedgehog pathway activator is palmorfamine. 46. ​​A method for producing a population of osteoblasts or bone-like organoids, comprising: a) deriving hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, cartilage-like tissue, or cartilage according to any one of the methods described in any one of aspects 1 to 13, 17 to 20, 22 to 26, or 28 to 45; and b) culturing the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, or hypertrophic cartilage in orbital culture with an osteogenic culture medium to generate osteoblasts or bone-like organoids expressing COL1A1 and COL1A2; The method includes: 47. The method of claim 46, wherein the osteogenic differentiation culture medium comprises β-glycerophosphate, ascorbic acid 2-phosphate, sodium ascorbate, and dexamethasone. 48. The method of claim 46 or 47, wherein the osteogenic differentiation culture medium comprises a WNT agonist for about the first 3-7 days of culture. 49. The method of claim 48, wherein the WNT agonist is CHIR99021. 50. The method of any one of claims 1 to 45, further comprising the step of decellularizing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells, cartilage-like tissue, or cartilage to produce a decellularized scaffold. 51. The method of any one of aspects 46 to 49, further comprising a step of decellularizing the bone-like organoid to produce a decellularized scaffold. 52. The method of any one of aspects 46 to 49, further comprising the step of degrading extracellular matrix and isolating the osteoblasts. 53. A chondrocyte, chondrocyte-like cell, cartilage-like tissue, cartilage, or a combination thereof, produced according to the method of any one of aspects 1 to 45. 54. An articular (non-hypertrophic) chondrocyte or articular chondrocyte-like cell, articular cartilage-like tissue, articular cartilage, or a combination thereof, produced according to the method of any one of aspects 15 to 45. 55. An osteoblast or bone-like organoid produced according to the method of any one of aspects 46 to 49 or 52. 56. A decellularized scaffold prepared according to the method of claim 50 or 51. 57. The method of any one of aspects 1 to 45, further comprising administering to the subject chondrocytes, chondrocyte-like cells, cartilage-like tissue, or cartilage, or a combination thereof. 58. The method of any one of aspects 46 to 49 or 52, further comprising administering osteoblasts or bone-like organoids, or a combination thereof, to a subject. 59. The method of claim 57 or 58, wherein the sclerotome cells are derived from the subject. 60. A composition comprising a homogenous population of iPSC-derived chondrocytes, wherein the cells express collagen 2 (COL2A1) and ACAN in a ratio ranging from 20:1 to 5:1, preferably at a ratio of about 10:1, and do not substantially express COL10A1. 61. A composition comprising a homogenous population of iPSC-derived hypertrophic chondrocytes, wherein the cells express collagen 2 (COL2A1) and COL10A1 in a ratio ranging from 1:1 to about 2.5:1. 62. A method of treating a cartilage or osteochondral defect in a subject in need thereof comprising the steps of: a) producing a composition comprising chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof produced according to any one of aspects 1 to 44, articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage according to any one of aspects 15 to 45, and / or osteoblasts or bone-like organoids according to aspects 46 to 49 or 52; and b) administering the composition to the subject. 63. A method for testing a candidate chondrogenic or osteogenic modulating substance, comprising: a) carrying out the method according to any one of aspects 1 to 49, wherein said test substance is included in any one or more of the incubation steps of said method; b) evaluating the effect of the test substance on the proliferation, maintenance, and / or differentiation of chondrocytes or osteoblasts compared to a control population generated in the absence of the test substance; and c) identifying the test substance as a candidate chondrogenic or osteogenic regulator if it increases or decreases proliferation compared to a control and / or affects the maintenance or differentiation of chondrocytes or osteoblasts. A method comprising: 64. The method of any one of aspects 1 to 52, wherein the method of any one of aspects 1 to 52 is performed using a cell derived from a subject having a bone or cartilage disease or disorder, or a cell engineered to have a mutation associated with a bone or cartilage disease or disorder, or a cell engineered to correct a mutation associated with a bone or cartilage disease or disorder. 65. The method of embodiment 64, wherein the bone disorder is osteogenesis imperfecta. 66. The method of embodiment 64, wherein the cartilage disorder is associated with a mutation in COL2A1. 67. The method of any one of aspects 1 to 52, wherein the cell is derived from a subject having a bone or cartilage disease or disorder, or has been engineered to have a mutation associated with a bone or cartilage disease or disorder, or has been engineered to correct a mutation associated with a bone or cartilage disease or disorder. 68. Use of chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof produced according to any one of aspects 1 to 45, articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage according to any one of aspects 15 to 45, and / or osteoblasts or bone-like organoids according to aspects 46 to 49 or 52 for modeling disease or testing therapeutics for a disease or disorder of bone or cartilage. [Brief description of the drawings]

[0023] [Figure 1]FIG. 1 shows the differentiation of committed iPSCs into skeletal cells. (A) Schematic representation of the differentiation stages of iPSCs into articular and hypertrophic chondrocytes and then into osteoblast-like cells. The process recapitulates embryonic development with the first 6 days of differentiation into sclerotome including aggregation of monolayer cells into a pellet on day 4. Culture media and conditions are shown below and above the timeline. The culture format is diagrammed below. (B) Gene expression of stage-specific markers OCT4, MIXL1, MSGN1, MEOX1, and PAX1, and chondrocyte markers SOX9 and COL2A1 during 6 days of differentiation into sclerotome in iPSC line MCRIi019-A. N=4 technical replicates. [Figure 1-1] Figure 1.1 shows gene expression of stage-specific markers, OCT4, MIXL1, MSGN1, MEOX1, and PAX1, and chondrocyte markers, SOX9 and COL2A1, during 6 days of differentiation into sclerotome in iPSC line MCRIi018-B. N=3 technical replicates. [Diagram 2]FIG. 2 shows optimization of sclerotome for chondrocyte differentiation. (A) iPSC line MCRIi018-B was differentiated into sclerotome and cell pellets formed on day 4. At the end of day 6, sclerotome pellets were supplemented with 20 ng / ml BMP4 or 20 ng / ml FGF2 for 14 days. Toluidine blue stained pellet sections at days 20, 34, and 48. FGF2 supplemented pellets differentiated into chondrocytes by day 34, while BMP4 treated pellets remained small and had isolated toluidine positive areas. Scale bar is 500 μm. (B) Relative mRNA expression of cartilage genes COL2A1, ACAN, and COL10A1. N = 2 or 3 technical replicates. (C) Immunostaining for collagen II and collagen X. FGF2 treated pellets had extensive collagen II extracellular matrix deposition by day 48. BMP4-treated pellets contained only small patches of cells expressing collagen II and collagen X. Scale bar is 200 μm. (D) Rotary culture affects chondrocyte maturation. iPSC line MCRIi001-A-2 differentiated into sclerotomes, forming cell pellets on day 4. At the end of day 6, 20 ng / ml FGF2 was added to the sclerotome pellets for 14 days. At the end of days 6, 13, 20, or 27, some pellets were transferred to rotary culture and some were left in static culture. Toluidine blue stained pellets at days 34 and 48. Collagen X immunostaining at day 48 shows that pellets grown in rotary culture from days 13 and 20 matured into hypertrophic chondrocytes expressing collagen X, whereas static pellets and pellets transferred to rotary culture on day 6 did not produce collagen X. Scale bar is 500 μm. [Figure 2-1-1]Figure 2.1 shows enhanced chondrocyte differentiation in feeder-free iPSC lines. Four iPSC lines, MCRIi018-B (feeder-free), RM3.5 (feeder-dependent), MCRIi001-A and MCRIi001-A-2 (syngeneic feeder-dependent lines), were differentiated into sclerotomes and cell pellets formed on day 4. At the end of day 6, sclerotome pellets were supplemented with 20ng / ml FGF2 for 14 days and then harvested for RNAseq. N = 3 technical replicates. (A) Principal component analysis. (B) Volcano plot shows a large number of genes differentially expressed in the feeder-free vs. feeder-dependent comparison, with signature cartilage genes COL2A1, COL9A1, COL11A1, ACAN, and SOX9 being more highly expressed in the feeder-free lines. Fewer genes were differentially expressed in the feeder-dependent comparison, and only a few genes were differentially expressed between the syngeneic lines. [Figure 2-1-2] Figure 2.1 shows enhanced chondrocyte differentiation in feeder-free iPSC lines. Four iPSC lines, MCRIi018-B (feeder-free), RM3.5 (feeder-dependent), MCRIi001-A and MCRIi001-A-2 (syngeneic feeder-dependent line), were differentiated into sclerotomes and cell pellets formed on day 4. At the end of day 6, sclerotome pellets were supplemented with 20ng / ml FGF2 for 14 days and then harvested for RNAseq. N = 3 technical replicates. (C) MCRIi001-A-2 was adapted to feeder-free conditions. The feeder-dependent and feeder-free versions were then differentiated into chondrocytes as before and pellets were transferred to rotational culture at the end of day 6. Toluidine blue stained pellets at days 34, 48, and 62. At the end of differentiation, pellets from feeder-dependent MCRIi001-A-2 contained a mixture of cartilage and non-cartilage tissue, whereas pellets from feeder-free MCRIi001-A-2 were primarily cartilage with only a thin layer of non-cartilage tissue on the outside of the pellet. Scale bar, 500 μm. [Figure 2-2]Figure 2.2 shows that rotary culture affects chondrocyte maturation. iPSC line MCRIi019-A differentiated into sclerotomes and cell pellets formed on day 4. At the end of day 6, sclerotome pellets were supplemented with 20ng / ml FGF2 for 14 days. Some pellets were transferred to rotary culture at the end of days 6, 13, 20, or 27, and some were left in static culture. MCRIi019-A pellets stained with toluidine blue on days 34 and 48. At day 48, pellets grown in rotary culture from day 6 were surrounded by a layer of non-cartilage tissue, while pellets grown in static culture had reduced toluidine blue staining, suggesting proteoglycan degradation. Scale bar is 500μm. Collagen X immunostaining on day 48 shows that pellets transferred to rotary culture on day 13 have a collagen X-rich ECM. Scale bar is 200μm. [Figure 3-1] Figure 3 shows maturation into growth cartilage plates. Chondronoids maintained in chondrogenic medium spontaneously mature into hypertrophy, which can be enhanced by T3. (A) Toluidine blue stained chondronoid sections (MCRIi019-A) at days 34, 48, and 69. Some pellets were treated with T3 for 3 weeks before harvesting at day 69. Cells visibly enlarged between days 48 and 69, suggesting that they were maturing into hypertrophic chondrocytes. Scale bar, 500 μm. (B) Immunostaining for collagen II and collagen X. Chondronoids contain extensive collagen II-rich ECM throughout the time course. Collagen X was not evident at day 48, but was deposited in the ECM by day 69. Scale bar, 200 μm. (C) Electron microscopy of chondronoids at day 52, showing an extensive network of collagen II fibrils in the ECM. N = 3 independent differentiation experiments. [Figure 3-2]Figure 3 shows maturation into growth cartilage plates. Chondronoids maintained in chondrogenic medium spontaneously mature into hypertrophy, which can be enhanced by T3. (D) Heatmap showing changes in mRNA expression of selected cartilage, hypertrophic cartilage, and bone proteins. Hierarchical clustering shows that markers of hypertrophic cartilage and bone are more highly expressed with time and expression is further enhanced by T3, while canonical cartilage genes are downregulated by time and T3 treatment. N = 3 independent differentiation experiments. [Figure 3-1-1] Figure 3.1 shows maturation into growth cartilage plates. Chondronoids maintained in chondrogenic medium spontaneously mature into hypertrophy, which can be enhanced by T3. (A) Toluidine blue stained chondronoid sections (MCRIi018-B) at days 34, 48, and 69. Some pellets were treated with T3 for 3 weeks before harvesting at day 69. Cells visibly enlarged between days 48 and 69, suggesting that they were maturing into hypertrophic chondrocytes. Scale bar is 500 μm. (B) Immunostaining for collagens II and X. Chondronoids contain extensive ECM rich in collagen II throughout the time course. Collagen X was not evident at day 48, but was deposited in the ECM by day 69. Scale bar is 200 μm. N = 3 independent differentiation experiments. [Figure 3-1-2] Figure 3.1 shows maturation into growth cartilage plates. Chondronoids maintained in chondrogenic medium spontaneously mature into hypertrophy, which can be enhanced by T3. (C) Heatmap showing changes in mRNA expression of selected cartilage, hypertrophic cartilage, and bone proteins in iPSC line MCRIi018-B during chondrocyte maturation into hypertrophy. Hierarchical clustering shows that markers of hypertrophic cartilage and bone are more highly expressed with time and expression is further enhanced by T3, while canonical cartilage genes are downregulated by time and T3 treatment. N = 3 independent differentiation experiments. [Figure 3-2-1]Figure 3.2 shows the most highly expressed core matrisome genes. The list of 20 core matrisome genes with the highest RPKM values ​​at 48 days, 69 days, and 69 days + T3 was combined and gene expression (log2 RPKM) in all samples was plotted. (A) MCRIi019-A. (B) MCRIi018-B. Of the 27 most highly expressed genes in each cell line, 25 were identical and gene expression patterns at 48 days, 69 days, and 69 days + T3 were similar. [Figure 4] Figure 4 shows enrichment of gene sets associated with hypertrophy. Gene set enrichment analysis (GSEA) of RNAseq D69T3 vs. D48 identified MSigDB gene sets associated with cartilage maturation. (A) Hallmark gene sets show reduced cell division in hypertrophic chondrocytes and enrichment of hypoxic and apoptotic pathways. (B) Enriched sets from MSigDB C2 (validated) gene set included NABA_CORE_MATRISOME and other matrisome gene sets. These matrisome gene sets have highly upregulated and highly downregulated members, reflecting dynamic changes in the ECM during chondrocyte hypertrophy. (C) Heatmap showing relative expression of NABA_SECRETED_FACTORS that were differentially expressed (Adj.P. value < 0.05, LogFC ≥ 1 or ≤ -1) in D69T3 vs. D48 and expressed with average RPKM > 4 in one or more cell lines / days / treatments. Genes in the GOBP_BLOOD_VESSEL_MORPHOGENESIS, KEGG_TGF_BETA_SIGNALING_PATHWAY, and KEGG_WNT_SIGNALING_PATHWAY gene sets are shown on the right. [Diagram 5]Dynamically regulated transcription factors during hypertrophic maturation. Differentially expressed (adj. P. value ≦0.05, LogFC ≧1 or ≦-1) in RNAseq D69T3 vs. D48 comparisons and expressed with an average RPKM >10 in one or more cell lines / days / treatments are shown, grouped by direction of change and their known or unknown role in cartilage development. Points indicate the average log2 RPKM for each group. N=3 biological replicates. [Figure 5-1] Figure 5.1 shows interactions between differentially expressed transcription factors during hypertrophy maturation. (A) STRING analysis (https: / / string-db.org) identified multiple protein-protein interactions and interaction nodes between transcription factors upregulated during hypertrophy (adj. P. value ≦0.05, LogFC ≧1 or ≦-1, expressed with average RPKM >4 in one or more cell lines / days / treatments). (B) STRING analysis of transcription factors downregulated in hypertrophy. [Figure 6]FIG. 6 shows that TGFβ3 induces an articular chondrocyte phenotype. iPSC line MCRIi019-A was differentiated into sclerotomes, and cell pellets formed on day 4. At the end of day 6, all sclerotome pellets were supplemented with 20 ng / ml FGF2 for 14 days, and some pellets were treated with 10 ng / ml TGFβ3 from day 13. On day 20, pellets were transferred to rotary culture. (A) Histology and immunostaining at day 48. Untreated pellets contained large hypertrophic chondrocytes with deposition of extracellular matrix including collagen II and collagen X, but did not express the articular cartilage protein PRG4. Chondrocytes in TGFβ3-treated pellets were small, and the extracellular matrix contained collagen II and PRG4, but not collagen X. Scale bar is 500 μm. (B) Graph showing mRNA expression (log2 RPKM) of the 20 most highly expressed core matrisome components in TGFβ3-treated chondronoids at day 48. Expression in untreated chondronoids is shown for comparison. (C) Expression (mean log2 RPKM) of the 20 most upregulated and 20 most downregulated core matrisome genes in TGFβ3-treated chondronoids at day 48 (logFC, adj. P. value < 0.05, mean RPKM > 10 in at least one treatment group). (D) Graph showing mRNA expression (log2 RPKM) of the 20 most highly expressed transcription factors in TGFβ3-treated chondronoids at day 48. Expression in untreated chondronoids is shown for comparison. (E) Expression (mean log2 RPKM) of the 20 most upregulated and 20 most downregulated transcription factor genes in TGFβ3-treated chondronoids at day 48 (logFC, adj. P. value < 0.05, mean RPKM >10 in at least one treatment group). RNAseq data N = 4 technical replicates. [Figure 7]FIG. 7 shows transdifferentiation of hypertrophic chondrocytes into osteoblasts. (A) In vivo transdifferentiation. The iPSC line MCRIi001-A-BFP differentiated into sclerotomes, forming pellets on day 4. Starting at the end of day 6, cultures were supplemented with 20 ng / ml FGF2 for 14 days. Starting on day 35, cultures were treated with 10 nM T3 for 7 days, and then on day 42, hypertrophic chondroneoids were implanted subcutaneously into immunodeficient mice and harvested 13 weeks later. The implants were decalcified, sectioned, and stained with Safranin O for cartilage proteoglycans and fast green to highlight bone. Scale bar in left image is 1000 μm. Box indicates the magnified area in the middle image. The right image shows the same area from an adjacent section immunostained with human-specific Ku80 antibody. Scale bars in middle and right images are 100 μm. (B) In vitro transdifferentiation. MCRIi018-B differentiated into sclerotomes and formed pellets on day 4. At the end of day 6, pellets were supplemented with 20 ng / ml FGF2 for 2 weeks and then transferred to rotary culture. Starting on day 38, cultures were treated with 10 nM T3 for 14 days (D52 + T3) and then transferred to osteogenic medium for an additional 3 weeks (D73 osteo) or to osteogenic medium supplemented with 3 μM CHIR99021 for 7 days followed by culture in osteogenic medium alone (D73 osteo + CHIR). Toluidine blue staining was reduced in osteo + CHIR treated organoids, collagen I was deposited in the ECM in both osteogenic conditions, and positive von Kossa staining indicates that calcium was deposited in the ECM in both osteogenic conditions. Scale bar is 500 μm. (C) Organoids were fixed and then micro-CT scanned. Representative images are shown, which are the samples shown in red in the two graphs. Scale bar is 200 μm. The graphs show that organoids grown under the two conditions are similar in size, but significant calcium phosphate mineral is only evident in organoids grown in osteogenic conditions without the addition of CHIR99021. [Figure 7-1]Figure 7.1 shows that T3 treatment primes osteoblastic transdifferentiation. MCRIi001-A-2 differentiated into sclerotomes and formed pellets on day 4. Starting at the end of day 6, pellets were supplemented with 20ng / ml FGF2 for 14 days and then transferred to rotary culture and matured until day 68. Some pellets were then treated with 10nM T3 for 14 days. Starting at day 82, transdifferentiation was induced for 3 weeks in osteogenic medium alone (osteo) or for 7 days in osteogenic medium supplemented with 3μM CHIR99021 followed by culture in osteogenic medium alone (osteo + CHIR). Toluidine blue staining shows a greater loss of proteoglycans in CHIR99021 treated organoids. In both osteogenic differentiation conditions, there was more extensive and intense von Kossa staining in T3 pretreated organoids compared to those without T3 treatment. In pellets pretreated with T3, there was more intense von Kossa staining in pellets transdifferentiated in osteogenic medium alone compared to pellets transdifferentiated in osteogenic medium supplemented with CHIR99021 for the first 7 days. Scale bar, 500 μm. [Figure 7-2] Figure 7.2 shows further evidence of transdifferentiation in vitro. Starting at day 38, MCRIi018-B chondronoids were treated with 10 nM T3 for 14 days (T3) and then transferred to osteogenic medium for an additional 3 weeks. A, In osteogenic conditions, relative expression (RPKM) of cartilage genes COL2A1 and ACAN is decreased and osteoblast / osteocyte genes COL1A1, BGLAP, SPP1, and DMP1 are significantly increased. B, Immunostaining shows that collagen I and BGLAP are deposited in the ECM in osteogenic conditions (scale bar is 200 μm). Calcium phosphate mineral is deposited in osteogenic medium (μCT, scale bar is 500 μm). [Figure 8]Figure 8 shows gene expression changes during transdifferentiation of hypertrophic chondrocytes to osteoblasts. MCRIi018-B were differentiated into hypertrophic chondrocytes (T3) and then transferred to osteogenic medium for an additional 3 weeks (D73 O) or to osteogenic medium supplemented with 3 μM CHIR99021 for 7 days followed by culture in osteogenic medium alone (D73 OC). (A) Graph showing mRNA expression (RPKM) of key hypertrophic cartilage markers COL2A1, ACAN, and COL10A1, as well as genes COL1A1, SPP1, and IBSP highly expressed in preosteoblasts and osteoblasts. (B) Graph showing mRNA expression (log2 RPKM) of the 20 most highly expressed core matrisome components in organoids treated with D73 OC. Expression in samples treated with D73 O and D52 T3 is shown for comparison. (C) Expression (mean log2 RPKM) of the 20 most upregulated and 20 most downregulated core matrisome genes in organoids treated with D73 OC (logFC, adj. P value < 0.05, mean RPKM >10 in at least one treatment group). (D) Graph showing mRNA expression (log2 RPKM) of the 20 most highly expressed transcription factors in organoids treated with D73 OC. Expression in samples treated with D73 O and D52 T3 is shown for comparison. (E) Expression (mean log2 RPKM) of the 20 most upregulated and 20 most downregulated transcription factor genes in organoids treated with D73 OC (logFC, adj. P value < 0.05, mean RPKM >10 in at least one treatment group). RNAseq data n = 4 technical replicates. [Figure 8-1]Figure 8.1 shows gene expression changes during transdifferentiation of hypertrophic chondrocytes to osteoblasts. MCRIi019-A were differentiated into hypertrophic chondrocytes (T3) and then transferred to osteogenic medium for an additional 3 weeks (D72 O) or to osteogenic medium supplemented with 3 μM CHIR99021 for 7 days followed by culture in osteogenic medium alone (D72 OC). (A) Graph showing mRNA expression (RPKM) of key hypertrophic cartilage markers COL2A1, ACAN, and COL10A1, as well as genes COL1A1, SPP1, IBSP, and BGLAP highly expressed in preosteoblasts and osteoblasts. (B) Graph showing mRNA expression (log2 RPKM) of the 20 most highly expressed core matrisome components in D72 OC-treated organoids. Expression in D72 O and D51 T3-treated samples is shown for comparison. (C) Expression (mean log2 RPKM) of the 20 most upregulated and 20 most downregulated core matrisome genes in organoids treated with D72 OC (logFC, adj. P. value < 0.05, mean RPKM >10 in at least one treatment group). (D) Graph showing mRNA expression (log2 RPKM) of the 20 most highly expressed transcription factors in organoids treated with D72 OC. Expression in samples treated with D72 O and D51 T3 is shown for comparison. (E) Expression (mean log2 RPKM) of the 20 most upregulated and 20 most downregulated transcription factor genes in organoids treated with D72 OC (logFC, adj. P. value < 0.05, mean RPKM >10 in at least one treatment group). RNAseq data n = 4 technical replicates. [Figure 9]Figure 9 shows the expression of genes that characterize osteoblast lineage cells in vivo. MCRIi018-B were differentiated into hypertrophic osteoblasts (T3) and then transferred to osteogenic medium for an additional 3 weeks (O) or to osteogenic medium supplemented with 3 μM CHIR99021 for 7 days followed by culture in osteogenic medium alone (OC). The graph shows the expression (log2 RPKM) of genes that define osteoblast precursors, osteoblasts, and more mature osteoblast clusters in scRNAseq of cells isolated from mouse bone (Ayturk et al., 2020). The log fold change in expression (logFC) relative to hypertrophic chondrocytes is shown on the right. Genes that characterize mature osteoblasts are highly expressed in osteogenic culture conditions (O and OC) and many are highly upregulated compared to hypertrophic chondrocytes (T3). RNAseq data N = 4 technical replicates. [Figure 9-1]Figure 9.1 shows gene expression during transdifferentiation of hypertrophic chondrocytes into osteoblasts. MCRIi018-B differentiated into hypertrophic chondrocytes (T3) were then transferred to osteogenic medium for 3 weeks (D73 O) or osteogenic medium supplemented with 3 μM CHIR99021 for 7 days followed by culture in osteogenic medium alone for 2 weeks (D73 OC). (A) Expression of key in vivo markers of skeletal lineage cells. Gene lists were derived from single-cell RNAseq data from the distal femoral and proximal tibial epiphysis of young mice and include genes that characterize hypertrophic chondrocytes, preosteoblasts, and osteoblast clusters (Haseeb et al., 2021). In osteogenic culture conditions (O and OC), all genes follow the expression patterns identified during transdifferentiation in vivo, except for two genes MGP and SOX4. Nine of the 11 genes are more highly upregulated in OC-treated organoids than in O-treated organoids (compared to T3-treated hypertrophic chondrocytes). All five chondrocyte marker genes are more downregulated in OC-treated organoids than in O-treated organoids. (B) Expression of key osteoblast transcription factors. Gene list was taken from a recent review (Chan et al., 2021). All of these osteogenic transcription factors are expressed in osteogenic culture conditions (O and OC), and 12 of 13 are upregulated compared to hypertrophic chondrocytes (T3). All of these osteogenic transcription factors were expressed at similar or higher levels in organoids exposed to CHIR pulses (OC) than when differentiated in osteogenic medium alone (O). RNAseq data N = 4 technical replicates. [Figure 10-1]Figure 10 shows disease modeling using chondrocyte and osteoblast organoids differentiated from human iPSCs. Panels A and B: Using gene-edited iPSC lines with a genetic cartilage disease (achondroplasia) due to the COL2A1 p.G1113C mutation and syngeneic controls (Lilianty J, Bateman JF, Lamande SR. Stem Cell Res. 2021 Aug 25; 56:102515), iPSCs were differentiated into mature chondrocytes using our protocol. Collagen II extracellular matrix was assessed by collagen II immunohistochemistry (A) and electron microscopy (B). Both methods demonstrated reduced collagen II extracellular matrix in achondroplasia mutants. Scale bars = 200 μm (A) and 500 nm (B). Arrows indicate pathogenic intracellular accumulation of mutant misfolded collagen II in achondroplasia iPSC-derived cartilage organoids (A). In panel B, defective formation of cartilage collagen II extracellular matrix in achondroplasia-induced iPSC-derived cartilage organoids is visualized by electron microscopy. Scale bar = 500 nm. [Figure 10-2] FIG. 10 shows disease modeling using chondrocyte and osteoblast organoids differentiated from human iPSCs. Panels C-F: Using gene-edited iPSC lines with inherited osteogenesis imperfecta due to COL1A1 p.W1312C mutation and syngeneic controls (Howden S, et al., Stem Cell Res. 2019 Jul;38:101453), iPSCs were differentiated into osteoblasts using our protocol. Osteogenesis imperfecta iPSC-derived osteoblasts show reduced collagen I extracellular matrix formation by immunohistochemistry (C) and reduced mineralization by von Kossa staining (D). Scale bar = 500 μm. Reduced mineralization (bone formation) of osteogenesis imperfecta bone organoids is more dramatically shown by micro-CT analysis (E). Scale bar = 100 μm. Quantification of micro-CT data is shown in (F). N=5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] definitionDefinitions of common terms in cell and molecular biology and biochemistry can be found in The Merck Manual of Diagnosis and Therapy, 20th edition (ISBN 9780911910421, 0911910425), published by Merck Sharp & Dohme Corp., 2018; The Encyclopedia of Molecular Cell Biology and Molecular Medicine (ISBN 3527305424, 9783527305421), published by Blackwell Science Ltd., 2008; and Molecular Biology and Biotechnology: a Comprehensive Desk Reference (ISBN 1- 56081-569-8), published by Robert A. Meyers (ed.), published by VCH Publishers, Inc., 1995; Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2016 (ISBN 9780815345510, 0815345518); Lewin's Genes XI published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN 0124199542); Laboratory Methods in Enzymology: RNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN: 9780124200371, 0124200370);Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385);Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in and Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe (eds.), John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737); Immunological Methods, Ivan Lefkovits, Benvenuto Pemis (eds.), Elsevier Science, 2014 (ISBN: 9781483269993, 148326999X), the contents of which are all incorporated herein by reference in their entireties.

[0025] As used in this specification and the appended claims, singular and singular terms, such as "a" and "the," optionally include plural referents unless the context clearly dictates otherwise. For example, "a" cell includes one cell, one or more cells, or multiple cells.

[0026] As used herein, the term "about" means, unless stated to the contrary, ±10%, more preferably ±5%, more preferably ±1% of the specified value. Thus, when used in conjunction with a specified value, for example, "about X" should be understood to mean the specified value itself or the specified value ±10%, more preferably ±5%, more preferably ±1% of the specified value.

[0027] The term "and / or," e.g., "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be interpreted as providing explicit support for both meanings or either meaning.

[0028] It will be understood that throughout this specification the term "comprise" or variations thereof, such as "comprises" or "comprising" implies the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0029] Numeric ranges are inclusive of the numbers that define the range. Every numerical upper limit given throughout this specification is intended to include all of the lower limits, as if such lower numerical limits were expressly written herein. Every numerical lower limit given throughout this specification will include all of the upper limits, as if such upper numerical limits were expressly written herein. Every numerical range given throughout this specification will include all of the narrower numerical ranges that are subsumed within the broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0030] The headings provided herein are not intended to limit the disclosure.

[0031] Throughout this specification, references to specific genes or proteins are used interchangeably, and a person of ordinary skill in the art will understand in context whether the reference is intended to be a reference to a specific gene or the protein encoded by that gene.

[0032] The terms "human pluripotent stem cells" and "hPSCs" refer to cells that are derived from, obtained from, or arise from human tissue and exhibit pluripotency. hPSCs may be human embryonic stem cells or human induced pluripotent stem cells.

[0033] Human pluripotent stem cells can be derived from the inner cell mass or reprogrammed from a number of fetal or adult somatic cell types using Yamanaka factors. hPSCs can be generated using somatic cell nuclear transfer.

[0034] The terms "human embryonic stem cell," "hES cell," and "hESC" refer to cells derived from, obtained from, or arising from a human embryo or blastocyst, which are self-renewing, pluripotent or totipotent, and have the ability to give rise to all of the cell types present in the adult animal. Human embryonic stem cells (hESC) can be isolated, for example, from human blastocysts obtained from human in vivo preimplantation embryos, from in vitro fertilized embryos, or from one-cell human embryos expanded to the blastocyst stage.

[0035] The terms "induced pluripotent stem cells" and "iPSCs" refer to cells derivable from, obtained from, or arising from any type of human adult somatic cell that has been reprogrammed to a pluripotent state through expression of exogenous genes, such as transcription factors, including a preferred combination of OCT4, SOX2, KLF4, and c-MYC. hiPSCs exhibit a level of pluripotency equivalent to hESCs, but can be derived from patients for autologous therapy, with or without simultaneous genetic correction prior to differentiation and cell delivery.

[0036] More generally, the methods disclosed herein may be applied to any pluripotent stem cells derived from any patient, or to hPSCs that are subsequently modified using gene editing to generate models of mutants, or to mutant hPSCs that have been corrected using gene editing, which may be by CRISPR, TALEN, or ZF nuclease technology.

[0037] As used herein, the term "cell culture" refers to any in vitro culture of cells. The term "culturing" refers to the process of growing and / or maintaining and / or manipulating cells. This term includes continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other population of cells maintained in vitro, including oocytes and embryos. As used herein, the terms "primary cell culture" and "primary culture" refer to cell cultures obtained directly from cells in vivo, for example, from animal or human tissue specimens or biopsies. These cultures can be derived from adult as well as fetal tissue.

[0038] A "progenitor cell" is a cell that can differentiate along one or more developmental pathways, with or without self-renewal. Typically, progenitor cells are unipotent or oligopotent, and are capable of at least limited self-renewal.

[0039] The terms "differentiate," "differentiating," and "differentiated" refer to the progression of a cell from an early or earlier stage in a developmental pathway to a later or more mature stage in a developmental pathway. It will be understood that "differentiated" in this context does not mean or imply that a cell has fully differentiated and lost pluripotency or the ability to progress further along a developmental pathway or along other developmental pathways. Differentiation may involve the division of a cell.

[0040] As is well understood in the art, the differentiation stage or state of a cell is characterized by the expression and / or non-expression of one of a number of markers. In this context, "marker" refers to a nucleic acid or a protein encoded by the genome of a cell, cell population, lineage, compartment, or subset, whose expression or pattern of expression changes throughout development. Expression of a nucleic acid marker may be detected or measured by any technique known in the art, including, but not limited to, amplification of nucleic acid sequences (e.g., polymerase chain reaction) and hybridization of nucleic acids (e.g., microarrays, northern hybridization, in situ hybridization). Expression of a protein marker may be detected or measured by any technique known in the art, including, but not limited to, flow cytometry, immunohistochemistry, immunoblotting, protein arrays, protein profiling (e.g., 2D gel electrophoresis).

[0041] Such terms are common and well understood by those skilled in the art when characterizing the phenotype of a cell. By way of further guidance, when a cell is said to be positive for a given marker, e.g., a given gene or gene product, or to express or contain expression of the marker, the skilled artisan will conclude the presence or evidence of a distinctive signal for the marker when performing an assay that can detect or quantify the marker in or on the cell. Preferably, the presence or evidence of a distinctive signal for the marker will be concluded based on a comparison of the assay results obtained for the cell with the results of the same assay performed on a negative control (e.g., a cell known not to express the marker) and / or a positive control (e.g., a cell known to express the marker). In the case of measurement methods that allow for quantitative assessment of a marker, a positive cell may generate a signal for the marker that is at least 1.5 times higher, such as at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, or even higher, than the signal generated for the marker by a reference cell (e.g., a negative control cell) or the average signal generated for the marker by a population of reference or negative control cells. Further, a positive cell may generate a signal for the marker that is 3.0 or more standard deviations, such as 3.5 or more, 4.0 or more, 4.5 or more, or 5.0 or more standard deviations, than the average signal generated for the marker by a population of reference or negative control cells.

[0042] As used herein, "sclerotome cells" or "sclerotome" are used interchangeably and refer to an iPSC-derived population enriched for cells that are functionally and phenotypically similar to the portions of somites in vertebrate embryos that give rise to bone or other skeletal tissue. In some embodiments, "sclerotome cells" or "sclerotome" express any one of PAX1, SOX9, FOXC2, PAX9, NKX3.2 / BAPX1, and TWIST1. In some embodiments, sclerotome cells express PAX1, SOX9, and FOXC2, and may also express one or more additional markers selected from PAX9, NKX3.2 / BAPX1, and TWIST1.

[0043] As used herein, "chondrocyte precursor cells" and "chondrocyte precursors" and "cartilage precursors" are used interchangeably and refer to precursor cells derived from sclerotome cells and specifically pre-positioned to differentiate into chondrocytes. In some embodiments, "chondrocyte precursor cells" and "chondrocyte precursors" and "cartilage precursors" may express some or all of the genes expressed by chondrocytes. In some embodiments, "chondrocyte precursor cells", "chondrocyte precursors" and "cartilage precursors" express about 10-50 fold less COL2A1 and about 50-200 fold less ACAN (relative to housekeeping genes) when compared to chondrocytes.

[0044] As used herein, "chondrocyte," "chondrocytes," and "chondrocyte cells" refer to cells derived from chondrocyte precursors. In some embodiments, a "chondrocyte" expresses COL2A1 and ACAN in a COL2A1:ACAN ratio ranging from about 10:1 (or 20:1 to 5:1), and does not substantially express COL10A1. In some embodiments, a "chondrocyte" expresses COL2A1 and ACAN, and one or more additional genes selected from the group consisting of COL11A1, COL11A2, COL9A1, COL9A2, COL9A3, MATN1, and MATN3. "Chondrocyte-like cell" refers to a cell that substantially reflects the phenotype of, or is functionally equivalent to, a chondrocyte.

[0045] As used herein, "hypertrophic chondrocytes" and "hypertrophic cartilage" refer to cells that express COL2A1 and COL10A1. In some embodiments, hypertrophic chondrocytes and hypertrophic cartilage-like tissue, hypertrophic cartilage, or combinations thereof, express COL2A1 and COL10A1 in a ratio ranging from about 1:1 to about 2.5:1. "Hypertrophic chondrocyte-like cells" and "hypertrophic cartilage-like tissue" refer to cells and tissues that substantially reflect the phenotype of, or are functionally equivalent to, hypertrophic chondrocytes.

[0046] As used herein, "articular chondrocytes" and "articular cartilage" refer to cells that express PRG4. In some embodiments, "articular chondrocytes" and "articular cartilage" express COL2A1, ACAN, and PRG4, and optionally one or more additional genes selected from the group consisting of ASPN, CILP, CILP2. In some embodiments, "articular chondrocytes" and "articular cartilage" also express one or more of EMILIN1, EMILIN3, FBLN1, and FBLN3. "Articular chondrocyte-like cells" and "articular cartilage-like tissue" refer to cells and tissue, respectively, that substantially reflect the phenotype of articular chondrocytes or are functionally equivalent to articular chondrocytes or articular cartilage.

[0047] As used herein, "osteoblasts" or bone-like organoids express COL1A1 and COL1A2. In some embodiments, "osteoblasts" or bone-like organoids also express one or more of MEPE, IBSP (bone sialoprotein 2), SPP1 (osteopontin), and DMP1.

[0048] As used herein, the terms "culture medium" and "cell culture medium" refer to a medium suitable for supporting the growth of cells in vitro (i.e., cell cultures, cell lines, etc.). The term is not intended to be limited to any particular culture medium. For example, the definition is intended to encompass maintenance media as well as other media for differentiation or specialization of cells. Indeed, the term is intended to encompass any culture medium suitable for cell culture and growth of the cells of interest.

[0049] As used herein, "tissue" refers to an aggregation of cells. In some embodiments, the cells in a tissue are in close contact or fused.

[0050] As used herein, "scaffold" refers to synthetic scaffolds such as polymeric scaffolds and porous hydrogels, non-synthetic scaffolds such as preformed extracellular matrix layers, dead cell layers, and decellularized tissues, as well as any other type of preformed scaffold that is integral to the physical structure of the engineered tissue and cannot be removed from the tissue without damaging / destroying said tissue. In further embodiments, the decellularized tissue scaffold comprises decellularized native tissue or decellularized cellular material produced by cultured cells in any manner, such as a layer of cells that have been killed or decellularized leaving behind the extracellular matrix (ECM) that they produced while alive.

[0051] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject for which diagnosis, treatment, or therapy is desired, particularly humans.

[0052] The terms "treatment", "treating", "treating" and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely stabilizing or curing the disease and / or adverse effects caused by the disease. "Treatment", as used herein, includes any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease or condition from arising in a subject prone to, but not yet diagnosed as having, the disease or condition, (b) arresting the symptoms of the disease, i.e., arresting its progression, or (c) alleviating the symptoms of the disease, i.e., causing regression of the disease or condition.

[0053] The terms "reduce", "reduced", "reduction", "to a lesser extent", or "inhibit" are all used herein to mean a decrease or lowering of a characteristic, level, or other parameter in a statistically significant amount. In some embodiments, "reduced", "reduction", "reduce", or "inhibit" typically means a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduce" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" means 100% inhibition compared to a reference level. A decrease may include a decrease, preferably to a level accepted to be within the normal range for individuals without a given disorder.

[0054] The terms "increased", "increase", "enhance", or "enhance", or "activate", or "greatly" are all used herein generally to mean an increase in a characteristic, level, or other parameter in a statistically significant amount. For the avoidance of doubt, the terms "increased", "increase", "greatly", "enhance", or "activate" can mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase between 10-100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 1000-fold, or more compared to a reference level.

[0055] As used herein, "homogeneous" is intended to mean a population of cells that has a uniform appearance and has uniform expression of key markers indicative of the population. The term "homogeneous" is intended to encompass a population of cells that is primarily of the same phenotype and may contain up to 5% or up to 10% cells of a different phenotype. As an example, a composition comprising a homogeneous population of iPSC-derived chondrocytes may comprise 90%, 95% or more iPSC-derived chondrocytes. As another example, a composition comprising a homogeneous population of iPSC-derived hypertrophic chondrocytes may comprise 90%, 95% or more iPSC-derived hypertrophic chondrocytes. As a further example, a composition comprising a homogeneous population of iPSC-derived articular chondrocytes may comprise 90%, 95% or more iPSC-derived articular chondrocytes.

[0056] As used herein, a "reference level" refers to the level of a marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., a cell, tissue, or biological sample obtained from a healthy subject, or a biological sample obtained from a subject at a previous time, e.g., a cell, tissue, or biological sample obtained from a patient before being diagnosed with a disease, or a biological sample that has not been contacted with an agent or composition disclosed herein). Alternatively, a reference level can refer to the level of a given marker or parameter in a subject, organ, tissue, or cell prior to administration of a treatment, e.g., by administration of an agent or transplant composition.

[0057] As used herein, "control" or "suitable control" refers to an untreated, otherwise identical cell, subject, organism, or population (e.g., a cell, tissue, or biological sample that has not been contacted with an agent or composition described herein) to a cell, tissue, biological sample, or population that has been contacted with or treated with a given treatment. For example, a suitable control can be a cell, tissue, organ, or subject that has not been contacted with an agent described herein or administered a cell described herein.

[0058] In one or more embodiments described herein, evaluating the expression of various genes comprises comparing fold changes. In one embodiment, fold changes are used to measure changes in the expression levels of genes. In one embodiment, the expression of genes can be expressed as relative expression compared to housekeeping genes. In one embodiment, fold changes are measured by RPKM. As used herein, the term "RPKM" refers to "Reads Per Kilobase per Million mapped reads". The term RPKM refers to a method of quantifying gene expression from RNA sequencing data by normalizing for total read length and number of sequencing reads. In one embodiment, the calculation of RPKM provides a normalization for the comparison of gene coverage values. The RPKM value corrects for differences in both the sequencing depth of the samples and the gene length. In one example, the RPKM can be calculated by the following formula: numReads / (geneLength / 1000×totalNumReads / 1,000,000) Here, "numReads" means the number of reads mapped to the gene sequence, "geneLength" means the length of the gene sequence, and "totalNumReads" means the total number of mapped reads for the sample.

[0059] The terms "agonist" or "activator" are used interchangeably and as used herein refer to, for example, an activator of a pathway or signaling molecule. An agonist of a molecule can retain substantially the same, or a subset, of the biological activities of that molecule (e.g., FGF). For example, an FGF agonist, or "FGF activator," or "FGF pathway activator" refers to a molecule that selectively activates FGF signaling.

[0060] The term "inhibitor" as used herein refers to, for example, a selective inhibitor of a pathway or signaling molecule. An inhibitor or antagonist of a molecule (e.g., an inhibitor of BMP4) can inhibit one or more of the activities of the naturally occurring form of the molecule. For example, a BMP4 inhibitor is a molecule that selectively inhibits BMP4 signaling.

[0061] Various embodiments are described hereafter. It should be noted that a particular embodiment is not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. An aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and may be practiced with any other embodiment. References throughout this specification to "one embodiment," "embodiment," or "exemplary embodiment" mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. That is, the appearance of the phrases "in one embodiment," "in an embodiment," or "exemplary embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, while some embodiments described herein include some features and not other features included in other embodiments, it is meant that combinations of features of different embodiments are within the scope of the invention. For example, in the appended claims, any of the claimed embodiments may be used in any combination. Any example or embodiment herein should be considered as applying mutatis mutandis to any other example or embodiment, unless specifically stated otherwise.

[0062] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent methods and systems as described herein are clearly within the scope of the present disclosure.

[0063] Throughout this specification, unless specifically stated otherwise or unless the context requires otherwise, reference to a single step, composition of matter, group of steps, or group of composition of matter should be considered to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of composition of matter.

[0064] The present disclosure will now be described by the following non-limiting examples and with reference to the accompanying drawings. Although the examples herein relate to humans and the terminology is primarily directed to humans, the concepts described herein are applicable to other animals. These and other aspects and features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the disclosure as set forth hereinafter.

[0065] The reference herein to a patent document or other matter offered as prior art should not be considered an admission that the document or matter was publicly known or that the information it contains was part of the common general knowledge at the priority date of any claim.

[0066] Chondrocyte precursors and chondrocytes Described herein are methods for producing chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof, from sclerotome cells derived from a population of stem cells. Products of such methods are also described herein. Cartilage or cartilage-like tissue produced by the novel methods developed by the inventors results in a product that is more phenotypically accurate and of higher quality than those produced by previously described methods.

[0067] Cartilage ECM has a characteristic composition of collagen types and non-collagenous components. Although all of these are important, it is the distribution of collagen types that provides the most distinctive signature of cartilage tissue. The pattern of collagen expression alone will likely be widely accepted as the defining feature of cartilage tissue.

[0068] Articular cartilage exhibits the following collagen expression pattern (% of total collagen): collagen II: 90-95% (main fibril component), collagen XI 3% (associated with collagen II fibrils), collagen IX: 1% (associated with collagen II fibrils), collagen X <1%, collagen VI: 0-1%. Other proteoglycans, glycoproteins, and proteins important in development and articular cartilage include aggrecan (ACAN), link protein (HAPLN1), biglycan (BGN), decorin (DCN), fibromodulin (FBN), chondroadherin (CHAD), CILP, COMP, matrilin 1 (MATN1), matrilin 3 (MATN3). In addition, articular cartilage contains other ECM components including PRG4 and elastic fiber components ELN, LTBP2, TGFBI, MFAP4, and MFAP5, among others.

[0069] Growth cartilage plates have many of the same ECM components as articular cartilage, but in different ratios. Collagen composition: Growth cartilage plates have a low percentage of collagen II (and associated collagens IX and XI) as a percentage of total collagen. Collagen X is expressed only by growth cartilage plates and is therefore a sentinel marker for this type of cartilage. It becomes the predominant collagen in growth cartilage plates. Various cartilage non-collagenous protein components, such as ACAN, MATN1, and COMP, are decreased. Other non-collagenous proteins, such as IBSP, MGP, and SPP1, are upregulated.

[0070] The inventors have discovered that by utilizing the novel protocol described herein, it is possible to produce 3D aggregates of chondrocytes (chondrocyte organoids or "chondroids") with collagen II (COL2A1) as the major component and small amounts of collagen II associated collagens IX and XI (COL9A1, COL9A2, COL9A3, COL11A1, COL11A2). The chondroids have an organized structure of this collagen II matrix and show the characteristic morphology of chondrocytes and collagen II fibrils. Importantly, at this stage of development, collagen X or collagen I are absent, which may indicate poor chondrocyte formation (collagen I) or hypertrophy (collagen X). Collagen X is also upregulated especially in osteoarthritic cartilage. Thus, having collagen X may be detrimental to the use of this cartilage for cartilage repair. These findings indicate that the cartilage or cartilage-like material produced by these methods is of high quality and phenotypically accurate.

[0071] Thus, one aspect of the invention includes a method of producing chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, the method comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte progenitor cells; and b) culturing the three-dimensional aggregates of chondrocyte precursor cells generated in step a) in the absence of an activator of the FGF pathway to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells expressing COL2A1 and ACAN. Including, The method includes transferring the three-dimensional aggregates of sclerotome cells or the three-dimensional aggregates of chondrocyte precursor cells into orbital culture.

[0072] In an embodiment, the culturing of the three-dimensional aggregates in step a) and / or b) is carried out in the absence of an antagonist of TGF-beta and / or in the absence of an inhibitor of the BMP pathway.In another embodiment, the culturing of the three-dimensional aggregates in step a) is carried out in the absence of an antagonist of TGF-beta and / or in the absence of an inhibitor of the BMP pathway.

[0073] In an embodiment, the culture of the three-dimensional aggregates in steps a) and b) is a non-adherent, low-adherent or suspension culture.

[0074] In an embodiment, the cultivation of the three-dimensional aggregates in steps a) and b) is performed in a pellet culture format. For clarity, steps a) and b) are not performed in a monolayer culture format.

[0075] In an embodiment, the culturing of the three-dimensional aggregates in step b) is carried out in orbital culture.

[0076] The inventors have discovered that culturing the sclerotome with a pulse of an FGF pathway activator produces chondrocytes with a more favorable ECM profile. In an embodiment, the duration of the culture of step a) in the presence of an FGF pathway activator is for a period of at least 7 days. In another embodiment, the duration of the culture of step a) in the presence of an FGF pathway activator is for a period of 7 to about 42 days. In another embodiment, the duration of the culture of step a) in the presence of an FGF pathway activator is for a period of 7 to about 21 days. In another embodiment, the duration of the culture of step a) in the presence of an FGF pathway activator is for a period of about 10 to 17 days. In another embodiment, the duration of the culture of step a) in the presence of an FGF pathway activator is for a period of about 14 days.

[0077] In the methods described herein, agonists or activators, inhibitors, or cell culture components may be added at the beginning of a particular period of time, or may be added repeatedly during the period, for example, for medium changes. For example, activators of the FGF pathway may be added with replacement of culture medium during the above-mentioned periods.

[0078] In various embodiments of the present invention and other embodiments described herein, the FGF pathway activator is selected from the group consisting of FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20, and FGF23. In a preferred embodiment, the FGF pathway activator is FGF2.

[0079] In another embodiment of the various aspects and other embodiments described herein, the FGF pathway activator is present in an amount of about 1 ng / ml to about 100 ng / ml. In another embodiment, the FGF pathway activator is present in an amount of about 10 ng / ml to about 50 ng / ml. In another embodiment, the FGF pathway activator is present in an amount of about 20 ng / ml.

[0080] In an embodiment, the culture period in step b) in the absence of an activator of the FGF pathway is at least 7 days in duration. In another embodiment, the culture period in step b) is from about 7 to about 28 days in duration. In another embodiment, the culture period in step b) is from about 7 to about 21 days in duration. In another embodiment, the culture period in step b) is from about 10 to 17 days in duration. In another embodiment, the culture period in step b) is from about 14 days in duration.

[0081] In an embodiment, the culturing in step b) is carried out in the absence of any growth factors.

[0082] In embodiments, the 3D aggregates of chondrocyte precursor cells are cultured in step b) for a sufficient period of time to achieve expression of collagen II (COL2A1) and aggrecan (ACAN). In some embodiments, the 3D aggregates of chondrocyte precursor cells are cultured in step b) for a sufficient period of time to achieve expression of collagen II (COL2A1) and aggrecan (ACAN) in a ratio ranging from 20:1 to 5:1 relative to each other. In some embodiments, the ratio of collagen II (COL2A1) to aggrecan (ACAN) is about 10:1.

[0083] In an embodiment, the 3D aggregates of chondrocyte precursor cells are cultured for a sufficient period of time to achieve in step b) an increase in the relative expression of collagen II (COL2A1) and aggrecan (ACAN) over housekeeping genes of at least 10-fold and at least 20-fold, respectively. In some embodiments, the increase in the relative expression of collagen II (COL2A1) and aggrecan (ACAN) over housekeeping genes is at least 40-fold and at least 100-fold, respectively. In one embodiment, the housekeeper gene is GAPDH.

[0084] In another embodiment, COL1A1, which is underexpressed relative to cartilage markers COL2A1 and ACAN in cartilage precursors, is not increased in expression relative to GAPDH during transition of the cartilage precursors to chondrocytes.Thus, in another embodiment, the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof, do not express collagen COL1A1 at a level greater than about 0.2% of the level of COL2A1.

[0085] In another embodiment, COL1A2, which is underexpressed relative to cartilage markers COL2A1 and ACAN in cartilage precursors, does not increase in expression relative to GAPDH during transition of the cartilage precursors to chondrocytes.Thus, in another embodiment, the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof do not express collagen COL1A2 at a level greater than about 0.2% of the level of COL2A1.

[0086] In another embodiment, the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof express one or more additional genes selected from the group consisting of COL11A1, COL11A2, COL9A1, COL9A2, COL9A3, MATN1, and MATN3. In another embodiment, the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof express COL2A1, ACAN, COL11A1, COL11A2, COL9A1, COL9A2, COL9A3, MATN1, and MATN3.

[0087] In another embodiment, the chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, do not express appreciable levels of collagen COL10A1.

[0088] In another embodiment, the 3D aggregates of chondrocyte precursor cells are cultured for a period and under conditions sufficient to produce a 3D aggregate of chondrocytes or chondrocyte-like cells in step b), wherein the collagen expression pattern in the 3D aggregates of chondrocytes or chondrocyte-like cells is about 30-50% COL2A1, about 5-40% COL11A1 and COL11A2, respectively, and about 1-30% COL9A1, COL9A2, COL9A3, respectively. In another embodiment, the collagen expression pattern in the 3D aggregates of chondrocytes or chondrocyte-like cells is about 40-50% COL2A1, about 10-30% COL11A1 and COL11A2, respectively, and about 5-15% COL9A1, COL9A2, COL9A3, respectively. In another embodiment, the collagen expression pattern in 3D aggregates of chondrocytes or chondrocyte-like cells is about 40% to about 50% COL2A1, about 15% to about 25% COL11A1, about 5% to about 20% COL11A2, about 5% to about 30% COL9A1, about 1% to about 10% COL9A2, and about 1% to about 10% COL9A3.

[0089] Through detailed studies described herein, the inventors have surprisingly found that the transfer of 3D aggregates of chondrocyte precursors to orbital rotating culture affects the rate of maturation into chondrocytes and in particular the composition of the resulting chondrocytes. That is, orbital rotating culture affects the rate and specificity of differentiation. Differentiation of 3D aggregates of sclerotoma cells cultured in static conditions versus rotating culture showed that rotating culture retards chondrocyte development or slows hypertrophy. Surprisingly, the inventors have found that this effect on chondrocyte development can be overcome by altering the timing of the transfer to rotating culture. As detailed in the examples described herein, it was found that a slower transfer to rotating culture resulted in hypertrophied chondrocytes of more uniform composition with significantly enlarged morphology and expression of collagen X, while an earlier transfer to rotating or static culture resulted in a composition with a higher number of non-chondrocytes. Thus, the use of rotating culture and the timing of such use can be exploited to vary the culture product obtained depending on the desired outcome.

[0090] Thus, in one embodiment, the method comprises transferring the 3D aggregates of sclerosing cells to orbital rotation culture. In another embodiment, the method comprises transferring the 3D aggregates of sclerosing cells to orbital rotation culture 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days after the start of step a) of the method. In another embodiment, the method comprises slowing the transfer of the 3D aggregates of sclerosing cells to orbital rotation culture. In another embodiment, the method comprises transferring the 3D aggregates of sclerotome cells to orbital rotation culture after 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In another embodiment, the method comprises transferring the 3D aggregates of sclerotome cells to orbital rotation culture any time after day 5, after day 6, or after day 7. In another embodiment, the method comprises transferring the 3D aggregates of sclerotome cells to orbital rotation culture any time between day 7 and day 14. In a further embodiment, the method comprises transferring the 3D aggregates of chondrocyte precursors (e.g., the product of step a)) to orbital rotation culture. In another embodiment, the method comprises transferring the 3D aggregates of chondrocyte precursor cells to orbital culture 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after the start of step b) of the method.

[0091] In one embodiment, the 3D aggregate of sclerotome cells is a dense aggregate of cells. In one embodiment, the 3D aggregate of cells is comprised of about 10,000 cells to about 1,000,000 cells. In another embodiment, the 3D aggregate of sclerotome cells is comprised of about 5×10 4 Cells ~ approx. 5 x 10 5 In another embodiment, the 3D aggregate of sclerotome cells comprises about 1×10 5 cells ~ approx. 3 x 10 5 In another embodiment, the 3D aggregate of sclerotome cells comprises about 1×10 5 ~5×10 5 , preferably about 2 x 105 It consists of cells.

[0092] Articular chondrocytes and articular cartilage In one embodiment, the method is for producing articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, and step c) comprises culturing three-dimensional aggregates of said chondrocytes or chondrocyte-like cells with an agonist of TGF-beta to produce articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof.

[0093] Another aspect of the invention includes a method of producing articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, the method comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte progenitor cells; b) culturing the three-dimensional aggregates of chondrocyte precursor cells with an activator of the FGF pathway and an agonist of TGF-beta to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells expressing COL2A1 and ACAN; c) culturing the three-dimensional aggregates of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta for an extended period of time to produce articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof. Including, The method includes transferring the three-dimensional aggregates of sclerotome cells, the three-dimensional aggregates of chondrocyte precursor cells, or the three-dimensional aggregates of chondrocytes or chondrocyte-like cells into orbital culture.

[0094] In an embodiment, the method is for generating articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, and step c) comprises culturing three-dimensional aggregates of said chondrocytes or chondrocyte-like cells with an agonist of TGF-beta to produce PRG4-expressing articular (non-hypertrophic) chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof.

[0095] In an embodiment, the TGF-beta agonist is selected from TGFb1, TGFb2, TGFb3, and / or combinations thereof, hi an embodiment, the TGF-beta agonist is TGFb3.

[0096] In an embodiment, the step of culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta is for a period of at least 2 weeks. In an embodiment, the step of culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta is for a selected period of about 2 weeks to about 10 weeks. In an embodiment, the step of culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta is for a selected period of about 2 weeks to about 7 weeks. In an embodiment, the step of culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta is for a period selected from about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, and about 7 weeks. In an embodiment, the step of culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta is for a period of about 5 weeks.

[0097] In an embodiment, the culture period in step a) in the presence of an FGF pathway activator is for a period of at least 2 to 21 days. In another embodiment, the culture period in step a) in the presence of an FGF pathway activator is for a period of 2 to about 14 days. In another embodiment, the culture period in step a) in the presence of an FGF pathway activator is for a period of about 5 to 10 days. In another embodiment, the culture period in step a) in the presence of an FGF pathway activator is for a period of about 7 days.

[0098] In an embodiment, the culture period in step b) in the presence of an FGF pathway activator and an agonist of TGF-beta is at least 2 days and up to 21 days. In another embodiment, the culture period in step b) in the presence of an FGF pathway activator and an agonist of TGF-beta is 2 to about 14 days. In another embodiment, the culture period in step b) in the presence of an FGF pathway activator and an agonist of TGF-beta is about 5 to 10 days. In another embodiment, the culture period in step b) in the presence of an FGF pathway activator and an agonist of TGF-beta is about 7 days.

[0099] In an embodiment, the culture period in step c) in the presence of an agonist of TGF-beta is at least 2 to 42 days in duration. In another embodiment, the culture period in step c) in the presence of an agonist of TGF-beta is at least 14 to about 35 days in duration. In another embodiment, the culture period in step c) in the presence of an agonist of TGF-beta is at least 21 to about 35 days in duration. In another embodiment, the culture period in step c) in the presence of an agonist of TGF-beta is at least 28 days in duration.

[0100] In an embodiment, the culturing of the three-dimensional aggregates in step a) and / or in step b) is performed in the absence of an agonist of TGF-beta and / or in the absence of an inhibitor of the BMP pathway.

[0101] In an embodiment, the cultivation of the three-dimensional aggregates in steps a), b) and c) is carried out in a pellet culture format. For clarity, steps a), b) and c) are not carried out in a monolayer culture format.

[0102] In an embodiment, the culturing of the three-dimensional aggregates in step c) is carried out in orbital culture.

[0103] In another embodiment, the articular chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof express one or more markers selected from COL2A1, ACAN, and PRG4, and optionally ASPN, CILP, CILP2, EMILIN1, EMILIN3, FBLN1, and FBLN3.

[0104] In another embodiment, the chondrocyte or chondrocyte-like cell aggregates are cultured with TGF-beta3 for a period and under conditions sufficient to produce a population of articular chondrocytes or chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, wherein the collagen expression pattern in the articular chondrocytes or chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof is about 70-95% collagen 2 (COL2A1) and about 1% to about 30% COL9A1 and COL9A2, respectively. In some embodiments, the collagen expression pattern in the articular chondrocytes or chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof is about 80-95% collagen 2 (COL2A1) and about 1% to about 10% COL9A1 and COL9A2, respectively. In some embodiments, collagen 2 (COL2A1) represents about 85% to about 95% of the total collagen. In some embodiments, collagen 2 (COL2A1) represents about 85% to about 95% of total collagen, and COL9A1 and COL9A2 together represent about 5% to about 15%.

[0105] Hypertrophic chondrocytes and hypertrophic cartilage In an embodiment, the method is for generating hypertrophic chondrocytes or hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof, the method comprising the steps of: a) culturing a three-dimensional aggregate of scleroblast cells with an activator of the FGF pathway to produce a three-dimensional aggregate of chondrocyte progenitor cells; and b) culturing the three-dimensional aggregate of chondrocyte progenitor cells generated in step a) in the absence of said activator of the FGF pathway to produce a three-dimensional aggregate of chondrocytes or chondrocyte-like cells expressing COL2A1 and ACAN; and c) culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with triiodothyronine (T3) to produce hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof expressing COL10A1, and comprising transferring the three-dimensional aggregate of scleroblast cells or the three-dimensional aggregate of chondrocyte progenitor cells into orbital culture.

[0106] In embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with T3 for an extended period of time. In some embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with T3 for a period ranging from about 2 days to about 4 weeks. In some embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with T3 for a period of at least 1 week. In some embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with T3 for a period of at least 2 weeks. In embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with T3 for a period selected from about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with T3 for a period of from about 1 week to about 4 weeks. In embodiments, the three-dimensional aggregate of chondrocytes or chondrocyte-like cells is cultured with T3 for a period of from about 2 weeks to about 4 weeks.

[0107] In an embodiment, the culture period of step a) in the presence of an FGF pathway activator is for a period of at least 7 days. In another embodiment, the culture period of step a) in the presence of an FGF pathway activator is for a period of 7 to about 42 days. In another embodiment, the culture period of step a) in the presence of an FGF pathway activator is for a period of 7 to about 21 days. In another embodiment, the culture period of step a) in the presence of an FGF pathway activator is for a period of about 10 to 17 days. In another embodiment, the culture period of step a) in the presence of an FGF pathway activator is for a period of about 14 days.

[0108] In an embodiment, the culture period of step b) in the absence of an activator of the FGF pathway is for a period of at least 7 days. In another embodiment, the culture period of step b) is for a period of about 7 to about 42 days. In another embodiment, the culture period of step b) is for a period of about 7 to about 28 days. In another embodiment, the culture period of step b) is for a period of about 7 to about 21 days. In another embodiment, the culture period of step b) is for a period of about 10 to 17 days. In another embodiment, the culture period of step b) is for a period of from about 14 days.

[0109] In an embodiment, the culturing in step b) is carried out in the absence of any growth factors.

[0110] In an embodiment, the culturing of the three-dimensional aggregates in step a), step b) and / or step c) is performed in the absence of an antagonist of TGF-beta and / or in the absence of an inhibitor of the BMP pathway. In an embodiment, the culturing of the three-dimensional aggregates in step a) is performed in the absence of an antagonist of TGF-beta and / or in the absence of an inhibitor of the BMP pathway.

[0111] In an embodiment, the culturing of the three-dimensional aggregate in step a), step b) and / or step c) is performed in the absence of an activator of the BMP pathway and / or an activator of the Wnt pathway.In an embodiment, the culturing of the three-dimensional aggregate in step c) is performed in the absence of an activator of the BMP pathway and / or an activator of the Wnt pathway.

[0112] In an embodiment, the cultivation of the three-dimensional aggregates in steps a), b) and c) is carried out in a pellet culture format. For clarity, steps a), b) and c) are not carried out in a monolayer culture format.

[0113] In an embodiment, the culturing of the three-dimensional aggregates in steps b) and c) is carried out in orbital culture.

[0114] In another embodiment, the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof express COL2A1 and COL10A1.In another embodiment, the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof express COL2A1 and COL10A1 in a ratio ranging from about 1:1 to about 2.5:1.

[0115] In another embodiment, the chondrocyte or chondrocyte-like cell aggregates are cultured with T3 for a period and under conditions sufficient to produce a population of hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof, wherein the collagen expression pattern in the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof is about 30-50% collagen 2 (COL2A1) and about 30-50% COL10A1, and about 1% to about 10% COL9A1 and COL9A2, respectively. In some embodiments, the collagen expression pattern in the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof is about 40-50% collagen 2 (COL2A1) and about 40-50% COL10A1, and about 1% to about 5% COL9A1 and COL9A2, respectively. In some embodiments, collagen 2 (COL2A1) and COL10A1 represent about 90% to about 99% of the total collagen. In some embodiments, collagen 2 (COL2A1) and COL10A1 represent about 95% to about 99% of the total collagen, and COL9A1 and COL9A2 represent about 1% to about 5%.

[0116] Formation of osteoblasts and bone organoids Through detailed studies described herein, the inventors have discovered that chondrocytes matured into hypertrophic chondrocytes prepared according to the above-mentioned method can transition into osteoblasts to produce mineralized extracellular matrix, which can occur in vivo through transplantation of hypertrophic chondrocytes or in vitro.

[0117] Thus, in another embodiment, there is provided a method of producing a population of osteoblasts or bone-like organoids, the method comprising: a) producing hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, cartilage-like tissue, or cartilage according to the methods described herein; and b) culturing the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, or hypertrophic cartilage with an osteogenic culture medium to generate osteoblasts or bone-like organoids expressing COL1A1 and COL1A2; Includes.

[0118] In one embodiment, the culture of hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, or hypertrophic cartilage-like tissue is an orbital culture. In another embodiment, the culture is for an extended period of time. In another embodiment, the culture is for a period of up to about 3 weeks.

[0119] In one embodiment, the osteogenic differentiation culture medium comprises β-glycerophosphate, ascorbic acid 2-phosphate, sodium ascorbate, and dexamethasone, hi another embodiment, the osteogenic differentiation culture medium further comprises an activator of the WNT pathway for about the first 3-7 days of culture. In another embodiment, the WNT pathway activator is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidyl]amino]ethyl]amino]-3-pyridinecarbonitrile), Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10 b, Wnt-11, Wnt-16b, RSPO coagonist, lithium chloride, TDZD8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), BIO-acetoxime ((2'Z,3'E)-6-bromoindirubin-3'-acetoxime), A1070722 (1-(7-methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea), HLY78 (4-ethyl-5,6-dihydro-5-methyl-[1,3]dioxolo[4,5-j]phenanthridine), CID 11210285 hydrochloride (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride), WAY-316606, (hetero)arylpyrimidines, IQ1, QS11, SB-216763, and DCA. In a preferred embodiment, the Wnt pathway activator is CHIR99021.

[0120] In one embodiment, the osteogenic differentiation culture medium does not contain activators of the BMP pathway IL1A, IL1B, IL6, and / or TNF.

[0121] Hard knots Methods for generating sclerotome starting from pluripotent stem cells, leading to the primitive streak, paraxial mesoderm, early somites, and then sclerotome have been previously described (see, e.g., Loh et al., 2016). The methods of the present invention can employ sclerotome cells produced by any known method.

[0122] In one embodiment, the sclerotome cells employed in the methods described herein are obtained by a method comprising the steps of: i) culturing a population of multipotent progenitor cells with a composition comprising an activator of the TGF-beta pathway, an activator of the Wnt pathway, an activator of the FGF pathway, and an inhibitor of P13K for a period of about 24 hours; ii) culturing the cells of step i) with a composition comprising an inhibitor of the TGF-beta pathway, an activator of the Wnt pathway, an activator of the FGF pathway, and an inhibitor of the BMP pathway for a period of about 24 hours; iii) culturing the cells of step ii) with a composition comprising an inhibitor of the Wnt pathway, an inhibitor of the BMP pathway, and an inhibitor of the MEK / ERK pathway for a period of about 24 hours; and iv) contacting the cells of step iii) with a composition comprising an inhibitor of the Wnt pathway and an activator of the Hedgehog pathway for a period of about 72 hours to generate sclerotome cells.

[0123] In one embodiment, the activator of the TGF-beta pathway is selected from the group consisting of activin A, TGF-beta 1, TGF-beta 2, TGF-beta 3, IDE1 / 2 (IDE1 (1-[2-[(2-carboxyphenyl)methylene]hydrazide]heptanoic acid), IDE2 (heptanedioic acid-1-(2-cyclopentylidenehydrazide)), and Nodal. In a preferred embodiment, the activator of the TGF-beta pathway is activin A.

[0124] In an embodiment, the composition in step iii) used to culture the cells in step ii) does not comprise an activator of the TGF-beta pathway and / or an activator of the FGF pathway.

[0125] In an embodiment, the composition in step iv) used to culture the cells in step iii) does not comprise an inhibitor of the TGF-beta pathway, an activator of the FGF pathway, and / or an inhibitor of the BMP pathway.

[0126] In one embodiment, the activator of the Wnt pathway is CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10 b, Wnt-11, Wnt-16b, RSPO coagonist, lithium chloride, TDZD8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), BIO-acetoxime ((2'Z,3'E)-6-bromoindirubin-3'-acetoxime), A1070722 (1-(7-methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea), HLY78 (4-ethyl-5,6-dihydro-5-methyl-[1,3]dioxolo[4,5-j]phenanthridine), CID 11210285 hydrochloride (2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride), WAY-316606, (hetero)arylpyrimidines, IQ1, QS11, SB-216763, and DCA. In a preferred embodiment, the Wnt pathway activator is CHIR99021.

[0127] In one embodiment, the inhibitor of the PI3K pathway is AS 252424 (5-[[5-(4-fluoro-2-hydroxyphenyl)-2-furanyl]methylene]-2,4-thiadiazolidinedione), AS 605240 (5-(6-quinoxalinylmethylene)-2,4-thiazolidine-2,4-dione), AZD 6482 ((-)-2-[[(1R)-1-[7-methyl-2-(4-morpholinyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl]ethyl]amino]benzoic acid), BAG 956 (α,α,-dimethyl-4-[2-methyl-8-[2-(3-pyridinyl)ethynyl]-1H-imidazo[4,5-c]quinolin-1-yl]-benzeneacetonitrile), CZC 24832 (5-(2-amino-8-fluoro[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(1,1-dimethylethyl)-3-pyridinesulfonamide), GSK 1059615 (5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidenedione), KU 0060648 (4-ethyl-N-[4-[2-(4-morpholinyl)-4-oxo-4H-1-benzopyran-8-yl]-1-dibenzothienyl]-1-piperazineacetamide), LY 294002 hydrochloride (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride), 3-methyladenine (3-methyl-3H-purin-6-amine), PF 04691502 (2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one), PF 05212384 (N-[4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea), PI 103 hydrochloride (3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride), PI 828 (2-(4-morpholinyl)-8-(4-aminophenyl)-4H-1-benzopyran-4-one), PP 121 (1-cyclopentyl-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), quercetin, TG 100713 (3-(2,4-diamino-6-pteridinyl)-phenol), wortmannin, PIK90, and GDC-0941. In a preferred embodiment, the inhibitor of the PI3K pathway is PIK90.

[0128] In one embodiment, the inhibitor of the TGF-beta pathway is A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), GW 788388 (4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), LY 364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), RepSox (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), SB-505124 (2-[4-(1,3-benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine), SB 525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), SD208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine), ITD1 (4-[1,1'-biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester), DAN / Fc, antibodies against TGF-beta and TGF-beta receptors, TGF-beta inhibitory nucleic acids. In a preferred embodiment, the inhibitor of the TGF-beta pathway is A-83-01.

[0129] In one embodiment, the inhibitor of the BMP pathway is selected from the group consisting of chordin, soluble BMPR1a, soluble BMPR1b, noggin, LDN-193189, and dorsomorphin. In a preferred embodiment, the inhibitor of the BMP pathway is LDN-193189.

[0130] In one embodiment, the inhibitor of the Wnt pathway is C59 (4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide), DKK1, IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide), Ant1.4Br, Ant 1.4CI, niclosamide, apicularen, bafilomycin, XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one), IWR-1 (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide), NSC668036 (N-[(1,1-dimethylethoxy)carbonyl]-L-alanyl-(2S)-2-hydroxy-3-methylbutanoyl-L-alanine-(1S)-1-carboxy-2-methylpropyl ester hydrate) The inhibitor is selected from the group consisting of: 2,4-diamino-quinazoline, quercetin, ICG-001 ((6S,9aS)-hexahydro-6-[(4-hydroxyphenyl)methyl]-8-(1-naphthalenylmethyl)-4,7-dioxo-N-(phenylmethyl)-2H-pyrazino[1,2-a]pyrimidine-1(6H)-carboxamide), PKF115-584, BML-284 (2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine), FH-535, iCRT-14, JW-55, JW-67, antibodies against Wnt and Wnt receptors, and Wnt inhibitory nucleic acids. In a preferred embodiment, the inhibitor of the Wnt pathway is C59.

[0131] In one embodiment, the inhibitor of the MEK / ERK pathway is AP 24534 (3-(2-imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]-benzamide), PD173074 (N-[2-[[4-(diethylamino)butyl]amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea), FIIN 1 hydrochloride (N-(3-((3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(4-(diethylamino)butylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)methyl)phenyl)acrylamide), PD 161570 (N-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea), SU 5402 (2-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-4-methyl-1H-pyrrole-3-propanoic acid), SU 6668 (5-[1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid), PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide), BIX 02189 ((3Z)-3-[[[3-[(dimethylamino)methyl]phenyl]amino]phenylmethylene]-2,3-dihydro-N,N-dimethyl-2-oxo-1H-indole-6-carboxamide), FR 180204 (5-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine), Pluripotin (N-[3-[7-[(1,3-dimethyl-1H-pyrazol-5-yl)amino]-1,4-dihydro-1-methyl-2-oxopyrimide[4,5-d]pyrimidin-3(2H)-yl]-4-methylphenyl]-3-(trifluoromethyl)benzamide), TCS ERK 11e (4-[2-[(2-chloro-4-fluorophenyl)amino]-5-methyl-4-pyrimidinyl]-N-[(1S)-1-(3-chlorophenyl)-2-hydroxyethyl]-1H-pyrrole-2-carboxamide), TMCB (2-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)acetic acid), XMD 8-92 (2-[[2-ethoxy-4-(4-hydroxy-1-piperidinyl)phenyl]amino]-5,11-dihydro-5,11-dimethyl-6H-pyrimido[4,5-b][1,4]benzodiazepin-6-one), SU5402, AZD4547, BGJ398, AL 8697, AMG 548, CMPD-1, DBM 1285 dihydrochloride, EO 1428, JX 401, ML 3403, RWJ 67657, SB 202190, SB-203580, SB 239063, SB 706504, Scio-469, SKF 86002 dihydrochloride, SX 011, TA 01 (4-(2-(2,6-difluorophenyl)-4-(fluorophenyl)-1H-imidazol-5-yl)pyridine), TA 02 (4-(2-(2-fluorophenyl)-4-(fluorophenyl)-1H-imidazol-5-yl)pyridine), TAK 715, VX-702, and VX-745. In a preferred embodiment, the inhibitor of the MEK / ERK pathway is PD0325901.

[0132] In one embodiment, the activator of the Hedgehog pathway is selected from the group consisting of Hedgehog family ligands (Hh, Shh, Ihh, Dhh, etc.) and fragments thereof, benzothiophene smoothened agonists, SAG (Hh-Ag1.3), SAG21k (3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-((1r,4r)-4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide), Hh-Ag1.1, Hh-Ag1.5, and palmorphamin. In a preferred embodiment, the activator of the Hedgehog pathway is palmorphamin.

[0133] In another embodiment, the sclerotome cells express any one of PAX1, SOX9, FOXC2, PAX9, NKX3.2 / BAPX1, and TWIST1.

[0134] In another embodiment, sclerotome cells are converted into 3D aggregates (high density pellets) after 4 days and then maintained intact in culture medium. In another embodiment, pre-pelleted sclerotome stage cells can be frozen in medium containing 10% DMSO, stored in liquid nitrogen, and harvested for subsequent use in the methods described herein.

[0135] iPSC Any population of pluripotent stem cells, including a population of human embryonic stem cells (hESCs) or a population of induced pluripotent stem cells (iPSCs), can be used as starting material to derive sclerotome cells (e.g., to obtain iPSC-derived chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage) for use in the methods of the present invention. In a preferred embodiment, the population of pluripotent progenitor cells is a population of human iPSCs. Through detailed studies described herein, the inventors have discovered that key chondrocyte genes, SOX9, COL2A1, COL9A1, COL11A1, and ACAN, are more highly expressed in iPSC cells grown in feeder-free conditions and can differentiate more efficiently. Thus, in a further preferred embodiment, the population of pluripotent progenitor cells (employed to generate sclerotome) is iPSCs derived from feeder-free cell culture.

[0136] In another embodiment, cells obtained from a subject can be subjected to a method to generate patient-specific iPSCs that can then be differentiated using the methods described herein. There are over 430 skeletal genetic disorders caused by mutations in over 360 different genes. Thus, in one embodiment, the iPSCs used in the methods described herein are selected from the group consisting of the following cartilage and bone disorders: FGFR3 chondrodysplasias, collagen type 2, collagen type 11, sulfation disorders, perlecan, aggrecan, filamin and related disorders, TRPV4, ciliary disorders with large skeletal lesions, multiple epiphyseal dysplasias and pseudoachondroplasias, metaphyseal dysplasias, spondyloepiphyseal dysplasias (SMD), spondyloepiphyseal (stem) dysplasias (SE(M)D), severe spondylodysplasias, acrometaphyseal dysplasias, distal intermedius dysplasias, mesophyseal dysplasias, and pediculosis. The tissue samples are obtained from patients with a disorder selected from one of the following: interpeduncular and proximal limb dysplasia, ankylotic limb dysplasia and related disorders, narrow bone dysplasia group, chondrodysplasia punctata (CDP) group, neonatal osteosclerotic dysplasia, osteopetrosis and related disorders, osteogenesis imperfecta and low bone density group, abnormal mineralization group, lysosomal storage disease with skeletal lesions (dysostosis multiplex group), developmental abnormalities of skeletal components group, cleidocranial dysplasia and related disorders, and dysostosis with prominent craniofacial lesions.

[0137] composition In another embodiment, there is also provided an isolated population of chondrocytes or chondrocyte-like cells, or cartilage-like tissue or cartilage, or a combination thereof, produced by the methods described herein. In one embodiment, the isolated population of chondrocytes or chondrocyte-like cells, or cartilage-like tissue or cartilage, or a combination thereof, may be provided as a composition, optionally comprising a suitable carrier.

[0138] In one embodiment, a composition is provided comprising a homogenous population of iPSC-derived chondrocytes, the cells expressing collagen 2 (COL2A1) and ACAN in a ratio ranging from 20:1 to 5:1, preferably about 10:1, and substantially no expression of COL10A1. In some embodiments, the homogenous population of iPSC-derived chondrocytes may also express COL9A1, COL9A2, COL9A3, COL11A1, and COL11A2. In some embodiments, COL2A1 represents about 30% to about 50% of the expressed collagens, as determined by RNAseq analysis and expressed as % of RPKM of total collagens. In some embodiments, COL2A1 represents about 30% to about 50% of the total collagen, COL11A1 represents about 10% to about 30%, COL11A2 represents about 5% to about 20%, COL9A1 represents about 5% to about 30%, COL9A2 represents about 1% to about 10%, and COL9A3 represents about 1% to about 10%. In some embodiments, COL2A1 represents about 40% to about 50% of the total collagen, COL11A1 and COL11A2 each represent about 5% to about 40%, and COL9A1, COL9A2, and COL9A3 each represent about 5% to about 15%. In another embodiment, COL2A1 represents about 45% of total collagen, COL11A1 represents about 21%, COL11A2 represents about 10%, COL9A1 represents about 13%, COL9A2 represents about 6%, and COL9A3 represents about 4%. The compositions of these embodiments may be suitable for repairing or replacing articular cartilage. Thus, in another embodiment, a composition according to the above embodiment is provided for use in repairing or replacing articular cartilage in a subject in need thereof. In another embodiment, a method of repairing or replacing articular cartilage in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a composition according to the above embodiment. In another embodiment, a use of a composition according to the above embodiment for the manufacture of a medicament for repairing or replacing articular cartilage in a subject in need thereof is provided.

[0139] In another embodiment, a composition is provided comprising a homogenous population of iPSC-derived articular chondrocytes, the cells expressing one or more markers selected from COL2A1, ACAN, and PRG4, and optionally ASPN, CILP, CILP2, EMILIN1, EMILIN3, FBLN1, and FBLN3. In some embodiments, the homogenous population of iPSC-derived articular chondrocytes expresses COL2A1, COL9A1, and COL9A2. In some embodiments, collagen 2 (COL2A1) represents about 70% to about 95% of the expressed collagen as determined by RNAseq analysis and expressed as % of RPKM of total collagen. In some embodiments, collagen 2 (COL2A1) represents about 70% to about 95% of the total collagen, and COL9A1 and COL9A2 represent about 1% to about 30%, respectively. In some embodiments, collagen 2 (COL2A1) represents about 80% to about 95% of the total collagen, and COL9A1 and COL9A2 represent about 1% to about 10%, respectively. In some embodiments, collagen 2 (COL2A1) represents about 85% to about 95% of the total collagen, and COL9A1 and COL9A2 represent about 5% to about 15%, respectively. The compositions of these embodiments may be suitable for repairing or replacing articular cartilage. Thus, in another embodiment, a composition according to the above embodiments is provided for use in repairing or replacing articular cartilage in a subject in need thereof. In another embodiment, a method of repairing or replacing articular cartilage in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a composition according to the above embodiments. In another embodiment, a use of a composition according to the above embodiments for the manufacture of a medicament for repairing or replacing articular cartilage in a subject in need thereof is provided.

[0140] In another embodiment, a composition is provided comprising a homogenous population of iPSC-derived hypertrophic chondrocytes, the cells expressing collagen 2 (COL2A1) and COL10A1 in a ratio ranging from 1:2.5 to about 2.5:1. In some embodiments, the ratio of collagen 2 (COL2A1) to COL10A1 ranges from about 1.5:1 to 1:1.5. In some embodiments, the homogenous population of iPSC-derived hypertrophic chondrocytes may also express COL9A1 and COL9A2. In some embodiments, collagen 2 (COL2A1) and COL10A1 represent about 90% to about 99% of the expressed collagen as determined by RNAseq analysis and expressed as % of RPKM of total collagen. In some embodiments, collagen 2 (COL2A1) and COL10A1 represent about 90% to about 99% of the total collagen, and COL9A1 and COL9A2 represent about 1% to about 10%. In some embodiments, collagen 2 (COL2A1) and COL10A1 represent about 95% to about 99% of the total collagen, and COL9A1 and COL9A2 represent about 1% to about 5%. In some embodiments, collagen 2 (COL2A1) represents about 30% to about 50% of the total collagen, COL10A1 represents about 30% to about 50%, and COL9A1 and COL9A2 represent about 1% to about 10% each. In some embodiments, collagen 2 (COL2A1) represents about 40% to about 50% of the total collagen, COL10A1 represents about 40% to about 50%, and COL9A1 and COL9A2 represent about 1% to about 5% each. The compositions of these embodiments may be suitable for repairing or replacing hypertrophic cartilage. Thus, in another embodiment, a composition according to the above embodiments is provided for use in repairing or replacing hypertrophic cartilage in a subject in need thereof. In another embodiment, there is provided a method of repairing or replacing hypertrophic cartilage in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition according to the above embodiments. In another embodiment, there is provided a use of a composition according to the above embodiments for the manufacture of a medicament for repairing or replacing hypertrophic cartilage in a subject in need thereof.

[0141] In another embodiment, an isolated population of osteoblasts or bone-like organoids produced by the methods described herein is also provided. In another embodiment, a composition is provided comprising an isolated population of osteoblasts or bone-like organoids derived from iPSCs, said cells expressing COL1A1 and COL1A2. In some embodiments, the isolated population of osteoblasts or bone-like organoids derived from iPSCs may also express one or more of MEPE, IBSP (bone sialoprotein 2), SPP1 (osteopontin), and DMP. The compositions of these embodiments may be suitable for bone repair or replacement. Thus, in another embodiment, a composition according to the above embodiment is provided for use in bone repair or replacement in a subject in need thereof. In another embodiment, a method of repairing or replacing bone in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a composition according to the above embodiment. In another embodiment, a use of a composition according to the above embodiment for the manufacture of a medicament for repairing or replacing bone in a subject in need thereof is provided.

[0142] Further embodiments relate to compositions comprising an isolated population of chondrocytes or chondrocyte-like cells, or cartilage-like tissue or cartilage, or combinations thereof, and / or an isolated population of osteoblasts or bone-like organoids produced by the methods described herein, and a carrier, such as a polymer, a hydrogel, a bone scaffold, a bone replacement scaffold, etc. Other carriers include carriers comprising one or more of the group consisting of, for example, sodium hyaluronate, hyaluronic acid and its derivatives, gelatin, collagen, chitosan, alginate, buffered PBS, dextran, and polymers. For example, the carrier may be a carrier suitable for use in transplantation applications, such as a pharmaceutical grade carrier. The carrier may be suitable for stabilizing the cells for transportation and / or storage. The cells may be, for example, deep-frozen, and / or the tissue may be transported at room temperature and / or at any temperature between room temperature and about 4°C. In an embodiment, the carrier is pharmaceutical grade.

[0143] In embodiments, the isolated population is comprised in a composition comprising a diluent or carrier, optionally a pharmaceutical diluent, hi embodiments, the diluent is culture medium, optionally comprising a cryopreservative such as glycerol and / or DMSO, and serum and albumin, such as human serum albumin.

[0144] According to another embodiment, the three-dimensional aggregates of chondrocytes or chondrocyte-like cells, cartilage-like tissue or cartilage, or a combination thereof, or osteoblasts or bone-like organoids produced by the methods described herein may be decellularized to produce a decellularized scaffold.

[0145] In one embodiment, the composition may be, for example, a slurry comprising dissociated cells, for example, for administration to a subject. In an embodiment, the composition may include other cells, for example, endothelial cells and / or fibroblasts, for example, for growth plate cell / cartilage transplantation.

[0146] Further aspects include cartilage or bone tissue products comprising the cells and / or tissues described herein and scaffolds or membranes. For example, during implantation applications, chondrocytes may be administered to the damaged area in combination with a membrane (e.g., tibial periosteum or biomembrane) or pre-seeded on a scaffold matrix. In an embodiment, the scaffold is a bone substitute. In another embodiment, different types of chondrocytes or chondrocyte-like cells, cartilage-like tissue or cartilage, or combinations thereof may be combined. For example, articular cartilage produced by the method of the present invention may be placed as a laminate with chondrocytes in proliferative / dormant zones, which can further mature, hypertrophy, and develop into bone.

[0147] Thus, the cells and tissues, compositions, and scaffolds produced according to the methods disclosed herein can be used, for example, to treat a subject suffering from a joint or bone injury or disorder, or to reduce or ameliorate symptoms associated with said joint or bone injury. In one embodiment, a method is provided for treating a cartilage or osteochondral disorder or defect in a subject in need thereof, comprising: a) deriving a composition comprising chondrocytes, chondrocyte-like cells, cartilage-like tissue, or a combination thereof, or osteoblasts or bone-like organoids, or a combination of said chondrocytes, chondrocyte-like cells, cartilage-like tissue, or a combination thereof and said osteoblasts or bone-like organoids, according to the methods described herein, and b) administering the composition to the subject.

[0148] According to another embodiment, there is provided a composition comprising chondrocytes, chondrocyte-like cells, cartilage-like tissue, or a combination thereof, or osteoblasts or bone-like organoids produced by the methods described herein, or a combination of said chondrocytes, chondrocyte-like cells, cartilage-like tissue, or a combination thereof with said osteoblasts or bone-like organoids, for use in treating cartilage injury or defect or osteochondral injury or defect in a subject in need thereof.

[0149] According to another embodiment, there is provided the use of chondrocytes, chondrocyte-like cells, cartilage-like tissue, or a combination thereof, or osteoblasts or bone-like organoids produced by the methods described herein, or a composition comprising said chondrocytes, chondrocyte-like cells, cartilage-like tissue, or a combination thereof and said osteoblasts or bone-like organoids, in the manufacture of a medicament for treating cartilage injury or defect, or osteochondral injury or defect in a subject in need thereof.

[0150] Thus, another embodiment includes a method of ameliorating symptoms and / or treating a subject in need thereof comprising administering a population of cells and / or tissues described herein and / or inserting / implanting a product comprising said cells.

[0151] Uses of the cells, tissues, and products are also provided in another aspect. In embodiments, the disclosure provides for the use of a population of cells and / or tissues or compositions or products described herein to ameliorate symptoms and / or treat a subject in need thereof.

[0152] In embodiments, for example, the cells and tissues, compositions, and scaffolds for the uses or methods described herein to be administered to a subject are derived from autologous cells.

[0153] Screening Methods Candidate substances may be screened for their ability to promote, inhibit, maintain, or play other roles in endochondral development or osteochondral diseases or disorders using the methods described herein.

[0154] In another embodiment, a method of testing a candidate chondrogenic or osteogenic modulating agent is provided, the method comprising: a) carrying out a method of generating chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, or osteoblasts or bone-like organoids as described herein, wherein the test substance is included in any one or more of the culture steps of the method; b) evaluating the effect of the test substance on the proliferation, maintenance, and / or differentiation of chondrocytes or osteoblasts compared to a control population generated in the absence of the test substance; and c) identifying the test substance as a candidate chondrogenic or osteogenic regulator if it increases or decreases proliferation compared to a control and / or affects the maintenance or differentiation of chondrocytes or osteoblasts. Includes.

[0155] The chondrocytes, chondrocyte-like cells, cartilage-like tissues, or combinations thereof, or osteoblasts or bone-like organoids produced by the methods described herein, or cells employed to derive such cells / tissues, may be engineered to stably express a reporter gene operably linked to the promoter of a gene typically expressed in chondrocytes (e.g., articular chondrocytes-PRG4, or hypertrophic chondrocytes collagen X), for example, to provide model cells that can be used to test for candidate substances. Numerous reporter genes are known in the art, including, for example, fluorescent proteins (e.g., GFP, RFP, dsRed, etc., luciferase). Reporter gene assays are versatile and sensitive methods that can be used to assay a large number of candidate substances in high-throughput drug screening programs.

[0156] In one embodiment, the screening method may be performed using cells derived from a subject with a bone or cartilage disease or disorder. In another embodiment, the screening method may be performed using gene-edited cells engineered to have one or more mutations associated with the disease or to correct one or more mutations associated with the disease. In one embodiment, the bone or cartilage disorder is selected from the following cartilage and bone disorder groups: FGFR3 chondrodysplasia group, collagen type 2 group, collagen type 11 group, sulfation disorder group, perlecan group, aggrecan group, filamin group and related disorders, TRPV4 group, ciliary disorders with large skeletal lesions, multiple epiphyseal dysplasia and pseudoachondroplasia group, metaphyseal dysplasia, spondyloepiphyseal dysplasia (SMD), spondyloepiphyseal (stem) dysplasia (SE(M)D), severe spondylodysplasia dysplasia, acrodysplasia, distal mid-limb dysplasia, spondyloepiphyseal ... The disease is selected from one of the following: dysplasia, intermediate and proximal limb dysplasia, ankylotic dysplasia and related disorders, narrow bone dysplasia group, chondrodysplasia punctata (CDP) group, neonatal osteosclerotic dysplasia, osteopetrosis and related disorders, osteogenesis imperfecta and low bone density group, abnormal mineralization group, lysosomal storage disease with skeletal involvement (dysostosis multiplex group), developmental abnormalities of skeletal components group, cleidocranial dysplasia and related disorders, and dysostosis with prominent craniofacial involvement. In one embodiment, the disease is osteogenesis imperfecta. In another embodiment, the cartilage disorder is associated with a mutation in COL2A1.

[0157] kit Also provided herein are kits that include one or more of the cells or tissues generated by the methods described herein, products or compositions comprising the generated cells or tissues, optionally including an additional therapeutic agent (optionally the cells include a reporter system or other modification according to the methods described herein), a combination of at least two selected from agonists, inhibitors, vehicles, devices, or other components that can be used in the methods described herein, and instructions for use, e.g., how to generate the cells, how to assay or administer the cells, tissues, compositions, or products, as well as a vial or other container for containing one of the above cells, tissues, compositions, products, agonists, inhibitors, vehicles, etc. EXAMPLES

[0158] Herein, we have optimized the protocol for differentiation of sclerotome into chondrocytes. From the cartilage precursor stage to 3D cartilage organoids, the "chondronoids" are guided towards articular chondrocytes in chondrogenic medium containing TGFβ3, or directed along the growth plate chondrocyte pathway to become hypertrophic chondrocytes. Hypertrophy can be stimulated and enhanced by thyroxine (T3). We show that when transplanted into immunodeficient mice, hypertrophic chondrocytes in T3-treated organoids can recapitulate growth plate maturation and endochondral bone formation, including transition to human osteoblasts in the forming bone. Furthermore, when cultured in vitro with osteogenic medium, hypertrophic chondronoids could show transdifferentiation into osteoblasts and extensive mineralization. In the sequential steps of this novel in vitro model of human endochondral bone formation, we performed a large-scale transcriptional study to identify the gene signatures of these developmental stages during cartilage maturation, hypertrophy, and transdifferentiation into osteoblasts. We identified a transcription factor network involved in this maturation and changes in the extracellular matrix (matrisome) during this developmental sequence. This in vitro endochondral model system will facilitate studies exploring normal growth plate cartilage development as well as mechanistic studies of genetic disorders of cartilage and bone development and homeostasis. Furthermore, homogenous articular cartilage chondronoids will be valuable for the study of osteoarthritis and regenerative medicine approaches to articular cartilage repair.

[0159] Materials and Methods Human induced pluripotent stem cell lines and maintenance Five independent, fully characterized and validated control human induced pluripotent stem cell (iPSC) lines were used: feeder-dependent fibroblast-derived RM3.5 (Kao et al., 2016), peripheral blood mononuclear cell-derived MCRIi001-A (Vlahos et al., 2019), and a gene-edited subclone expressing a SOX9-tdTomato reporter, MCRIi001-A (Nur Patria et al., 2020), and the fibroblast-derived feeder-independent lines MCRIi018-B (Howden et al., 2019), and MCRIi019-A (Kung et al., 2020). Feeder-dependent cells were routinely grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) in knockout DMEM / F-12 containing 20% ​​knockout serum replacement, 2 mM GlutaMax, 1% non-essential amino acid solution, 0.1 mM β-mercaptoethanol (all from Thermo Fisher Scientific), and 50 ng / ml FGF2 (PeproTech) at 37°C, 5% CO2. Medium was changed daily and cells were passaged (1:6 split) approximately every 3 days in PBS, 0.5 mM EDTA. Feeder-free cells were routinely grown on Matrigel (Corning)-coated plates in Essential 8 (E8) medium (Thermo Fisher Scientific). Medium was changed daily and cells were passaged (1:4–1:6) every 3–4 days in PBS, 0.5 mM EDTA.

[0160] Differentiation of sclerotome Undifferentiated hiPSCs at 70-90% confluency were dissociated into fine clumps using 0.5 mM EDTA in PBS and typically passaged into 6-well plates pre-seeded with MEFs or pre-coated with Matrigel (approximately 2 × 10 per well for RM3.5, MCRIi0018-B, MCRIi019-A). 5 Approximately 0.8 × 10 cells per well for MCRIi-001-A and MCRIi001-A-2 5Cells) and cultured for 24–48 h in appropriate growth media. Differentiation into sclerotomes was performed as described (Loh et al., 2016), modified by replacing CDM2 basal medium with APEL2 (StemCell Technologies). On day 0, medium was replaced with anterior primitive streak induction medium consisting of APEL2 containing 30 ng / ml activin A (R&D Systems), 4 μM CHIR99021 (Tocris), 20 ng / ml FGF2 (PeproTech), and 100 nM PIK90 (Merck Millipore). After 24 hours, the medium was replaced with APEL2 containing 3 μM CHIR99021, 20 ng / ml FGF2, 1 mM A8301 (Tocris), and 0.25 mM LDN193189 (Cayman Chemical) to induce paraxial mesoderm for 24 hours. Early somitic development was then induced with APEL2 containing 1 mM A8301, 0.25 mM LDN193189, 3 mM C59 (Tocris), and 5 mM PD0325901 (Selleck Chemicals). After 24 hours, sclerotome induction was initiated with APEL2 containing 1 mM C59 and 2 mM palmorphamin (Sigma-Aldrich). After the first day of sclerotome induction (day 4), dissociate cells from the monolayer culture using 0.025% trypsin / EDTA, resuspend in sclerotome differentiation medium, and culture at 2 × 10 5 Aliquots of 300 μl of cells were dispensed into low-attachment round-bottom 96-well plates (Corning) and pelleted by centrifugation at 400 g for 3 min in a swing-out rotor. The pellets were incubated in sclerotome differentiation medium for an additional 48 h to complete sclerotome differentiation in a 96-well static culture format. Importantly, cells at the sclerotome stage prior to pelleting could be frozen in medium containing 10% DMSO, stored under liquid nitrogen, and retrieved for subsequent studies, allowing the accumulation of well-structured sclerotomes for differentiation consistency and large-scale experiments.

[0161] Chondrocyte Differentiation and Maturation Differentiation of sclerotomes into chondrocytes was performed in APEL2 medium containing 5% protein-free hybridoma medium (PFHM II; Thermo Fisher) and 20 ng / ml FGF2. In some experiments, BMP4 (R&D Systems) was included at 20 ng / ml. Pellets were routinely transferred to 6 cm non-adherent dishes (Greiner) at the end of day 6 or day 20, 15–20 pellets per dish in 5 ml of medium with orbital rotation at 60 rpm. Medium was changed every 2–3 days. From day 20 (2 weeks after FGF2 treatment), differentiation / maturation continued in APEL2 / 5% PFHM, with medium changed every 2–3 days for the duration of the indicated chondrogenic differentiation experiment. In some experiments, pellets were treated with FGF2 for 4 weeks after sclerotome.

[0162] Triiodothyronine (T3) is important for chondrocyte hypertrophy (Aghajanian et al., 2017). Therefore, in some experiments, differentiated chondrocytes were treated with 10 nM T3 (Sigma-Aldrich) for 10–21 days to induce chondrocyte hypertrophy. From day 20 (2 weeks after FGF2 treatment), differentiation / maturation was continued in APEL2 / 5% PFHM (without FGF2) with medium changes every 2–3 days. T3 was added when the pellet had a consistency and the cartilage was histologically homogenous (this ranged from 4–6 weeks after sclerotome / chondrocyte differentiation). In some experiments, to remove small amounts of non-cartilage tissue that occasionally remained on the surface of the chondrocyte pellet, the pellet was briefly treated with 0.25% trypsin / EDTA for 4 min at 37 °C with agitation. These were allowed to recover in chondrocyte medium (APEL2 / 5% PFHM) for one week before analysis and / or T3 treatment and continued maturation.

[0163] In some experiments, differentiated chondrocytes were treated with 10ng / ml TGFβ3 (R&D Systems) up to day 34 to generate articular cartilage. From day 6, differentiation / maturation was continued for 7 days in APEL2 / 5% PFHM (with FGF2). From day 13, 10ng / ml TGFβ3 was added (i.e., FGF2 and TGFβ3) and the medium was changed every 2-3 days. On day 20, pellets were transferred to rotary culture and cultured with 10ng / ml TGFβ3 (R&D Systems) (without FGF2) for an additional 4 weeks.

[0164] osteogenic differentiation Hypertrophic chondrocytes can transdifferentiate into osteoblasts and osteocytes in vivo (Yang et al., 2014; Zhou et al., 2014). To recapitulate this later stage of endochondral bone formation in vitro, hypertrophic chondrocyte pellets in orbital cultures were rinsed with PBS and then cultured for up to 3 weeks in DMEM, high glucose, GlutaMAX supplement, pyruvate (ThermoFisher), 20% FBS (ThermoFisher), 10 mM β-glycerophosphate, 50 μg / ml ascorbic acid 2-phosphate, 50 μg / ml sodium ascorbate, 100 nM dexamethasone (osteogenic differentiation medium). Some cultures were supplemented with 10 μM CHIR99021 for the first 7 days. Medium was changed every 2–3 days.

[0165] In vivo implantation to generate human heterotopic bone Cartilage organoids were treated with T3 for 1 week starting on day 35 and then subcutaneously implanted into NBSGW mice (McIntosh et al., 2015). Organoid grafts were harvested after 13 weeks. All animal experimental procedures were approved by the Murdoch Children's Research Institute Animal Ethics Committee (approval number A863).

[0166] Histological analysis Cultured in vivo grafted pellet organoids were fixed overnight at 4°C in neutral buffered formalin in Confix (Australian Biostain), then decalcified, washed in 70% ethanol, and embedded in paraffin. After fixation, some in vivo grafted pellets were washed in water for 10 min, then decalcified and embedded in 14% EDTA, pH 7.4, 4°C for 5 days. Serial 5 μm sections cut from the center of the pellets were placed on Superfrost Plus slides (Menzel-Glaser) and heated at 55°C overnight. Sections were treated with xylene to remove the paraffin wax, and rehydrated in an ethanol series containing 100%, 90%, 70%, 50% ethanol, and then water. Sections were stained with toluidine blue to detect cartilage proteoglycan matrix or safranin O / fast green for cartilage and bone according to standard protocols. Osteogenic pellet sections were also stained with von Kossa to detect mineralization. Images were captured using a Leica DM 2000 LED microscope with Leica Application Suite (LAS) software version 4.9.0 or a Zeiss Axio Imager Z2 with Zen 3.1 Blue Edition software.

[0167] immunostaining Formalin-fixed, paraffin-embedded sections were immunostained for collagen II (MAB8887; Sigma-Aldrich; 1:150 dilution), collagen I (LF68; Kerafast ENH018-FP; 1:100), collagen X (Cameron et al., 2015) (1:100), PR4 (clone 9G3; Merck) (1:100), and Ku80 (Cell Signaling) (1:300). For collagen II staining, antigens were retrieved with 2 mg / ml porcine pepsin (Sigma) freshly prepared in 1 M Tris / HCl, pH 2.0 for 30 min at 37°C, followed by digestion with 0.2% hyaluronidase (Sigma-Aldrich) in PBS for 30 min at 37°C. Collagen X antigen retrieval involved heating to 60°C for 30 min in 10 mM Tris / 1 mM EDTA / 0.05% Tween-20, pH 9.0 (Tris / EDTA), cooling to room temperature for 30 min, followed by hyaluronidase digestion and staining for collagen I and Ku80, followed by antigen retrieval in Tris / EDTA at 60°C as above. Sections to be stained with Ku80 were then permeabilized with 0.05% Triton X-100. Sections were blocked with 3% BSA in PBS for 1 h at room temperature, then incubated with primary antibodies in 1% BSA in PBS for 16 h at 4°C. Appropriate species-specific Alexa Fluor 488 and 594 secondary antibodies (ThermoFisher) were diluted 1:200 in 1% BSA in PBS with 2.5 μg / ml DAPI, and sections were incubated for 1 h at room temperature. Slides were washed with PBS and coverslipped with Shandon Immumount (ThermoFisher). Images were captured as above. For collagen X subcellular colocalization studies, sections were treated with 0.2% hyaluronidase for 30 min at 37°C and then permeabilized with 0.05% Triton X-100. Collagen X antibodies were used at 1:100, 58K (PA1-9000; ThermoFisher) at 1:50, GRP94 (PA5-18534; ThermoFisher) at 1:200, and LAMP2 (ab25631; Abcams) at 1:50.Images were captured with a Zeiss LSM900 Airyscan 2 confocal microscope and Zen 3.1 Blue Edition software.

[0168] Transmission electron microscope Pellets were washed with PBS and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate at 4°C. Samples were post-fixed in 1% aqueous osmium tetroxide, dehydrated in an alcohol series, and embedded in Epon 812. Seventy nanometer ultrathin sections were cut and viewed on a Tecnai F30 with an extraction voltage of 200 kV. Photomicrographs were acquired using a Gatan UltraScan 1000.

[0169] Micro-CT Pellets were fixed in formalin, stored in 70% ethanol at 4°C, and then scanned using a microCT (μCT50, Scanco Medical AG) with a 0.5 mm Al filter at 70 kVp energy, 200 μA intensity, 300 ms integration time, and 3 μm voxel (native) resolution. Bone morphometric parameters were calculated using a common threshold of 210 / 1000 for bone tissue within the volume of interest.

[0170] Quantitative RT-PCR RNA was extracted from monolayer cultures using TRIzol (Qiagen) according to the manufacturer's instructions. Typically, 100-200 ng of total RNA was used for synthesis of cDNA in a total volume of 20 μl using the QuantiTect® Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. Quantitative RT-PCR (qPCR) was performed in triplicate with the Brilliant III Ultra-Fast SYBR Green QRT-PCR Kit (Agilent) using 10 μl reactions consisting of 1× Brilliant III Ultra-Fast SYBR Green QRT-PCR Master Mix, 1 μl of cDNA, and 1 μM of each primer. Amplification was performed in a 384-well plate format on a LightCycler 480 Instrument II (Roche Life Sciences) using a thermocycling protocol that included 40 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and amplification at 72°C for 20 s. Gene-specific primers were designed using Primer-BLAST (NCBI). To avoid amplification from genomic DNA, primer sets were designed across multiple exons, and PCR products were analyzed by agarose gel electrophoresis to confirm a single product corresponding to the predicted cDNA amplicon for each gene. Analysis was performed using LightCycler 480 software (release 1.5.1.62). Human beta-actin (ACTB) was routinely used as a housekeeping reference gene. Graphs were generated using GraphPad Prism 9.

[0171] RNAseq of bulk populations For RNAseq, organoids (2–3 pooled) were flash frozen in liquid nitrogen, pulverized using a liquid nitrogen-cooled tissue grinder, and RNA was extracted with TRIzol and subsequently purified using Direct-zol RNA Microprep Kit spin columns (Zymo Research) according to the manufacturer's instructions. RNA samples were quality controlled and sequenced at the Translational Genomics Unit, Murdoch Children's Research Institute. Libraries were constructed using the Illumina Stranded mRNA Prep kit and sequenced using a NextSeq 500, yielding approximately 20 × 10 RNA per sample. 675 bp paired-end reads were sequenced using a NovaSeq 6000 to obtain 150 bp paired-end reads. Reads were aligned to hg38 using the Bpipe (Sadedin et al., 2012) RNAseq pipeline incorporating FastQC quality control, adaptor trimming with Trimmomatic v.0.35 (Bolger et al., 2014), mapping with STAR 2.7.3a (Dobin et al., 2013), gene-wide read summarization with featureCounts (Liao et al., 2014), and MultiQC (Ewels et al., 2016) to summarize the analysis. The EdgeR Bioconductor package (Law et al., 2016) was used for downstream analysis and identification of differentially expressed genes. Genes with an expression level of at least 1 count per million in at least three samples were kept for further analysis. Data were TMM normalized and Boom transformed. Differential expression was identified by robust paired moderated t-tests using limma (Law et al., 2016). For graphical visualization, the gplots, tidyverse, ggplot2, and EnhancedVolcano packages were used. For gene set enrichment analysis, the Broad Institute's MSigDB gene sets (https: / / www.gsea-msigdb.org / gsea / msigdb / ) and clusterProfiler (Yu et al., 2012) and enrichplot packages were used. Protein interactomes were visualized using the online STRING tool (https: / / string-db.org / ).

[0172] Example 1 Creation of sclera Our goal was to develop and optimize an efficient and reproducible method to differentiate human iPSCs into chondrocytes that can mature towards hypertrophy and then transdifferentiate into osteoprogenitors or chondrocytes that are committed towards an articular cartilage phenotype. The first 6 days of our protocol (Figure 1A) are based on a published method for generating sclerotomes (Loh et al., 2016). We found this method to be robust and reproducible. We monitored the expression of stage-specific markers using qRT-PCR in two iPSC lines and found that the pluripotent marker OCT4 was gradually downregulated, while MIXL1, MSGN1, MEOX1, and PAX1, markers of primitive streak, paraxial mesoderm, somitic mesoderm, and sclerotome, respectively, were upregulated and downregulated at the appropriate developmental stages (Figure 1B, Figure 1.1). The key chondrocyte transcription factor SOX9 and cartilage collagen COL2A1 were upregulated in the somitic mesoderm and sclerotome (Figure 1B, Figure 1.1).

[0173] Example 2 FGF2 promotes differentiation of sclerotome into chondrocytes. It is widely recognized that high-density pellet culture is advantageous for maintaining the chondrocyte phenotype ex vivo. Therefore, we cultured 2 × 10 cells at the end of day 4. 5 The cells / pellet were transferred to pellet culture. In our initial experiments, we added 20 ng / ml BMP4 to pellet cultures for 2 weeks after sclerotome to promote chondrocyte differentiation ( Adkar et al., 2019 ; Loh et al., 2016 ), but found that key cartilage genes were not highly expressed (data not shown).

[0174] We then added 20 ng / ml BMP4 or 20 ng / ml FGF2 for 14 days after sclerotization and compared extended culture without additional growth factors. Because changing the culture medium on cell pellets in 96-well plates was time-consuming and proved difficult to automate without damaging the pellets, we also planned to compare chondrocyte differentiation in stationary pellets and pellets transferred to rotary culture at the end of day 6. However, BMP4-treated pellets were fragile and could not be maintained in rotary culture. In static culture, FGF2-treated pellets showed strong toluidine blue staining throughout at day 34, reflecting the accumulation of extracellular matrix proteoglycans typical in cartilage. On the other hand, BMP4-treated pellets were smaller and only isolated areas were toluidine blue positive (Figure 2A). FGF2-treated pellets in rotary culture also developed into organoids that stained overall with toluidine blue, but differentiation was delayed compared to FGF2-treated stationary pellets (Figure 2A). In FGF2-treated cultures, expression of COL2A1 and ACAN genes was consistently higher than in BMP4-treated cultures, whereas the hypertrophic cartilage gene COL10A1 was expressed earlier in BMP4-treated cultures (Figure 2B). By day 48, FGF2-treated pellets, both stationary and rotated, had extensive and uniform collagen II extracellular matrix deposition, whereas BMP4-treated pellets had only small spots positive for collagen II (Figure 2C). Similarly, extracellular staining for collagen X was intense in FGF2-treated stationary pellets, especially in areas where cells were larger and appeared hypertrophied. In BMP4-treated pellets, collagen X was only seen in small localized areas (Figure 2C).

[0175] Based on these data, subsequent differentiation experiments involved establishing pellet cultures at the end of day 4 and adding 20 ng / ml FGF2 for 14–28 days after sclerotome differentiation.

[0176] Example 3 The cell line and culture format influence post-sclerotization chondrocyte differentiation. To assess variability in chondrocyte differentiation among iPSC lines, four iPSC lines were differentiated in parallel into sclerotomes, then treated for FGF2 for 14 days, and pellets were harvested for RNAseq. One iPSC line, MCRIi018-B, was grown in feeder-free conditions, while three lines, MCRIi001-A, MCRIi001-A-2, and RM3.5c, were feeder-dependent lines. MCRIi001-A and MCRIi001-A-2 are isogenic lines, with MCRIi001-A-2 modified to express a heterozygous SOX9-TdTomato fluorescent reporter (Nur Patria et al., 2020). Principal component analysis showed that the samples formed three unique clusters according to cell line, with the two isogenic lines clustering close to each other (Figure 2.1A). Comparing gene expression in feeder-dependent and feeder-free lines, we found that over 1800 genes were differentially expressed between the feeder-free line MCRIi018-B and the feeder-dependent MCRIi001-A, and over 1000 genes were differentially expressed between MCRIi018-B and RM3.5c (logFC >1, adj. P. value < 0.00001, Figure 2.1B). Fewer genes (approximately 750) were differentially expressed in the feeder-dependent comparison, compared with only 26 in the syngeneic feeder-dependent line (Figure 2.1B). Key chondrocyte genes, SOX9, COL2A1, COL9A1, COL11A1, and ACAN, were more highly expressed in the feeder-free lines than either of the feeder-dependent lines (Figure 2.1B), suggesting that iPSC lines maintained in feeder-free conditions may differentiate more efficiently.

[0177] To test this, we adapted the feeder-dependent MCRIi001-A-2 to grow in feeder-free conditions, and then differentiated the feeder-free and feeder-dependent versions in parallel. Both feeder-dependent and feeder-free lines were able to differentiate into cartilage, but at day 62, pellets from feeder-dependent MCRIi001-A-2 iPSCs still contained a large amount of non-cartilage tissue (toluidine blue negative), whereas pellets from feeder-free MCRIi001-A-2 were almost entirely cartilage with only a thin layer of non-chondrocytes around the outside (Figure 2.1C). Subsequent differentiation experiments all used iPSC lines adapted to feeder-free conditions.

[0178] Our early comparison of chondrocyte differentiation in pellets cultured in static conditions versus rotary culture from day 6 showed that rotary culture delayed chondrocyte development (Figure 2). iPSC line MCRIi001-A-2 differentiated into sclerotomes and formed pellets on day 4. Some pellets were transferred to rotary culture on days 6, 13, 20, and 27, and some pellets were maintained in static culture. On day 34, pellets from rotary culture from day 6 had a cartilage center surrounded by a thick layer of non-cartilage tissue, while pellets from all other treatments had only a thin layer of non-chondrocytes on the outside (Figure 2.2). On day 48, all pellets had matured further, but pellets from rotary culture from day 6 retained a surface layer of non-chondrocytes. Pellets from rotary culture from days 13, 20, and 27 had significantly enlarged chondrocytes and collagen X staining, confirming that these were hypertrophic chondrocytes expressing collagen X (Figure 2.2A). This finding was confirmed in a second iPSC line, MCRIi019-A, where maximal collagen X expression at day 48 was in pellets transferred to rotary culture on day 13 (Figure 2.2A). Taken together, these experiments confirm that the novel differentiation protocol described herein works with multiple iPSC lines, with individual cell lines maturing into chondrocytes and hypertrophy at different rates, and that transfer to rotary culture affects chondrocyte maturation.

[0179] Example 4 Triiodothyronine enhances spontaneous chondrocyte hypertrophy. To further understand the changes that occur as iPSC-derived chondrocytes mature towards hypertrophy, we monitored differentiation in two iPSC lines, MCRIi019-A and MCRIi018-B. In these experiments, sclerotome pellets were transferred to rotary culture on day 6 and then supplemented with FGF2 for 28 days. Chondronoids were matured in chondrogenic medium for an additional 35 days. Although spontaneous hypertrophy and collagen X expression had been observed in previous experiments (Figure 2), triiodothyronine (T3) is essential for chondrocyte hypertrophy and osteoblast transdifferentiation in mouse secondary ossification centers (Aghajanian et al., 2017), and therefore we treated some pellets with T3 from day 48 to day 69. By day 48, the pellets were pure cartilage with extensive deposition of ECM rich in proteoglycans and collagen II (Figure 3A, Figure 3B, Figure 3C, Figure 3.1A, Figure 3.1B). By day 69, chondrocytes had enlarged, collagen X was abundant in the ECM, and spontaneous maturation to hypertrophy was clearly demonstrated (Figure 3B, Figure 3.1B).

[0180] Cartilage expresses a characteristic repertoire of extracellular matrix proteins that confer unique properties to the tissue and reflect subpopulations of chondrocytes. To obtain a comprehensive and unbiased assessment of ECM gene expression during in vitro iPSC-derived chondrocyte maturation, our RNAseq transcriptome data at day 48, day 69, and day 69+T3 were interrogated against the core matrisome gene set (Nabi et al., 2016). At all developmental stages from day 48 to hypertrophy in both cell lines, members of the cartilage collagen family (COL2A1, COL11A1, COL9A1, COL9A2, and COL9A3), which assemble together to form collagen fibril supramolecular assemblies, are among the 10 most highly expressed components (Figure 3.2). Similarly, other important cartilage ECM molecules aggrecan (ACAN) and HAPLN1, which links aggrecan to hyaluronan polymers, as well as cartilage matrilines (MATN1, MATN3), are strongly expressed throughout this developmental sequence. Concurrent with hypertrophy (day 69 and day 69+T3), expression of COL10A1 and IBSP, markers of core matrisome hypertrophy, is upregulated. Highly expressed sentinel cartilage genes that are downregulated by T3 include COL11A1, COL9A1, COL9A2, COL9A3, EPYC, MATN1, LUM, COMP, and EDIL3 (Figure 3.2), consistent with preparation for osteoblastic transdifferentiation.

[0181] Cartilage maturation to hypertrophy between days 48 and 69 was evident from histology and immunostaining, with chondronoids appearing similarly with and without T3 at day 69 (Figure 3B, Figure 3.1B). Despite this similar appearance, our RNAseq data showed that thousands of genes were differentially expressed between treatments at day 69. We examined the expression of selected cartilage, hypertrophic cartilage, and bone markers and found that hypertrophic cartilage markers, such as COL10A1, IHH, MEF2C, SP7, and SPP1, were upregulated between days 48 and 69 (Figure 3D, Figure 3.1C). T3 treatment enhanced the expression of late hypertrophic cartilage markers, including VEGFA, DMP1, HIF1A, MMP13, and ALPL, and downregulated canonical cartilage genes, such as COL2A1, COL9A1, COL11A1, SOX9, SOX6, and SOX5 (Figure 3D, Figure 3.1C). It was concluded that T3 enhances spontaneous maturation into hypertrophic chondrocytes in our chondroid cultures.

[0182] Gene set enrichment analysis (GSEA) was used to explore global transcriptome changes during the chondrocyte hypertrophy transition. Significantly enriched hallmark gene sets associated with cartilage maturation (MSigDB) showed reduced cell division and increased hypoxia and apoptosis in hypertrophic chondrocytes (Figure 4A). Other enriched gene sets that are important in cartilage development and arthritis, but are less well studied in growth plate maturation, include TNFA, MTORC1, KRAS, and JAK / STAT3 signaling (Figure 4A).

[0183] The enriched C2 (curated) gene set included core matrisome genes and three other matrisome-associated gene sets (Figure 4B). These matrisome gene sets had highly upregulated and highly downregulated members, reflecting the dynamic changes in the ECM during chondrocyte hypertrophy. We focused on secreted factors because these factors may provide further insight into the regulatory steps involved in initiating hypertrophic differentiation and chondrocyte transdifferentiation into osteoblasts. The upregulation of the TGFβ pathway indicated by GSEA analysis of the complete transcriptome dataset (Figure 4A) was confirmed by the upregulation of KEGG TGFβ pathway components TGFB1, TGFB2, BMP2, BMP6, BMP7, and LEFTY2 in the matrisome secreted factors (Figure 4C), consistent with studies showing that TGFβ signaling positively regulates hypertrophy and angiogenesis in growth plate cartilage (Sue Yoshi et al., 2012). Members of the angiogenesis and WNT signaling pathways in the secreted factor gene set are dynamically and differentially expressed during maturation to hypertrophy in our dataset. Upregulated angiogenesis factors (GOBP vascular morphogenesis gene set) include VEGFA, VEGFC, PDGFA, ANGPTL2, and ANGPT2, while SFRP1, WNT5A, ANGPT1, and VEGFB are downregulated (Figure 4C). The angiogenesis pathway has partial overlap with the KEGG WNT signaling pathway, with WNT11 and WIF1 being upregulated and GDF10, SFRP1, SFRP5, SFRP2, and WNT5A being downregulated with hypertrophy (Figure 4C).

[0184] The most highly expressed secreted factor gene upregulated during hypertrophy is FGFBP2. No role has been reported for this FGF-binding protein in cartilage and bone development, and our data suggest that it may regulate the biological activity of the FGF family during endochondral development. Other dynamically regulated secreted factors in the in vitro cartilage maturation model are members of the S100 calcium-binding protein family, including S100A2, S100A6, S100A4, S100P, S100B, and S100A16 (Figure 4C). S100 proteins have many roles in regulating cell and tissue function, including in cartilage the TGFβ and PI3 / AKT pathways, Ca 2+ It has been implicated in homeostasis and TRPV4-mediated mechanoenzymes (Diaz-Romero and Mesic, 2017). SCUBE proteins regulate growth factor signaling, and all three are differentially expressed during maturation and hypertrophy (Figure 4C). In zebrafish, Scube1 and Scube2 promote Vega signaling (Tsao et al., 2021), and loss-of-function mutations in SCUBE3 and the resulting impaired BMP signaling underlie human skeletal disorders (Lin et al., 2021).

[0185] Example 5 Expression of transcription factors during chondrogenic maturation of iPSCs Normal skeletal development is controlled by complex temporal and spatial transcription factor circuits that dictate cell fate decisions (Hate et al., 2017; Arseniy et al., 2009; Kroonenberg, 2003; Liu et al., 2017; Tsang et al., 2014). By analyzing the regulation of transcription factors (TFs) during differentiation of iPSCs into mature hypertrophic chondrocytes, we can determine how closely in vitro pathways recapitulate in vivo growth plate cartilage maturation, and therefore we used the human TF catalog (Lambert et al., 2018) to examine our RNAseq data at successive stages of chondrocyte maturation (day 48, day 69, and day 69+T3). To reduce the complexity of this analysis, we filtered the data to include TFs that were at least 2-fold up- or down-regulated (Adj. P. value < 0.05) and expressed with a mean RPKM >4 in at least one experimental group (cell line, day / treatment). Using these criteria, 118 TF genes were differentially expressed during chondrocyte maturation (Figure 5, Figure 5.1). The expression patterns of TFs with established roles in chondrocyte differentiation and maturation demonstrated that the in vitro process closely recapitulates their developmental regulation. Highly expressed TFs such as HIF1A (HIF1α), a master transcriptional mediator of the adaptive response to hypoxia, and EPAS1 (HIF2α), a regulator of VEGF expression, are upregulated with hypertrophy (Figure 5). The transition to hypertrophy is characterized by upregulation of SP7 (Osteria), RARG (retinoic acid receptor) (Shimon et al., 2019), and MEF2C, which are important for endochondral differentiation of mesenchymal progenitor cells (Dreher et al., 2020). Similarly, hypertrophic upregulation of CEBPB (C / EBPβ) is consistent with its suggested role in suppressing early chondrocyte differentiation and stimulating hypertrophic markers (Okuma et al., 2015). The coordinated expression of these (and other) TFs and their potential functional interactions in regulatory circuits of our iPSC chondrocyte differentiation and maturation pathways is highlighted by known and predicted protein interactions revealed by STRING analysis (Figure 5.1A).Of the 17 upregulated and most highly expressed TFs with established roles in chondrocyte maturation (Figure 5), 15 are interactors. Many have multiple predicted interaction partners, with HIF1A, CEBPB, EPAS1, FOXO1, FOSL2, MEF2C, ATF3, and SP7 present at obvious key regulatory nodes (Figure 5.1) (Liu et al., 2017).

[0186] SOX5 and SOX6, part of the SOX9 / SOX5 / SOX6 master chondrogenic trio (Liu et al., 2017), were coordinately downregulated along with SOX9 (LogFC = -0.83, adj. P value = 8.89 × 10 -7 ) (Figure 5), consistent with the transition to hypertrophy. GLI1 (Figure 5.1) and GLI3 (LogFC = -0.72, adj. P value = 3.95 × 10 -5 ) are also downregulated with hypertrophy. GLI factors act in concert with SOX9 to suppress Col10a1 expression in proliferating mouse chondrocytes (Leung et al., 2011). Meanwhile, elevated FOXA2 in hypertrophic chondrocytes competes with SOX9 binding to the Col10a1 promoter and activates collagen X expression (Tan et al., 2018). FOXA2 is upregulated in our iPSC-derived hypertrophic chondrocytes (Figure 5.1), suggesting that a similar co-regulatory network between SOX9-GLI cooperation in proliferating chondrocytes and SOX9-FOXA competition in hypertrophic chondrocytes is also relevant for human chondrocyte differentiation.

[0187] We also identified many TFs that are less recognized in cartilage development but were strongly up- or down-regulated with hypertrophy (Figure 5, Figure 5.1). TFs up-regulated in association with regulatory interaction nodes included ETS1, STAT3, DDIT3, and members of the KLF family (KLF2, KLF5, and KLF10). Up-regulated during maturation were several TFs related to the circadian rhythm pathway, namely NFIL3, NR1D1, BHLHE41, and NPAS2, in addition to NR3C1 (glucocorticoid receptor) and FOXO1 (Figure 5, Figure 5.1). Our data also identify other up-regulated transcription factors with potentially important but currently poorly described roles in chondrogenesis and chondrocyte maturation, including IRX3, IRX5, DLX3, and CXXC5.

[0188] Example 6 TGFβ3 drives iPSC-derived prechondrocytes towards an articular chondrocyte fate. Having shown that our iPSC differentiation protocol produces growth plate chondrocytes capable of transitioning into hypertrophic chondrocytes, we next tested whether the protocol could be manipulated to produce chondrocytes from an articular cartilage lineage. Lineage tracing experiments indicate that articular and growth plate chondrocytes originate from a common mesenchymal progenitor cell, and that specific fate decisions to form articular chondrocytes occur in early chondrocytes (Decker et al., 2015, 2014; Soda et al., 2010; Zhang et al., 2011). Members of the TGFβ superfamily have important roles in many chondrogenesis steps (Cleary et al., 2015; Pogue and Lyons, 2006; Wan and Cao, 2005; Wang et al., 2014), and TGFβ3 has been used in several iPSC chondrogenesis protocols (Dakar et al., 2019; Craft et al., 2015; Nakajima et al., 2018; O'Connor et al., 2020; Wu et al., 2021). A recent study demonstrated that mouse iPSC-derived chondrocyte precursors treated with TGFβ3 for 4 weeks expressed the sentinel articular chondrocyte marker PRG4 (O'Connor et al., 2020). Based on these data suggesting a role for TGFβ3 in early endochondral / articular cell fate decision and articular chondrocyte specification in vitro, iPSC-derived chondrocyte precursors were treated with TGFβ3 for 5 weeks starting on day 13.

[0189] At day 48, profound differences in cell morphology and gene expression were observed in TGFβ3-treated and untreated chondrocytes. Untreated D48 organoids contained large chondrocytes that strongly expressed collagen II and the hypertrophic marker collagen X, but did not express PRG4, a marker for articular chondrocytes (Figure 6A). In contrast, TGFβ3-treated cartilage precursors developed into a unique population of small chondrocytes that also expressed collagen II but did not express extracellular collagen X (Figure 6A), indicating that they did not develop down the endochondral chondrocyte pathway. High and uniform PRG4 immunostaining suggested that these TGFβ3-treated cells were articular chondrocytes (Figure 6A).

[0190] While the primary role of permanent articular cartilage is weight-bearing and promoting joint articulation, transitional growth plate cartilage serves primarily to drive longitudinal bone growth through endochondral ossification during prepuberty. These distinct functions are reflected in the composition of their extracellular matrices. Therefore, we compared mRNA expression at day 48 in chondronoids that were treated with TGFβ3 to promote articular cartilage development or left untreated to mature toward hypertrophy. Canonical cartilage components COL2A1, COMP, COL9A1, COL9A2, COL9A3, and COL11A1 were highly expressed in both TGFβ3-treated and untreated chondronoids, and either were not differentially expressed (COMP) or were downregulated by 2-fold or less (Figure 6B,C). In contrast, other core matrisome genes that were highly expressed in chondronoids treated with TGFβ3 were strongly upregulated compared to untreated chondronoids. These include the sentinel articular cartilage marker PRG4, the articular cartilage proteins ASPN, CILP, and the elastic fiber-associated protein CILP2 (Figure 6B, Figure 6C; Supplementary table 1). Other elastic fiber components ELN, LTBP2, TGFBI, and MFAP5 were among the 20 most highly upregulated genes (Figure 6C), consistent with an articular cartilage phenotype (Botanic et al., 2019). EMILIN1, EMILIN3, FBLN1, and FBLN3 are also elastic fiber components and are strongly upregulated (Supplementary table 1). Importantly, ECM proteins associated with cartilage hypertrophy and endochondral ossification, such as COL10A1, IBSP, SPP1, and MATN3, are among the most downregulated in chondrocytes differentiating along the articular cartilage pathway by TGFβ3 (Figure 6C). Other changes consistent with articular cartilage ECM included downregulation of ACAN and the associated link protein HAPLN (Figure 6C).

[0191] Compared to what is known about transcription factors (TFs) that are dynamically expressed in maturing growth plates, relatively little is known about TFs important for developing articular chondrocytes. Analysis of the expression of core matrisome genes in TGFβ3-treated chondronoids is consistent with known changes between joint and growth plate expression, suggesting that our dataset may be explored to identify key articular cartilage TFs. ERG, a TF enriched in articular cartilage (Iwamoto et al., 2007) and required for articular cartilage integrity (Otha et al., 2015), is highly expressed and upregulated in TGFβ3-treated chondronoids (Figure 6D, Figure 6E). Consistent with this, PRG4, an ERG target and articular cartilage ECM gene (Otha et al., 2015), is also highly upregulated in TGFβ3-treated pellets (Figure 6B, Figure 6C). Other ETS transcription factors, ELK3 and ERF, are upregulated and highly expressed (Figure 6D, Figure 6E). During development, Elk3 is expressed in mouse precartilage condensations, and expression is then restricted to the perichondrium and future joint regions (Ayad et al., 2001), but its function in articular cartilage has not been described. ERF is expressed in osteoblasts and is important for normal bone development (Raouf and Seth, 2000), but again, its role in articular cartilage has not been identified. Another highly expressed and highly upregulated TF is TRPS1 (Figure 6D, Figure 6E). Mutations in TRPS1 cause a skeletal dysplasia, trichorhinophallic syndrome, and Trps1 mutant mice have similar craniofacial defects, premature growth plate closure, and defects in long bone growth (Napierala et al., 2008). These features highlight a function for TRPS1 in growth plate chondrocytes, and Trps1 is expressed in developing joints (Napierala et al., 2008), but its role there has not been defined. Similarly, PLAGL1, highly expressed and upregulated in our TGFβ3-treated dataset, is abundant in chondrogenic tissues during development ( Tsuda et al., 2004 ) and KO mice have delayed ossification ( Varrault et al., 2006 ), but has no reported role in articular cartilage.Interestingly, MEOX1 and MEOX2 are not expressed at day 48 of chondrogenic differentiation without TGFβ3, but are highly upregulated with TGFβ3 (Figure 6D, Figure 6E; Supplementary table 1). Their role in articular cartilage development has not been characterized, but their early role in somite development is well known (Reijntjes et al., 2007). BHLHE40 is highly expressed and upregulated by TGFβ3 (Figure 6D, Supplementary table 1) and is a key molecular clock gene, again highlighting the central role of circadian rhythms in cartilage homeostasis (Goncalves and Meng, 2019; Gossan et al., 2013). AEBP1, the most highly expressed TF in TGFβ3-treated chondronoids, is also strongly upregulated (Figure 6D, Figure 6E). Alternative AEBP1 splicing produces mRNAs encoding the transcription factor AEBP1 and the secreted core matrisome protein ACLP, which binds collagen (Blackburn et al., 2018). Mutations in AEBP1 cause a form of Ehlers-Danlos syndrome with abnormalities of collagen fibrils (Blackburn et al., 2018; Vishwanath et al., 2020), and thus upregulation of AEBP1 in articular cartilage may be primarily related to its extracellular role.

[0192] Among the TFs most strongly downregulated in TGFβ3-treated chondronoids are MEF2C, RUNX2, SP7, and DLX5, well-known TFs involved in chondrocyte hypertrophy (Figure 6E). NR3C1, which has a known role in hypertrophy, is downregulated along with others identified in the hypertrophy pathway, including KLF2, KLF5, KLF10, DDIT3, DLX3, IRX5, ZNF277, and MXI1 (Supplementary Table 1). Together, the expression patterns of core matrisome and transcription factors mirror what is known about expression in growth plate and articular cartilage, revealing previously unknown changes in expression that may be important in tissue development and homeostasis.

[0193] Example 7 Transdifferentiation of hypertrophic chondrocytes into osteoblasts The fate of hypertrophic chondrocytes during endochondral ossification was long thought to involve cell death followed by remodeling of the hypertrophic cartilage and vascular invasion. However, it is now clear that an alternative fate is transdifferentiation into osteoblasts (Haseeb et al., 2021; Park et al., 2015; Tsang et al., 2014; Yang et al., 2014; Zhou et al., 2014). The ability of stem cell-derived chondrocytes to enter the transdifferentiation pathway in vitro has not been studied, and therefore it is unclear whether the complex regulatory pathways and environmental cues are fully recapitulated in these systems. This ability is important to model human diseases affecting the endochondral pathway at different stages of cartilage and bone development. This is essential to study osteoblast dysfunction in endochondral bone formation.

[0194] Although the regulatory mechanisms driving transdifferentiation are not fully understood, several key components have been identified, including expression of RUNX2 (Qin et al., 2020; Wang et al., 2017; Xing et al., 2019), PTPN11 (Wang et al., 2017), and SP7 (Xing et al., 2019) in hypertrophic chondrocytes. Conversely, sustained expression of SOX9 suppresses transdifferentiation of chondrocytes into osteoblasts (Lui et al., 2019). Although it is undoubtedly true that other important regulators will be identified in due course, the strong expression of RUNX2, SP7, and PTPN11 mRNA and reduced expression of SOX9 in our hypertrophic chondrocytes (Figure 3, Figure 3.1) suggests that they may be primed for transdifferentiation if provided with the right cues in vitro.

[0195] To test whether our iPSC-derived chondrocytes could transdifferentiate into osteoblasts, we first implanted hypertrophic cartilage organoids subcutaneously into immunodeficient mice. After 13 weeks, the organoids were harvested and histologically analyzed by Safranin O / Fast Green staining for cartilage and bone, respectively (Figure 7A). The implants contained some residual cartilage, with characteristic hypertrophic chondrocytes embedded in a Safranin O-positive ECM. However, many of the implants formed histologically recognizable bone (Figure 7A). To distinguish human iPSC-derived cells from mouse cells that may have migrated into the implants, we immunostained with a human-specific Ku80 antibody. Staining showed that the bone in the implants was derived from human cells (Figure 7A), clearly indicating that in vitro differentiated hypertrophic chondrocytes can transdifferentiate into bone cells if signaling cues are provided in vivo.

[0196] We next tested whether in vitro transdifferentiation and bone formation would be possible if culture conditions supporting osteogenesis were provided. Because β-catenin plays a key role in promoting chondrocyte transdifferentiation in mice (Jing et al., 2018), MCRIi018-B hypertrophied organoids were cultured in conventional osteogenic medium for 3 weeks or in osteogenic medium pulsed with CHIR99021 for 7 days to activate the Wnt / β-catenin pathway, followed by 2 weeks in osteogenic medium alone. We first compared the two osteogenic conditions using histology. There was a significant reduction in toluidine blue staining in organoids treated with CHIR99021, indicating that aggrecan degradation occurred in this osteogenic condition (Figure 7B). This is consistent with the remodeling of the cartilage matrix that occurs during endochondral ossification in vivo. Collagen I, the major collagen type in bone, was deposited in the ECM in both osteogenic conditions, but staining was more intense in organoids grown in osteogenic medium alone (Figure 7B). Osteogenic differentiation conditions were compared using von Kossa staining, which is commonly used to show matrix mineralization. Again, both osteogenic conditions induced matrix mineralization, but staining was stronger in organoids differentiated in osteogenic medium alone (Figure 7B). Furthermore, μCT analysis confirmed extensive deposition of calcium phosphate mineral in organoids cultured in osteogenic medium alone, suggesting a transition to bone-like organoids (Figure 7B). In contrast, organoids cultured in osteogenic medium pulsed with CHIR99021 showed little attenuation of μCT, despite showing positive von Kossa staining (Figure 7B). Although these findings seem contradictory, von Kossa staining detects phosphate and does not necessarily indicate the presence of calcium or hydroxyapatite, the calcium phosphate component of bone (Bonewald et al., 2003). This highlights that von Kossa staining should be interpreted with caution when assessing mineralized ECM.

[0197] We compared osteogenic differentiation conditions in a second iPSC line, MCRIi001-A-2, and found that aggrecan degradation was more extensive in osteogenic conditions pulsed with CHIR99021, and von Kossa staining was more intense in organoids cultured in osteogenic medium alone (Figure 7.1). To determine whether enhancing chondrocyte hypertrophy by T3 is important for the transition to osteoblast-like cells, we compared osteogenic differentiation in organoids pretreated with T3 and those without T3 pretreatment. In both osteogenic conditions, there was more extensive and more intense von Kossa staining in T3 pretreated organoids (Figure 7.1), confirming that T3 treatment is essential for optimal transition to bone-like organoids.

[0198] We next examined the changes in gene expression during culture in osteogenic conditions, focusing first on several well-recognized characteristic chondrocyte and osteoblast genes (Figure 7.2). Osteogenic conditions result in a strong upregulation of the key osteoblast marker genes COL1A1, BGLAP, SPP1, and DMP1, with a concomitant striking downregulation of the hypertrophic chondrocyte genes COL2A1 and ACAN (Figure 7.2A). Extracellular deposition of bone collagen I (COL1A1) and osteocalcin (BGLAP) proteins under osteogenic conditions was confirmed by immunohistochemistry (Figure 7.2B).

[0199] Both osteogenic conditions resulted in a significant downregulation of the hypertrophic cartilage genes COL2A1 and ACAN, whereas COL10A1 was reduced only in osteogenic medium pulsed with CHIR99021 (Figure 8A). SPP1 and IBSP, highly expressed in osteoblasts, were upregulated in both osteogenic conditions, as was the major bone collagen, COL1A1, but expression of COL1A1 was much higher in osteogenic medium pulsed with CHIR (Figure 8A). Other cartilage markers such as SOX9, COMP, IHH, MATN3, CEBPB, and cartilage collagens COL11A1, COL11A2, COL9A1, COL9A2, and COL9A3 were also more downregulated in osteogenic medium pulsed with CHIR, whereas bone markers COL3A1, COL5A1, and OMD were more highly expressed. A similar pattern of gene expression changes was seen in the second iPSC line, MCRIi019-A (Figure 8.1). Based on this initial study suggesting that the transition from hypertrophic chondrocytes to osteoblast-like cells was more efficient in CHIR-pulsed osteogenic conditions than in osteogenic medium alone, we focused on further analysis of gene expression in CHIR-pulsed osteogenic medium.

[0200] Similar to chondrocytes, preosteoblasts and osteoblasts express a characteristic set of extracellular matrix proteins, including collagens I (COL1A1 and COL1A2), III (COL3A1), and V (COL5A1 and COL5A2), the proteoglycans, biglycan (BGN), decorin (DCN), keratocan (KERA), and asporin (ASPN), the glycoproteins, osteonectin (SPARC), and thrombospondin 1 (THBS1), members of the SIBLING protein family, IBSP, SPP1, DMP1, and MEPE, and osteocalcin (BGLAP) (Lin et al., 2020). This pattern of gene expression changes was again similar when a second iPSC line, MCRIi019-A, was induced to transition to osteoblasts (Figure 8.1).

[0201] To provide further support for the transition from hypertrophic chondrocytes to pre-osteoblasts / osteoblasts in vitro, we compared gene expression in our differentiation to the expression of top markers defining osteoblast precursors, osteoblasts, and more mature osteoblast clusters in single-cell RNAseq of cells isolated from mouse bone (Ayturk et al., 2020). Osteoblast precursor marker genes are not significantly altered between T3-treated hypertrophic chondrocytes and organoids cultured in osteogenic conditions (Figure 9). In contrast, many osteoblast and mature osteoblast marker genes are highly upregulated in osteogenic conditions. For example, osteoblast markers POSTN, TNC, CCDC3, FAP, and SFRP4 are upregulated by 54-fold, 72-fold, 121-fold, 64-fold, and 4-fold, respectively, in organoids cultured in osteogenic conditions with CHIR pulses compared to hypertrophic chondrocytes (Figure 9). Markers of mature osteoblasts, COL1A1, COL1A2, IBSP, DMP1, and IFITM5, were upregulated 324-, 210-, 7-, 935-, and 105-fold, respectively, under osteogenic conditions with CHIR, and these five genes are the most highly expressed of all the marker genes in our dataset (Figure 9). The gene expression data are consistent with the proposal that hypertrophic chondrocytes transition to osteoblasts in vitro.

[0202] We next focused on the expression of 19 genes that characterize hypertrophic chondrocytes, preosteoblasts, and osteoblast clusters during in vivo mouse transdifferentiation and skeletal development (Haseeb et al., 2021). In osteogenic conditions, all genes except two, namely MGP and SOX4, follow the expression pattern identified during in vivo transdifferentiation (Figure 8.2A). Among the 11 genes that are upregulated during osteoblast transition, nine are more highly expressed in organoids exposed to CHIR pulses, and all five hypertrophic chondrocyte marker genes are more downregulated. Furthermore, all 13 well-characterized and well-recognized osteogenic transcription factors (Chan et al., 2021) are expressed in organoids cultured in osteogenic conditions, 12 of which are more highly expressed than in hypertrophic chondrocytes (Figure 8.2B).

[0203] This gene expression data suggests that our in vitro transdifferentiation protocol recapitulates in vivo development and provides an opportunity to further understand how transdifferentiation of hypertrophic chondrocytes to osteoblasts is regulated. The 20 most highly expressed transcription factors (TFs) in MCRIi018-B osteoblasts include 11 with known roles in osteoblast differentiation or bone development, namely SP7, MEF2A, ATF4, NFE2L1, VDR, HIF1A, XBP1, EGR1, CREB3L1, DLX3, and SKIL (Figure 8D) (Chan et al., 2021; Kim et al., 2010; Leupin et al., 2007; Pang et al., 2020; Price et al., 1998; Symoens et al., 2013; Tohmonda et al., 2011; Zhang et al., 2020). Other highly expressed TFs, such as YBX1, HMGN3, HMGA1, MAZ, CREB3, SON, TRAFD1, ELK3, and DRAP1, have not been implicated in osteoblast differentiation, but their high expression suggests that they may be important. Similarly, the 20 most highly upregulated TFs include many with known roles in osteoblasts and bone, namely PRRX1, SATB2, RARB, TBX2, MAFB, PRRX2, TWIST1, FOS, JUNB, and ZHX3 (Figure 8E) (Chan et al., 2021; Dobreva et al., 2006; Liu et al., 2018, 2018; Suehiro et al., 2011; ten Berge et al., 1998; Wagner, 2002; Zankl et al., 2012), while others, such as EGR3, CENPX, SNAI2, and ELK3, have unknown osteogenic roles. Our data also demonstrate that this hypertrophic chondrocyte-to-osteoblast differentiation protocol is reproducible; 10 of the 20 most highly expressed TFs in MCRIi018-B osteoblasts are also among the most highly expressed TFs in MCRIi019-A osteoblasts (Figure 8D, Figure 8.1D), and similarly, 10 of the 20 most highly expressed TFs are identical in osteoblasts derived from both iPSC lines (Figure 8E, Figure 8.1E).

[0204] Our data showing unique core matrisome expression patterns in articular chondrocytes, hypertrophic chondrocytes, and osteoblasts support recent evidence showing that expression of core matrisome genes is sufficient to populate scRNAseq data, as each cell type produces its own extracellular matrix (Sacher et al., 2021). Together, the mRNA expression signatures of core matrisome and transcription factor genes indicate that hypertrophic chondrocytes can transition into pre-osteoblasts / osteoblasts if culture conditions that support bone formation are provided, making it possible to model endochondral ossification in vitro.

[0205] Example 8 Modeling disease using chondrocyte and osteoblast organoids differentiated from human iPSCs Using a gene-edited iPSC line with a genetic cartilage disorder (hypoptosis), the COL2A1 p.G1113C mutation, and a syngeneic control (Lilianty J, Bateman JF, Lamande SR. Stem Cell Res. 2021 Aug 25; 56:102515), iPSCs were differentiated into mature chondrocytes using the methods described in Materials and Methods and Example 3. Collagen II extracellular matrix was assessed by collagen II immunohistochemistry (Figure 10A) and electron microscopy (Figure 10B). Both methods demonstrated reduced collagen II extracellular matrix in the hypochondrosis mutants.

[0206] Using a gene-edited iPSC line with hereditary osteogenesis imperfecta COL1A1 p.W1312C mutation and a syngeneic control (Howden S, et al., Stem Cell Res. 2019 Jul;38:101453), iPSCs were differentiated in vitro into osteoblasts using the methods described in Materials and Methods and Example 3. Osteogenesis imperfecta iPSC-derived osteoblasts show reduced collagen I extracellular matrix formation by immunohistochemistry (Figure 10C) and reduced mineralization by von Kossa staining (Figure 10D). The reduced mineralization (bone formation) in osteogenesis imperfecta bone organoids is more dramatically shown by micro-CT analysis (Figure 10E).

[0207] Consideration We have developed an iPSC differentiation protocol to produce chondrocytes that follow the normal mesodermal developmental pathway, maturing into sclerotome, chondrocyte precursors, and then articular cartilage, or alternatively, chondrocytes that can mature into hypertrophic chondrocytes and transition into osteoblasts to produce mineralized extracellular matrix. Thus, our protocol recapitulates in vitro key steps in growth plate development and endochondral ossification. At key stages, we have demonstrated widespread gene expression and identified unique gene expression patterns for extracellular matrix genes and transcription factors. These gene expression patterns reflect what is known about gene expression in vivo and can provide new insights into how cartilage development and endochondral bone growth are regulated.

[0208] We first optimized post-sclerotome chondrogenesis. Our optimized protocol involves forming pellets at the end of day 4, i.e. 24 hours after the start of differentiation from somitic mesoderm to sclerotome, to provide the cells with a 3D environment essential for chondrocytes to maintain their phenotype (Ecke et al., 2019). We found that differentiation was more uniform in iPSC lines maintained in feeder-free culture. An unreported innovation in chondrocyte differentiation is the use of orbital rotation culture, where multiple pellets are in a non-adherent dish. In addition to reducing the time and precision required to maintain pellets compared to individual pellet culture, it was found that rotation culture affected the rate and specificity of differentiation. Depending on the cell line, chondrocytes matured towards hypertrophy and most efficiently expressed collagen X, specific for hypertrophic cartilage, when pellets were transferred to rotation culture between days 13 and 20. Pellets transferred to rotary culture between days 13 and 27 had no evident non-chondrocytes within or around the pellet by day 48, making them an ideal tool for studying chondrocyte maturation and hypertrophy and for use in biomaterials for therapeutic applications.

[0209] Long-term culture with a post-sclerotization FGF2 pulse and TGFβ3 supplementation produced articular chondrocytes that expressed the sentinel articular chondrocyte marker PRG4 and a range of other articular cartilage extracellular matrix proteins such as CILP, ASPN, COL1A1, COL3A1, and elastic fiber components, consistent with what is known about the articular cartilage matrisome.

[0210] An alternative post-sclerotomy cell fate, growth plate hypertrophic chondrocytes, can be induced by FGF2 pulse followed by culture in our chondrogenic basal medium APEL2. While some hypertrophy markers, such as COL10A1, RUNX2, SPP1, SP7, MMP13, IHH, and MEF2C, are upregulated with spontaneous hypertrophy and may therefore be early hypertrophy markers, others, including ALPL, FOSL2, VEGFA, BMP7, and DMP1, are only upregulated after stimulation with T3 (triiodothyronine). T3 is required to downregulate the chondrogenic transcription factors SOX9 / SOX5 / SOX6 and the cartilage matrisome genes COL9A1, COMP, and MATN1. In mouse skeletal development, T3 is required to promote hypertrophy around the secondary ossification center of the tibia and to allow the transition of hypertrophic chondrocytes to osteoblasts (Aghajanian et al., 2017). Others have used T3 to promote hypertrophy in differentiation of iPSCs into chondrocytes (Pretemer et al., 2021). However, their protocol did not follow the normal post-sclerotome developmental pathway and included sequential and overlapping addition of dexamethasone, PDGF, TGFβ3, BMP4, T3, and β-glycerophosphate. Our gene expression data indicate that our differentiation program from cartilage precursors to chondrocytes and chondrocyte hypertrophy follows the known program for matrisome and transcription factor expression during growth plate maturation.

[0211] Our transcriptomic data suggest that hypertrophic chondrocytes are primed for transdifferentiation into osteoblasts. Hypertrophic cartilage core matrisome components COL10A1, SPP1, and DMP1 are upregulated, and genes in the angiogenic pathway, such as VEGFA, which promotes vascular invasion into hypertrophic cartilage, as well as genes in the TGFβ and WNT signaling pathways, are differentially expressed. Importantly, we show that both HIF-1a (HIF1A) and HIF-2a (EPAS1) are upregulated during hypertrophic differentiation in our chondroid in vitro model, consistent with a hypoxic / angiogenic switch required for normal in vivo endochondral ossification processes (Liu and Olsen, 2014; Schipani et al., 2009).

[0212] Based on this, we show that hypertrophic chondrocytes can transition to osteoblasts in vitro. When hypertrophic chondrocytes are supplied with the bone formation-promoting factors β-glycerophosphate, ascorbic acid, and dexamethasone, they switch on and / or upregulate characteristic osteoblast matrisome and transcription factor genes and downregulate cartilage-specific gene networks. This dynamic gene expression change during in vitro transition mirrors the reported changes during in vivo transdifferentiation of hypertrophic chondrocytes to osteoblasts in mice (Haseeb et al., 2021) and expression of osteoblast lineage cells in mouse bone (Ayturk et al., 2020). In osteogenic conditions, consistent with the transition to bone-like organoids, the organoids produce collagen I extracellular matrix and deposit calcium phosphate mineral. Importantly, promoting hypertrophy with T3 prior to osteogenic culture was found to be critical for matrix mineralization.

[0213] We have established an iPSC differentiation protocol that generates hypertrophic chondrocytes with plasticity for the forward transition to osteoblasts, thereby recapitulating endochondral ossification in vitro. First generating sclerotomes using a robust and reproducible method, then promoting chondrocyte maturation with minimal growth factor stimulation, means that chondrocytes follow a series of developmental pathways in vivo, making this system ideal for exploring how development is controlled and modeling genetic skeletal diseases. Stimulating the transdifferentiation of hypertrophic chondrocytes to osteoblasts in vitro is an important and significant advance that increases the understanding of this fundamental but newly recognized pathway and allows modeling of bone diseases in vitro. Selection of an alternative differentiation pathway from cartilage precursors to articular cartilage allows for the production of cartilage biomaterials for arthritis-related studies and tissue regeneration. While our protocol is reproducible in all cell lines tested, a key finding is that the rate of differentiation and maturation after sclerotomes varies slightly between cell lines, which results in significant changes in gene expression. This highlights the importance of minimizing variation between cell lines and comparing mutant lines with syngeneic controls for disease modeling.

[0214] (References) TIFF2024538786000001.tif217170TIFF2024538786000002.tif222170TIFF2024538786000003.tif232170TIFF202 4538786000004.tif232170TIFF2024538786000005.tif232170TIFF2024538786000006.tif228170TIFF20245387860 00007.tif232170TIFF2024538786000008.tif232170TIFF2024538786000009.tif229170TIFF2024538786000010.t if226170TIFF2024538786000011.tif221170TIFF2024538786000012.tif232170TIFF2024538786000013.tif158170

Claims

1. 1. A method for producing chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte precursor cells; and b) culturing the three-dimensional aggregates of chondrocyte precursor cells generated in step a) in the absence of the activator of the FGF pathway to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells that express COL2A1 and ACAN. Including, A method comprising the step of transferring a three-dimensional aggregate of sclerotome cells or a three-dimensional aggregate of chondrocyte precursor cells into orbital culture.

2. The chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof: i) expressing one or more additional genes selected from the group consisting of COL11A1, COL11A2, COL9A1, COL9A2, COL9A3, MATN1, and MATN3; and / or ii) expresses collagen 2 (COL2A1) and ACAN in a ratio ranging from 20:1 to 5:1, preferably at a ratio of about 10:1, and does not substantially express COL10A1; and / or iii) does not express collagen COL1A1 or COL1A2 at a level greater than about 0.2% of the level of COL2A1; The method of claim 1.

3. 2. The method of claim 1, further comprising culturing the three-dimensional aggregates of chondrocytes or chondrocyte-like cells with triiodothyronine (T3) to produce a population of hypertrophic chondrocytes or hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, hypertrophic cartilage, or a combination thereof, that express COL10A1, or COL2A1 and COL10A1, preferably in a ratio ranging from about 1:1 to about 2.5:

1.

4. 2. The method of claim 1, further comprising transferring the sclerotome cell aggregates to orbital culture at any time after day 7 of step a).

5. 2. The method of claim 1, further comprising culturing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells with TGF-beta3, preferably for a period of at least 2 weeks to about 7 weeks, to produce a population of PRG4-expressing articular chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof.

6. 1. A method for producing articular non-hypertrophic chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof, comprising: a) culturing three-dimensional aggregates of sclerotome cells with an activator of the FGF pathway to produce three-dimensional aggregates of chondrocyte precursor cells; b) culturing the three-dimensional aggregates of chondrocyte precursor cells with an activator of the FGF pathway and an agonist of TGF-beta to produce three-dimensional aggregates of chondrocytes or chondrocyte-like cells that express COL2A1 and ACAN; and c) culturing the three-dimensional aggregates of chondrocytes or chondrocyte-like cells with an agonist of TGF-beta for an extended period of time to produce articular non-hypertrophic chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof. Including, A method comprising transferring a three-dimensional aggregate of sclerotome cells, a three-dimensional aggregate of chondrocyte precursor cells, or a three-dimensional aggregate of chondrocytes or chondrocyte-like cells into orbital culture.

7. 7. The method of claim 6, wherein the articular chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage, or a combination thereof express one or more additional genes selected from the group consisting of COL2A1, ACAN, PRG4, and optionally ASPN, CILP, and CILP2.

8. The method according to claim 1 or 6, wherein the culturing in step a) and / or step b) is for a period of at least 7 days, a period of 7 to 21 days, a period of about 10 to 17 days, a period of about 14 days, or a period of about 7 days, and / or the culturing in step c) is for a period of about 2 to about 5 weeks.

9. 7. The method of claim 1 or 6, wherein the activator of the FGF pathway is selected from the group consisting of FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20, and FGF23, preferably the activator of the FGF pathway is FGF2.

10. The method further comprises culturing the hypertrophic chondrocytes, hypertrophic chondrocyte-like cells, hypertrophic cartilage-like tissue, or hypertrophic cartilage in orbital culture with an osteogenic culture medium to generate osteoblasts or bone-like organoids expressing COL1A1 and COL1A2; 10. The method of claim 9, wherein preferably the osteogenic differentiation culture medium comprises β-glycerophosphate, ascorbic acid 2-phosphate, sodium ascorbate, and dexamethasone, more preferably the osteogenic differentiation culture medium comprises a WNT agonist for the first approximately 3-7 days of culture, and even more preferably the WNT agonist is CHIR99021.

11. 10. The method of claim 1 or 6, further comprising decellularizing the three-dimensional aggregate of chondrocytes or chondrocyte-like cells, cartilage-like tissue, or cartilage to produce a decellularized scaffold.

12. 10. Chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or a combination thereof, produced according to the method of claim 1 or 6.

13. 11. An osteoblast or bone-like organoid produced according to the method of claim 10.

14. Osteoblasts or bone-like organoids produced according to the method of claim 11.

15. 1. A method for testing a candidate chondrogenic or osteogenic modulating substance, comprising: a) carrying out the method of claim 1 or 6, wherein the test substance is included in any one or more of the incubation steps of the method; b) assessing the effect of the test substance on the proliferation, maintenance, and / or differentiation of chondrocytes or osteoblasts compared to a control population generated in the absence of the test substance; and c) identifying the test substance as a candidate chondrogenic or osteogenic modulator if it increases or decreases proliferation compared to a control and / or affects the maintenance or differentiation of chondrocytes or osteoblasts. A method comprising:

16. A method according to claim 1 or 6, wherein the method is carried out using cells derived from a subject having a bone or cartilage disease or disorder, or cells engineered to have a mutation associated with a bone or cartilage disease or disorder, or cells engineered to correct a mutation associated with a bone or cartilage disease or disorder, preferably wherein the bone disorder is osteogenesis imperfecta and the cartilage disorder is associated with a mutation in COL2A1.

17. Use of chondrocytes, chondrocyte-like cells, cartilage-like tissue, cartilage, or combinations thereof produced in accordance with claim 1, and / or articular non-hypertrophic chondrocytes or articular chondrocyte-like cells, articular cartilage-like tissue, articular cartilage produced in accordance with claim 6, for disease modeling or testing of therapeutic agents for bone or cartilage diseases or disorders.

18. Use of osteoblasts or bone-like organoids produced according to claim 10 for disease modeling or testing of therapeutic agents for bone or cartilage diseases or disorders.

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