HPSC-derived articular chondrocyte compositions, systems and methods of use thereof

Abstract

Improved compositions and methods are provided for generating chondrocytes and cartilage tissue from human pluripotent stem cells. The methods include inducing chondrogenesis in monolayer cultures using one or more of FGF agonists, cAMP agonists, and TGFβ agonists. The articular cartilage tissue generated using the methods has a zonal organization similar to native cartilage tissue, including surface chondrocytes and midzone chondrocytes, with an increase in extracellular matrix components consistent with native cartilage tissue. These biochemical and mechanical properties make the cartilage tissue particularly suitable for tissue transplantation in vivo.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 320,334, filed March 16, 2022, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. R01AR073821 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present invention is generally in the field of chondrocytes and cartilage, and in particular, methods for producing articular chondrocytes and articular cartilage-like tissue from human pluripotent stem cells. [Background technology]

[0004] Degenerative joint disease, also known as osteoarthritis, is among the top five most costly medical conditions in the U.S. One of the challenges of repairing the articular cartilage that lines the joints is that this tissue is formed before birth and regeneration does not normally occur after birth.

[0005] Damage to articular cartilage often leads to joint degeneration or the development of osteoarthritis, which causes lifelong pain and limits the patient's ability to lead a normal daily life. Current treatments for damaged or degenerated cartilage are inadequate and focus primarily on pain management and joint replacement (Redman et al., 2005). Successfully repairing damaged areas of articular cartilage using human pluripotent stem cell-based therapies may provide an effective way to prevent or delay the onset of joint degeneration and improve the patient's quality of life. Current cell-based therapies approved for cartilage repair are limited to autologous chondrocyte implantation (ACI) and its modified form, matrix-induced autologous chondrocyte implantation (MACI). These procedures require an initial surgery to harvest a small piece of cartilage, from which chondrocytes can be isolated and expanded before reimplanting the cultured cells in a second procedure. ACI and MACI have been moderately successful, but have numerous drawbacks, such as the need for multiple surgical procedures, donor site morbidity, low numbers of cells retrieved, and dedifferentiation of chondrocytes upon expansion. In addition, the efficacy of such techniques does not exceed that of the current standard of care, microfracture, which involves drilling the subchondral bone and releasing cells from the medullary cavity to stimulate repair within the defect (Steadman et al., 2001). All of these procedures result in fibrocartilage-like repair tissue that lacks important biochemical and biomechanical properties of cartilage (LaPrade et al., 2008), which can lead to delamination and failure of the graft.

[0006] Human pluripotent stem cells (hPSCs) may provide a solution to many of the problems currently facing the treatment of damaged articular cartilage. The use of self-renewing human embryonic stem cells (hESCs) offers the opportunity to develop “off the shelf” cell sources and / or tissue grafts without the need for the numerous procedures currently required for cell-based therapies. Furthermore, cartilage defects can be repaired using tissues generated from induced pluripotent stem cells (hiPSCs) derived from the patient’s own cells, thus reducing the chance of immune rejection (Staerk et al., 2010; Takahashi et al., 2007). Although rigorous safety studies are necessary before routine clinical use of iPSCs, many approaches to treat human diseases using these cells are under development. Biotechnology companies and academic institutions based on regenerative medicine are developing clinical-grade iPSCs, and the first clinical trial of iPSC-derived platelets was approved in Japan in 2018 (Akabayashi et al., 2019). Clinical trials are also underway to establish the potential of hESC-derived progenitor cells to treat a number of conditions, including ischemic heart disease and age-related macular degeneration (Ilic et al., 2015). The progress of these studies clearly demonstrates the need and potential for hPSCs to be used clinically. Both hESCs and hiPSCs can be differentiated into either articular-like or hypertrophic growth plate-like chondrocyte phenotypes using chemically defined and precisely controlled directed differentiation methods (Craft et al., 2013; Craft et al., 2015). This protocol provides experience of signaling embryonic cells during cartilage development in utero, with induction of primitive streak-like mesoderm, followed by specification of chondrogenic mesoderm, and finally generation of chondrocyte precursors and articular tissue. Long-term exposure to transforming growth factor β3 (TGFβ1, TGFβ2, and TGFβ3) further induces the formation of articular cartilage-like tissue important for the maintenance of frictionless joint surfaces, marked by the expression of SOX9, COL2A1, and PRG4 (the last of which encodes the proteoglycan lubricin). Conversely, exposure of chondrocyte precursors to bone morphogenetic protein 4 (BMP4) induces the expression of hypertrophic chondrocyte genes, including COL10A1 and ALPL.When implanted subcutaneously in mice, TGFβ3-treated chondrocytes produced an articular cartilage-like matrix that resisted vascularization and ossification over a 12-week period in vivo, whereas tissue produced by hypertrophic BMP4-treated chondrocytes created a cartilaginous matrix that initiated remodeling and ossification (Craft et al., 2015).

[0007] In addition to hPSCs, other cell sources are being investigated for use in cell-based therapies, notably human mesenchymal stem cells (hMSCs). Human MSCs can be isolated from a number of postnatal tissues, including adipose, synovium, periosteum, and bone marrow, and are also capable of generating cartilage-like tissue when stimulated by TGFβ (De Bari et al., 2001a; De Bari et al., 2001b; Johnstone et al., 1998; Pittenger et al., 1999; Zuk et al., 2002). However, unlike hESCs and hiPSCs, hMSCs demonstrate a high level of donor-to-donor variability (Stoddart et al., 2012) and are unable to maintain a stable articular cartilage-like phenotype in vivo. Instead, these cells progress toward hypertrophy in response to chondrogenic induction (Johnstone et al., 1998; Mueller et al., 2013). The relative uniformity and phenotypic stability of hPSC-derived chondrocytes compared to hMSCs (Craft et al., 2015), together with their unlimited capacity for proliferation, make hPSC-derived chondrocytes an excellent candidate cell type for future cell-based therapies and research in cartilage repair as a prelude to repairing damaged tissue in patients.

[0008] There remains a need for methods of generating chondrocytes from hPSCs that reduce the number and / or duration of required steps and provide chondrocytes that have a reduced tendency to dedifferentiate following transplantation. Summary of the Invention [Problem to be solved by the invention]

[0009] It is therefore an object of the present invention to provide methods and reagents for producing cells that form articular cartilage.

[0010] It is a further object of the present invention to provide methods and reagents for producing cells capable of being expanded in cell culture to form articular cartilage.

[0011] It is a further object of the present invention to provide a method of treating a subject in need thereof using chemically induced chondrocytes.

[0012] It is a further object of the present invention to provide a method for expanding and preserving chemically induced chondrocytes. [Means for solving the problem]

[0013] Chemically induced chondrocytes or cartilage tissue modified using chemically induced chondrocytes, and extracellular matrix are disclosed. Cell culture media and methods for inducing chondrocytes from human pluripotent stem cells (hPSCs) are also disclosed. Typically, cell culture media is used in a four-step process for inducing chondrocytes from hPSCs. The steps include: Phase I, inducing the formation of primitive streak mesoderm from hPSCs; stage II, inducing the formation of paraxial mesoderm; Stage III, inducing conversion of paraxial mesoderm to chondrocytes; and Phase IV, inducing chondrocytes to form mature cartilage tissue It is.

[0014] In some embodiments, the cell culture medium useful for stage I is supplemented with an effective amount of a combination selected from an FGF agonist, such as FGF, a BMP4 agonist, such as BMP4, and a TGFβ agonist, such as activin A, in an amount effective to induce iPSC cell induction to form primitive streak mesoderm. In some embodiments, stage I includes the formation of embryoid bodies. In other embodiments, stage I does not include the formation of embryoid bodies. The cell culture medium is optionally supplemented with a Wnt agonist.

[0015] In some embodiments, cell culture medium useful for stage II is supplemented with an effective amount of a combination of small molecules and proteins selected from a BMP4 inhibitor and / or an FGF agonist, e.g., FGF, in an amount effective to induce the formation of paraxial mesoderm from primitive streak mesoderm. The cell culture medium for this stage is optionally supplemented with a Wnt inhibitor or a TGFβ inhibitor.

[0016] In some embodiments, cell culture media useful for stage III (i.e., induction of chondrocytes) is supplemented with an effective amount of a combination of small molecules and proteins selected from a TGFβ agonist, an FGF agonist, and a cyclic AMP agonist (e.g., forskolin) to induce conversion of paraxial mesoderm to chondrocytes. In a preferred embodiment, cell culture media useful for stage IV (i.e., induction of mature chondrocytes and cartilage tissue) is supplemented with a TGFβ agonist.

[0017] Methods for chemically inducing differentiation of hPSCs into mature chondrocytes are also provided. Small molecule / protein combinations are used in a four-step cell culture process to induce the formation of mature chondrocytes from hPSCs. Phase I includes hPSCs in cell culture medium supplemented with an effective amount of an FGF agonist, e.g., FGF, a BMP4 agonist, e.g., BMP4, and a TGFβ agonist, e.g., activin A, for 1-6 days, preferably about 1-3 days, to form primitive streak mesoderm. In some embodiments, the cell culture medium is also supplemented with a Wnt agonist. In some embodiments, phase I includes the formation of hPSCs in embryoid bodies. In other embodiments, phase I does not include the formation of hPSCs in embryoid bodies. Phase II comprises culturing a monolayer of primitive streak mesoderm cells in cell culture medium supplemented with an effective amount of a small molecule / protein combination selected from a BMP4 inhibitor and / or an FGF agonist (e.g., FGF) in an effective amount to induce the formation of paraxial mesoderm for about 8-14 days, preferably about 11 days. In some embodiments, the cell culture medium is also supplemented with a Wnt antagonist or a TGFβ antagonist. Phase III comprises culturing a monolayer of paraxial mesoderm cells in cell culture medium supplemented with an effective amount of a small molecule / protein combination selected from a TGFβ agonist, an FGF agonist and a cyclic AMP agonist, e.g., forskolin, for 14-40 days, preferably about 28 days, to obtain chondrocytes from the monolayer culture of paraxial mesoderm. Phase IV comprises culturing the chondrocytes in high density micromasses or encapsulated in a biomaterial to produce cartilage tissue in cell culture medium containing a TGFβ agonist.

[0018] Compositions of mature chondrocytes produced by the disclosed methods are provided.

[0019] Also disclosed are methods of using chemically induced chondrocytes to treat a subject in need thereof. The methods provide chondrocytes and / or cartilage tissue suitable for transplantation. The chondrocytes and / or cartilage tissue can be used to treat or prevent one or more diseases or disorders in a subject in need thereof. In some embodiments, the subject has osteoarthritis, osteochondritis dissecans, polychondritis, other cartilage diseases, or injuries or damage affecting cartilage. [Brief description of the drawings]

[0020] [Figure 1A] FIG. 1A is a schematic diagram showing human embryonic stem cell (HES)-derived micromass cultures that are serially reseeded to generate new cartilage tissue. [Figure 1B] Figures 1B and 1C are bar graphs showing the relative mRNA copy numbers of COL10A1 (Figure 1B) and PRG4 (Figure 1C) in culture after 12 weeks (12w) in response to TGFβ and BMP in samples including unpassaged micromass (P0), cultures that had been serially passaged once (P1), and cultures that had been serially passaged twice (P2) shown in Figure 1A. [Figure 1C] Figures 1B and 1C are bar graphs showing the relative mRNA copy numbers of COL10A1 (Figure 1B) and PRG4 (Figure 1C) in culture after 12 weeks (12w) in response to TGFβ and BMP in samples including unpassaged micromass (P0), cultures that had been serially passaged once (P1), and cultures that had been serially passaged twice (P2) shown in Figure 1A. [Figure 1D] FIG. 1D is a schematic diagram showing the differentiation of pluripotent stem cells into articular chondrocytes using micromasses compared to the use of monolayers at stage III of the differentiation process. [Figure 2A] FIG. 2A is a schematic diagram showing human embryonic stem cell (HES) derived micromass cultures that are serially reseeded and expanded (photomicrograph of expanded cells phenotype) to generate new cartilage tissue. [Figure 2B]Figures 2B and 2C are bar graphs showing the relative mRNA copy numbers of COL10A1 (Figure 2B) and PRG4 (Figure 2C) during 12 weeks (12w) of culture in response to TGFβ and BMP in samples including unpassaged micromass (P0) shown in Figure 2A, cultures that had been serially passaged once (E1), cultures of unpassaged micromass expanded once in monolayer before reseeding (E2), or cultures that had been expanded twice in monolayer before reseeding (E3). [Figure 2C] Figures 2B and 2C are bar graphs showing the relative mRNA copy numbers of COL10A1 (Figure 2B) and PRG4 (Figure 2C) during 12 weeks (12w) of culture in response to TGFβ and BMP in samples including unpassaged micromass (P0) shown in Figure 2A, cultures that had been serially passaged once (E1), cultures of unpassaged micromass expanded once in monolayer before reseeding (E2), or cultures that had been expanded twice in monolayer before reseeding (E3). [Figure 3A] 3A-3C show the relative gene expression (mRNA copy number relative to TBP) of COL2A1 (a cartilage gene) in monolayer cultures of paraxial mesoderm (day 14 of the induction protocol) after treatment for 2, 3, and 4 days with either a combination of TGFβ, FGF, and DMSO or a combination of TGFβ, FGF, and FSK with the specific components, DMSO or forskolin (FSK), added for 1 day only (1D hit), 2 days (2D hit), 3 days (3D hit), or 4 days (4D hit) (FIG. 3A); 3B is a bar graph showing the relative gene expression of COL2A1 in monolayer culture after 2 weeks in the presence of TGF-β, FGF and DMSO or TGF-β, FGF and FSK for the indicated time periods (1 day (1D HIT), 2 days (2D HIT), 3 days (3D HIT), 4 days (4D HIT) or continuously (CONT)); and the gene expression of SCX in monolayer culture after 2, 3, 4 days or 2 weeks in the presence of TGF-β, FGF and DMSO or TGF-β, FGF and FSK for the indicated time periods (FIG. 3C). [Figure 3B]3A-3C show the relative gene expression (mRNA copy number relative to TBP) of COL2A1 (a cartilage gene) in monolayer cultures of paraxial mesoderm (day 14 of the induction protocol) after treatment for 2, 3, and 4 days with either a combination of TGFβ, FGF, and DMSO or a combination of TGFβ, FGF, and FSK with the specific components, DMSO or forskolin (FSK), added for 1 day only (1D hit), 2 days (2D hit), 3 days (3D hit), or 4 days (4D hit) (FIG. 3A); 3B is a bar graph showing the relative gene expression of COL2A1 in monolayer culture after 2 weeks in the presence of TGF-β, FGF and DMSO or TGF-β, FGF and FSK for the indicated time periods (1 day (1D HIT), 2 days (2D HIT), 3 days (3D HIT), 4 days (4D HIT) or continuously (CONT)); and the gene expression of SCX in monolayer culture after 2, 3, 4 days or 2 weeks in the presence of TGF-β, FGF and DMSO or TGF-β, FGF and FSK for the indicated time periods (FIG. 3C). [Figure 3C] 3A-3C show the relative gene expression (mRNA copy number relative to TBP) of COL2A1 (a cartilage gene) in monolayer cultures of paraxial mesoderm (day 14 of the induction protocol) after treatment for 2, 3, and 4 days with either a combination of TGFβ, FGF, and DMSO or a combination of TGFβ, FGF, and FSK with the specific components, DMSO or forskolin (FSK), added for 1 day only (1D hit), 2 days (2D hit), 3 days (3D hit), or 4 days (4D hit) (FIG. 3A); 3B is a bar graph showing the relative gene expression of COL2A1 in monolayer culture after 2 weeks in the presence of TGF-β, FGF and DMSO or TGF-β, FGF and FSK for the indicated time periods (1 day (1D HIT), 2 days (2D HIT), 3 days (3D HIT), 4 days (4D HIT) or continuously (CONT)); and the gene expression of SCX in monolayer culture after 2, 3, 4 days or 2 weeks in the presence of TGF-β, FGF and DMSO or TGF-β, FGF and FSK for the indicated time periods (FIG. 3C). [Figure 4A]4A-4C are bar graphs showing relative gene expression of COL2A1 (FIG. 4A), SCX (FIG. 4B), and MKX (tendon gene, FIG. 4C) in two starting populations of day 14 paraxial mesoderm (meso) including 420 (days 3-5 of differentiation, i.e., at stage 2, with BMP inhibitors and FGF) and 420i mesoderm (days 3-5 of differentiation, i.e., at stage 2, 420 treated with the Wnt inhibitor IWP2). [Figure 4B] 4A-4C are bar graphs showing relative gene expression of COL2A1 (FIG. 4A), SCX (FIG. 4B), and MKX (tendon gene, FIG. 4C) in two starting populations of day 14 paraxial mesoderm (meso) including 420 (days 3-5 of differentiation, i.e., at stage 2, with BMP inhibitors and FGF) and 420i mesoderm (days 3-5 of differentiation, i.e., at stage 2, 420 treated with the Wnt inhibitor IWP2). [Figure 4C] 4A-4C are bar graphs showing relative gene expression of COL2A1 (FIG. 4A), SCX (FIG. 4B), and MKX (tendon gene, FIG. 4C) in two starting populations of day 14 paraxial mesoderm (meso) including 420 (days 3-5 of differentiation, i.e., at stage 2, with BMP inhibitors and FGF) and 420i mesoderm (days 3-5 of differentiation, i.e., at stage 2, 420 treated with the Wnt inhibitor IWP2). [Figure 4D] Figure 4D is a dot plot showing qPCR expression data of the tendon-related gene Mohawk Homeobox gene (MKX) in 420 and 420i paraxial mesoderm cells seeded in micromass cultures supplemented with TGFβ. qPCR was performed after 12 weeks of incubation in micromass culture. [Figure 5A] Figures 5A and 5B are bar graphs comparing the expression of COL2A1 (cartilage-associated gene, Figure 5A) and SCX (tendon-associated gene, Figure 5B) in 420 and 420i paraxial mesoderm cells. Cells were plated in serum-free monolayer cultures with no additional factors and qPCR was performed after 4 weeks of culture. N=3 per group. [Figure 5B] Figures 5A and 5B are bar graphs comparing the expression of COL2A1 (cartilage-associated gene, Figure 5A) and SCX (tendon-associated gene, Figure 5B) in 420 and 420i paraxial mesoderm cells. Cells were plated in serum-free monolayer cultures with no additional factors and qPCR was performed after 4 weeks of culture. N=3 per group. [Figure 5C] Figures 5C and 5D are bar graphs comparing the expression of SCX (Figure 5C) and COL2A1 (Figure 5D) in 420 and 420i paraxial mesoderm cells. Cells were seeded in serum-free monolayer cultures supplemented with either FGF alone or a combination of FGF, TGFβ and FSK. qPCR was performed after 4 weeks of culture. N=3 per group. [Figure 5D] Figures 5C and 5D are bar graphs comparing the expression of SCX (Figure 5C) and COL2A1 (Figure 5D) in 420 and 420i paraxial mesoderm cells. Cells were seeded in serum-free monolayer cultures supplemented with either FGF alone or a combination of FGF, TGFβ and FSK. qPCR was performed after 4 weeks of culture. N=3 per group. [Figure 6A]6A-6C are bar graphs showing relative gene expression of COL2A1 (cartilage gene, FIG. 6A), SCX (tendon gene, FIG. 6B), and MKX (tendon gene, FIG. 6C) in monolayer cultures after 2, 3, and 4 weeks in culture medium containing no additives (SFD), DMSO alone without TGF-β and FGF, or forskolin alone (30 μM, FSK30). [Figure 6B] 6A-6C are bar graphs showing relative gene expression of COL2A1 (cartilage gene, FIG. 6A), SCX (tendon gene, FIG. 6B), and MKX (tendon gene, FIG. 6C) in monolayer cultures after 2, 3, and 4 weeks in culture medium containing no additives (SFD), DMSO alone without TGF-β and FGF, or forskolin alone (30 μM, FSK30). [Figure 6C] 6A-6C are bar graphs showing relative gene expression of COL2A1 (cartilage gene, FIG. 6A), SCX (tendon gene, FIG. 6B), and MKX (tendon gene, FIG. 6C) in monolayer cultures after 2, 3, and 4 weeks in culture medium containing no additives (SFD), DMSO alone without TGF-β and FGF, or forskolin alone (30 μM, FSK30). [Figure 7A] 7A-7C are bar graphs showing relative gene expression of SOX9 (cartilage gene, FIG. 7A), COL2A1 (cartilage gene, FIG. 7B), and SCX (tendon gene, FIG. 7C) in monolayer cultures in culture medium containing i) TGFβ3, ii) TGFβ3 and FSK (T+FSK) or iii) TGFβ3, FSK and Creb binding protein inhibitor (CBPi) at concentrations of 0.1 μM, 0.5 μM, or 1.0 μM, with or without bFGF. [Figure 7B] 7A-7C are bar graphs showing relative gene expression of SOX9 (cartilage gene, FIG. 7A), COL2A1 (cartilage gene, FIG. 7B), and SCX (tendon gene, FIG. 7C) in monolayer cultures in culture medium containing i) TGFβ3, ii) TGFβ3 and FSK (T+FSK) or iii) TGFβ3, FSK and Creb binding protein inhibitor (CBPi) at concentrations of 0.1 μM, 0.5 μM, or 1.0 μM, with or without bFGF. [Figure 7C] 7A-7C are bar graphs showing relative gene expression of SOX9 (cartilage gene, FIG. 7A), COL2A1 (cartilage gene, FIG. 7B), and SCX (tendon gene, FIG. 7C) in monolayer cultures in culture medium containing i) TGFβ3, ii) TGFβ3 and FSK (T+FSK) or iii) TGFβ3, FSK and Creb binding protein inhibitor (CBPi) at concentrations of 0.1 μM, 0.5 μM, or 1.0 μM, with or without bFGF. [Figure 8A] 8A-8D show the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from T15 paraxial mesoderm progenitor micromasses, micromasses (C+), RAD16-I+3w micromasses [100 μl], and RAD16-I+3w micromasses [200 μl] (FIG. 8A); the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitor (T15) monolayers, monolayers (C+), RAD16-I+3w monolayers [100 μl], and RAD16-I+3w monolayers [200 μl] (FIG. 8B); 8C : PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) micromass, micromass (C+), RAD16-I+3w micromass [100 μl], and RAD16-I+3w micromass [200 μl] (FIG. 8C); PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) monolayer, monolayer (C+), RAD16-I+3w monolayer [100 μl], and RAD16-I+3w monolayer [200 μl] (FIG. 8D). mRNA copy number is relative to TBP and determined by qPCR-based expression analysis. T15 mesoderm is the starting paraxial mesoderm population and is the same for both micromass- and monolayer-derived chondrocytes. Bars represent the mean standard error. N is the number of samples per condition. [Figure 8B]8A-8D show the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from T15 paraxial mesoderm progenitor micromasses, micromasses (C+), RAD16-I+3w micromasses [100 μl], and RAD16-I+3w micromasses [200 μl] (FIG. 8A); the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitor (T15) monolayers, monolayers (C+), RAD16-I+3w monolayers [100 μl], and RAD16-I+3w monolayers [200 μl] (FIG. 8B); 8C : PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) micromass, micromass (C+), RAD16-I+3w micromass [100 μl], and RAD16-I+3w micromass [200 μl] (FIG. 8C); PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) monolayer, monolayer (C+), RAD16-I+3w monolayer [100 μl], and RAD16-I+3w monolayer [200 μl] (FIG. 8D). mRNA copy number is relative to TBP and determined by qPCR-based expression analysis. T15 mesoderm is the starting paraxial mesoderm population and is the same for both micromass- and monolayer-derived chondrocytes. Bars represent the mean standard error. N is the number of samples per condition. [Figure 8C]8A-8D show the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from T15 paraxial mesoderm progenitor micromasses, micromasses (C+), RAD16-I+3w micromasses [100 μl], and RAD16-I+3w micromasses [200 μl] (FIG. 8A); the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitor (T15) monolayers, monolayers (C+), RAD16-I+3w monolayers [100 μl], and RAD16-I+3w monolayers [200 μl] (FIG. 8B); 8C : PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) micromass, micromass (C+), RAD16-I+3w micromass [100 μl], and RAD16-I+3w micromass [200 μl] (FIG. 8C); PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) monolayer, monolayer (C+), RAD16-I+3w monolayer [100 μl], and RAD16-I+3w monolayer [200 μl] (FIG. 8D). mRNA copy number is relative to TBP and determined by qPCR-based expression analysis. T15 mesoderm is the starting paraxial mesoderm population and is the same for both micromass- and monolayer-derived chondrocytes. Bars represent the mean standard error. N is the number of samples per condition. [Figure 8D]8A-8D show the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from T15 paraxial mesoderm progenitor micromasses, micromasses (C+), RAD16-I+3w micromasses [100 μl], and RAD16-I+3w micromasses [200 μl] (FIG. 8A); the copy numbers of COL2A1 mRNA relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitor (T15) monolayers, monolayers (C+), RAD16-I+3w monolayers [100 μl], and RAD16-I+3w monolayers [200 μl] (FIG. 8B); 8C : PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) micromass, micromass (C+), RAD16-I+3w micromass [100 μl], and RAD16-I+3w micromass [200 μl] (FIG. 8C); PRG4 mRNA copy number relative to TBP in tissues generated from 3-week-old cells from paraxial mesoderm progenitors (T15) monolayer, monolayer (C+), RAD16-I+3w monolayer [100 μl], and RAD16-I+3w monolayer [200 μl] (FIG. 8D). mRNA copy number is relative to TBP and determined by qPCR-based expression analysis. T15 mesoderm is the starting paraxial mesoderm population and is the same for both micromass- and monolayer-derived chondrocytes. Bars represent the mean standard error. N is the number of samples per condition. [Figure 9A] 9A-9C are bar graphs showing relative mRNA copy numbers of COL10A1 (growth plate cartilage marker) (FIG. 9A), PRG4 (articular cartilage marker) (FIG. 9B), and COL2A1 (general cartilage marker) (FIG. 9C) in monolayer-derived chondrocytes (derived from either FGF treatment alone or the combination of TGFβ+FGF+FSK treatment) that generated articular or growth plate-like chondrocytes and cartilage tissue after 6 weeks of culture in micromass in the presence of TGFβ or BMP4. [Figure 9B]9A-9C are bar graphs showing relative mRNA copy numbers of COL10A1 (growth plate cartilage marker) (FIG. 9A), PRG4 (articular cartilage marker) (FIG. 9B), and COL2A1 (general cartilage marker) (FIG. 9C) in monolayer-derived chondrocytes (derived from either FGF treatment alone or the combination of TGFβ+FGF+FSK treatment) that generated articular or growth plate-like chondrocytes and cartilage tissue after 6 weeks of culture in micromass in the presence of TGFβ or BMP4. [Figure 9C] 9A-9C are bar graphs showing relative mRNA copy numbers of COL10A1 (growth plate cartilage marker) (FIG. 9A), PRG4 (articular cartilage marker) (FIG. 9B), and COL2A1 (general cartilage marker) (FIG. 9C) in monolayer-derived chondrocytes (derived from either FGF treatment alone or the combination of TGFβ+FGF+FSK treatment) that generated articular or growth plate-like chondrocytes and cartilage tissue after 6 weeks of culture in micromass in the presence of TGFβ or BMP4. [Figure 9D] Figures 9D-9F are bar graphs showing mRNA copy numbers of COL10A1 (Figure 9D), PRG4 (Figure 9E), and COL2A1 (Figure 9F) in monolayer-derived chondrocytes (derived from either FGF treatment alone or the combination of TGFβ+FGF+FSK treatment) that generated articular or growth plate-like chondrocytes and cartilage tissue after 12 weeks of culture in micromass (uM) in the presence of TGFβ or BMP4. [Figure 9E] Figures 9D-9F are bar graphs showing mRNA copy numbers of COL10A1 (Figure 9D), PRG4 (Figure 9E), and COL2A1 (Figure 9F) in monolayer-derived chondrocytes (derived from either FGF treatment alone or the combination of TGFβ+FGF+FSK treatment) that generated articular or growth plate-like chondrocytes and cartilage tissue after 12 weeks of culture in micromass (uM) in the presence of TGFβ or BMP4. [Figure 9F]Figures 9D-9F are bar graphs showing mRNA copy numbers of COL10A1 (Figure 9D), PRG4 (Figure 9E), and COL2A1 (Figure 9F) in monolayer-derived chondrocytes (derived from either FGF treatment alone or the combination of TGFβ+FGF+FSK treatment) that generated articular or growth plate-like chondrocytes and cartilage tissue after 12 weeks of culture in micromass (uM) in the presence of TGFβ or BMP4. [Figure 10A] Figures 10A and 10B are principal component (based on gene expression) and gene expression plots showing that hESC-derived articular and growth plate chondrocytes have unique transcriptional profiles similar to their corresponding fetal cartilage counterparts. Figure 10A is a principal component analysis (PCA) plot of hESC-derived and fetal cartilage RNA-seq expression data. Legend indicates cell type and sequencing batch. Figure 10B is a graph showing the top 100 differentially expressed genes up- or down-regulated in hESC-derived joints (top) compared to comparable log(2)FC values ​​in fetal cartilage (bottom). [Figure 10B] Figures 10A and 10B are principal component (based on gene expression) and gene expression plots showing that hESC-derived articular and growth plate chondrocytes have unique transcriptional profiles similar to their corresponding fetal cartilage counterparts. Figure 10A is a principal component analysis (PCA) plot of hESC-derived and fetal cartilage RNA-seq expression data. Legend indicates cell type and sequencing batch. Figure 10B is a graph showing the top 100 differentially expressed genes up- or down-regulated in hESC-derived joints (top) compared to comparable log(2)FC values ​​in fetal cartilage (bottom). [Figure 11A]Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11B] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11C] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11D]Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11E] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11F] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11G]Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11H] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11I] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11J]Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11K] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11L] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11M]Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11N] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11O] Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 11P]Figures 11A-11P are dot plots illustrating the results of validation of differential gene expression in hESC-derived articular and growth plate cartilage, and fetal epiphyseal and growth plate cartilage. Figures 11A-11H show quantitative RT-PCR data of differentially expressed genes (DEGs) in hESC-derived cartilage: FGF18 (Figure 11A), PTHLH (Figure 11B), MEOX1 (Figure 11C), CHI3L1 (Figure 11D), PTH1R (Figure 11E), FGFR3 (Figure 11F), PANX3 (Figure 11G) and ALPL (Figure 11H). N=5 independent experiments with 3-6 replicates per experiment. Figures 11I-11P show quantitative RT-PCR data of differentially expressed genes (DEGs) in fetal cartilage: FGF18 (Figure 11I), PTHLH (Figure 11J), MEOX1 (Figure 11K), CHI3L1 (Figure 11L), PTH1R (Figure 11M), FGFR3 (Figure 11N), PANX3 (Figure 11O) and ALPL (Figure 11P). Chondrocytes were isolated from the epiphysis and growth plate of the distal femur and proximal tibia (3 per site at E59, E67 and E72). *p<0.05, **p<0.01, ***p<0.001. [Figure 12A] Figure 12A is a histogram showing the expression and overlap in expression of a subset of transcription factors (TFs) between hESC-derived chondrocytes and human fetal chondrocytes. Figure 12A shows the direction of the top 20 differentially expressed transcription factors that were up- and downregulated when compared to hESC-derived articular and growth plate chondrocytes (top row) with comparable log(2)FC values ​​of fetal tissue samples (bottom row). [Figure 12B] Figures 12B and 12C are representative tables showing transcription factor motif enrichment within the epigenetic profiles of hESC-derived articular chondrocytes (AC) and growth plate cells (GPC). Homer de novo motif enrichment of putative enhancer sequences was biased towards either the TGFβ or BMP treatment lineage. The motifs best matching the de novo results are shown, the BMP4 Homer motif (Figure 12B) and the TGFβ Homer motif (Figure 12C). [Figure 12C]Figures 12B and 12C are representative tables showing transcription factor motif enrichment within the epigenetic profiles of hESC-derived articular chondrocytes (AC) and growth plate cells (GPC). Homer de novo motif enrichment of putative enhancer sequences was biased towards either the TGFβ or BMP treatment lineage. The motifs best matching the de novo results are shown, the BMP4 Homer motif (Figure 12B) and the TGFβ Homer motif (Figure 12C). [Figure 13A] Figure 13A is a graph illustrating results suggesting that gene expression variability can be attributed to different classes of regulatory elements (gene regulatory behavior). In Figure 13A, the logFC values ​​of genes were clustered by regulatory behavior. The significance bars indicate Tukey post-hoc corrected p-values. The proportion of significant differentially expressed (DE) genes in each cluster is shown. ns not significant; *p<0.05; **p<0.01; ***p<0.001. [Figure 13B]Figures 13B-13G are graphs confirming putative lineage-delineated transcription factors. Results of enrichment studies comparing the occurrence of indicated motifs in TGFβ or BMP-biased DARs to randomized background. The top 5 motifs (ordered by difference in enrichment results) are shown for each category. Figure 13B is a bar graph showing TFs differentially expressed (DE) in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. Figure 13C is a bar graph showing TFs DE in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. Figure 13D is an enrichment histogram for RELA motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DE in the corresponding lineage. Red lines indicate target settings and black bars indicate occurrence in randomized settings. FIG. 13E is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. FIG. 13F is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. FIG. 13G is an enriched histogram for RUNX2 motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DEs in the corresponding series. Red lines indicate target settings and black bars indicate occurrence in randomized settings. *p<0.05; NS, not significant. [Figure 13C]Figures 13B-13G are graphs confirming putative lineage-delineated transcription factors. Results of enrichment studies comparing the occurrence of indicated motifs in TGFβ or BMP-biased DARs to randomized background. The top 5 motifs (ordered by difference in enrichment results) are shown for each category. Figure 13B is a bar graph showing TFs differentially expressed (DE) in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. Figure 13C is a bar graph showing TFs DE in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. Figure 13D is an enrichment histogram for RELA motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DE in the corresponding lineage. Red lines indicate target settings and black bars indicate occurrence in randomized settings. FIG. 13E is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. FIG. 13F is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. FIG. 13G is an enriched histogram for RUNX2 motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DEs in the corresponding series. Red lines indicate target settings and black bars indicate occurrence in randomized settings. *p<0.05; NS, not significant. [Figure 13D]Figures 13B-13G are graphs confirming putative lineage-delineated transcription factors. Results of enrichment studies comparing the occurrence of indicated motifs in TGFβ or BMP-biased DARs to randomized background. The top 5 motifs (ordered by difference in enrichment results) are shown for each category. Figure 13B is a bar graph showing TFs differentially expressed (DE) in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. Figure 13C is a bar graph showing TFs DE in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. Figure 13D is an enrichment histogram for RELA motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DE in the corresponding lineage. Red lines indicate target settings and black bars indicate occurrence in randomized settings. FIG. 13E is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. FIG. 13F is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. FIG. 13G is an enriched histogram for RUNX2 motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DEs in the corresponding series. Red lines indicate target settings and black bars indicate occurrence in randomized settings. *p<0.05; NS, not significant. [Figure 13E]Figures 13B-13G are graphs confirming putative lineage-delineated transcription factors. Results of enrichment studies comparing the occurrence of indicated motifs in TGFβ or BMP-biased DARs to randomized background. The top 5 motifs (ordered by difference in enrichment results) are shown for each category. Figure 13B is a bar graph showing TFs differentially expressed (DE) in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. Figure 13C is a bar graph showing TFs DE in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. Figure 13D is an enrichment histogram for RELA motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DE in the corresponding lineage. Red lines indicate target settings and black bars indicate occurrence in randomized settings. FIG. 13E is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. FIG. 13F is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. FIG. 13G is an enriched histogram for RUNX2 motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DEs in the corresponding series. Red lines indicate target settings and black bars indicate occurrence in randomized settings. *p<0.05; NS, not significant. [Figure 13F]Figures 13B-13G are graphs confirming putative lineage-delineated transcription factors. Results of enrichment studies comparing the occurrence of indicated motifs in TGFβ or BMP-biased DARs to randomized background. The top 5 motifs (ordered by difference in enrichment results) are shown for each category. Figure 13B is a bar graph showing TFs differentially expressed (DE) in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. Figure 13C is a bar graph showing TFs DE in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. Figure 13D is an enrichment histogram for RELA motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DE in the corresponding lineage. Red lines indicate target settings and black bars indicate occurrence in randomized settings. FIG. 13E is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. FIG. 13F is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. FIG. 13G is an enriched histogram for RUNX2 motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DEs in the corresponding series. Red lines indicate target settings and black bars indicate occurrence in randomized settings. *p<0.05; NS, not significant. [Figure 13G]Figures 13B-13G are graphs confirming putative lineage-delineated transcription factors. Results of enrichment studies comparing the occurrence of indicated motifs in TGFβ or BMP-biased DARs to randomized background. The top 5 motifs (ordered by difference in enrichment results) are shown for each category. Figure 13B is a bar graph showing TFs differentially expressed (DE) in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. Figure 13C is a bar graph showing TFs DE in TGFβ-treated articular chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. Figure 13D is an enrichment histogram for RELA motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DE in the corresponding lineage. Red lines indicate target settings and black bars indicate occurrence in randomized settings. FIG. 13E is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around enhancer-centric DEGs. FIG. 13F is a bar graph showing TFs DE in BMP-treated growth plate chondrocytes examining motif occurrence in TGFβ or BMP-biased DARs around combo-centric DEGs. FIG. 13G is an enriched histogram for RUNX2 motif occurrence in BMP (left) and TGFβ (right) biased DARs around combo-centric gene DEs in the corresponding series. Red lines indicate target settings and black bars indicate occurrence in randomized settings. *p<0.05; NS, not significant. [Figure 14A]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14B]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14C]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14D]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14E]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14F]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14G]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14H]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14I]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14J]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14K]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14L]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14M]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14N]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14O]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14P]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14Q]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14R]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 14S]14A-14S are scatter dot plots showing putative targets of TF regulation in hESC-derived articular and growth plate chondrocytes. FIG. 14A illustrates the results that RELA is differentially expressed upon TGFβ treatment. FIG. 14B-14H illustrates the expression of selected genes with putative RELA binding motifs quantified by qRT-PCR. The selected genes are GLIPR2 (FIG. 14B), LOXL2 (FIG. 14C), PRG4 (FIG. 14D), DKK3 (FIG. 14E), TLR2 (FIG. 14F), LTBP2 (FIG. 14G), and COL15A1 (FIG. 14H). *p<0.05, **p<0.01, ***p<0.001. FIG. 14I illustrates the results that RUNX2 is differentially expressed upon BMP treatment. FIG. 14J-14S illustrates the expression of selected genes with putative RUNX2 binding motifs quantified by qRT-PCR. The selected genes are ATOH8 (Figure 14J), ACAN (Figure 14K), C16ORF72 (Figure 14L), COL10A1 (Figure 14M), RCL1 (Figure 14N), WNT10B (Figure 14O), GRP153 (Figure 14P), MAP4K3 (Figure 14Q), RXRA (Figure 14R) and SCUBE (Figure 14S). *p<0.05, **p<0.01, ***p<0.001. [Figure 15A] Figures 15A-15C are graphs showing TF interactions with putative regulatory elements, validated by ChIP-qPCR. Figures 15A and 15B illustrate that RELA results of ChIP-qPCR for differentially accessible peaks near target genes show enrichment for these sequences compared to a negative (gene desert) control (Untr12). BIRC3 is a positive RELA control. Figures 15A and 15B represent two pools of TGFβ-treated articular chondrocytes of ChIP. Figure 15C shows that RUNX2 results of ChIP-qPCR for differentially accessible peaks near target genes show enrichment for these sequences compared to a negative (gene desert) control (Untr12). DPF1 is a positive RUNX2 control. [Figure 15B]Figures 15A-15C are graphs showing TF interactions with putative regulatory elements, validated by ChIP-qPCR. Figures 15A and 15B illustrate that RELA results of ChIP-qPCR for differentially accessible peaks near target genes show enrichment for these sequences compared to a negative (gene desert) control (Untr12). BIRC3 is a positive RELA control. Figures 15A and 15B represent two pools of TGFβ-treated articular chondrocytes of ChIP. Figure 15C shows that RUNX2 results of ChIP-qPCR for differentially accessible peaks near target genes show enrichment for these sequences compared to a negative (gene desert) control (Untr12). DPF1 is a positive RUNX2 control. [Figure 15C] Figures 15A-15C are graphs showing TF interactions with putative regulatory elements, validated by ChIP-qPCR. Figures 15A and 15B illustrate that RELA results of ChIP-qPCR for differentially accessible peaks near target genes show enrichment for these sequences compared to a negative (gene desert) control (Untr12). BIRC3 is a positive RELA control. Figures 15A and 15B represent two pools of TGFβ-treated articular chondrocytes of ChIP. Figure 15C shows that RUNX2 results of ChIP-qPCR for differentially accessible peaks near target genes show enrichment for these sequences compared to a negative (gene desert) control (Untr12). DPF1 is a positive RUNX2 control. [Figure 16A] FIG. 16A shows COL2A1 mRNA copy number relative to TBP, and FIG. 16B shows PRG4 mRNA copy number relative to TBP in 6-week-old tissue generated from micromass or monolayer-derived cells by either reseeding in micromass culture (Reseeding uM) or encapsulating in RAD16-I [100 μl]. mRNA copy number is relative to TBP and determined by qPCR-based expression analysis. T15 mesoderm is the starting paraxial mesoderm population and is the same for both micromass- and monolayer-derived chondrocytes. Bars represent the mean standard error. n=number of samples per condition. [Figure 16B] FIG. 16A shows COL2A1 mRNA copy number relative to TBP, and FIG. 16B shows PRG4 mRNA copy number relative to TBP in 6-week-old tissue generated from micromass or monolayer-derived cells by either reseeding in micromass culture (Reseeding uM) or encapsulating in RAD16-I [100 μl]. mRNA copy number is relative to TBP and determined by qPCR-based expression analysis. T15 mesoderm is the starting paraxial mesoderm population and is the same for both micromass- and monolayer-derived chondrocytes. Bars represent the mean standard error. n=number of samples per condition. [Figure 16C] Figures 16C-16F depict mRNA copy numbers after 1 week, 2 weeks, 3 weeks, and 4 weeks in paraxial mesoderm at stage III (day 14), micromass cultures derived from paraxial mesoderm seeded into high density micromass at stage III (micromass), monolayer cultures derived from paraxial mesoderm seeded into monolayer at stage III (monolayer), and monolayer-derived micromass cultures derived from monolayer cells seeded into micromass culture, as indicated. [Figure 16D] Figures 16C-16F depict mRNA copy numbers after 1 week, 2 weeks, 3 weeks, and 4 weeks in paraxial mesoderm at stage III (day 14), micromass cultures derived from paraxial mesoderm seeded into high density micromass at stage III (micromass), monolayer cultures derived from paraxial mesoderm seeded into monolayer at stage III (monolayer), and monolayer-derived micromass cultures derived from monolayer cells seeded into micromass culture, as indicated. [Figure 16E] Figures 16C-16F depict mRNA copy numbers after 1 week, 2 weeks, 3 weeks, and 4 weeks in paraxial mesoderm at stage III (day 14), micromass cultures derived from paraxial mesoderm seeded into high density micromass at stage III (micromass), monolayer cultures derived from paraxial mesoderm seeded into monolayer at stage III (monolayer), and monolayer-derived micromass cultures derived from monolayer cells seeded into micromass culture, as indicated. [Figure 16F]Figures 16C-16F depict mRNA copy numbers after 1 week, 2 weeks, 3 weeks, and 4 weeks in paraxial mesoderm at stage III (day 14), micromass cultures derived from paraxial mesoderm seeded into high density micromass at stage III (micromass), monolayer cultures derived from paraxial mesoderm seeded into monolayer at stage III (monolayer), and monolayer-derived micromass cultures derived from monolayer cells seeded into micromass culture, as indicated. [Figure 17A] Figures 17A-17C depict COL10A1 mRNA copy numbers normalized to TBP in micromass cultures at the indicated time points and treatment regimens. Figure 17A depicts COL10A1 mRNA copy numbers in micromass cultures treated with TGFB3 for 2-24 weeks, and TGFB3-treated micromass cultures where gene expression was quantified 2, 6, 12 or 24 weeks after medium change to BMP4 supplementation at 2 weeks (BMP4@2w) and 4, 6, 8, 10, 12 weeks. Figures 17B and 17C depict PRG4 (Figure 17B) and COL10A1 (Figure 17C) copy numbers in micromass cultures after 2, 6, 12 and 24 weeks (w). [Figure 17B] Figures 17A-17C depict COL10A1 mRNA copy numbers normalized to TBP in micromass cultures at the indicated time points and treatment regimens. Figure 17A depicts COL10A1 mRNA copy numbers in micromass cultures treated with TGFB3 for 2-24 weeks, and TGFB3-treated micromass cultures where gene expression was quantified 2, 6, 12 or 24 weeks after medium change to BMP4 supplementation at 2 weeks (BMP4@2w) and 4, 6, 8, 10, 12 weeks. Figures 17B and 17C depict PRG4 (Figure 17B) and COL10A1 (Figure 17C) copy numbers in micromass cultures after 2, 6, 12 and 24 weeks (w). [Figure 17C]Figures 17A-17C depict COL10A1 mRNA copy numbers normalized to TBP in micromass cultures at the indicated time points and treatment regimens. Figure 17A depicts COL10A1 mRNA copy numbers in micromass cultures treated with TGFB3 for 2-24 weeks, and TGFB3-treated micromass cultures where gene expression was quantified 2, 6, 12 or 24 weeks after medium change to BMP4 supplementation at 2 weeks (BMP4@2w) and 4, 6, 8, 10, 12 weeks. Figures 17B and 17C depict PRG4 (Figure 17B) and COL10A1 (Figure 17C) copy numbers in micromass cultures after 2, 6, 12 and 24 weeks (w). [Figure 18A] Figure 18A shows examples of quantified amounts (μg per μg of DNA content) of sulfated (s) glycosaminoglycans (GAGs) in micromass cultures cultured in the presence of TGFβ3 or BMP4 for the indicated time periods (weeks). sGAG content increases over time in both cartilaginous tissues. [Figure 18B] Figure 18B depicts representative quantification of both sulfated GAGs and hydroxyproline (OH-Pro; an alternative biochemical quantification of collagen content) in TGFβ3-treated articular cartilage tissue cultured for 12 weeks. Values ​​were calculated as μg per μg of DNA content per culture. Error bars represent the standard error of the mean. [Figure 18C] Figure 18C depicts the relative differential expression levels of representative collagen genes in articular (TGFβ) and growth plate (BMP) cartilage micromass tissues. Values ​​represent average (n=6) numbers by RNA sequencing, and error bars indicate standard deviation. The graph depicts only genes found to be significantly differentially expressed, i.e., it is non-inclusive to all collagen genes expressed in these tissues. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] I. Definition The term "primitive streak-like mesoderm cell population" as used herein refers to a population of mesoderm cells that express brachyury and express the cell surface markers CD56 and PDGFRα. For example, a primitive streak-like mesoderm cell population can comprise at least 50%, at least 60%, at least 70%, at least 80%, or about 90% cells expressing CD56 and PDGFRα. Chondrogenic differentiation can be obtained by the methods of the disclosure using, for example, 50% CD56 / PDGFRα+ cells.

[0022] The term "paraxial mesoderm cells" refers to a population of mesoderm cells that express cell surface CD73, CD105 and / or PDGFRbeta. For example, a paraxial mesoderm cell population comprises at least 70% cells that express CD73, CD105 and / or PDGFRbeta.

[0023] The term "express" refers to transcription of a polynucleotide or translation of a polypeptide in a cell such that the level of the molecule is measurably higher in cells expressing the molecule than in cells that do not express the molecule. Methods for measuring the expression of a molecule are well known to those of skill in the art and include, but are not limited to, Northern blotting, RT-PCR, in situ hybridization, Western blotting, and immunostaining, such as FACS.

[0024] The term "expressing" is also presented as "+" and refers to detectable protein expression in relation to cellular protein levels, as measured, for example, by FACS analysis, relative to cells that do not express the protein.

[0025] The term "culturing" as used herein refers to incubating and / or passage in adherent, suspension or 3D culture. As used herein, the term "adherent culture" refers to a cell culture system in which cells are cultured on a solid surface, which may then be coated with an insoluble substrate, which may then be coated with another surface coat of the substrate, such as those listed below, or any other chemical or biological material that allows cells to grow or stabilize in culture. Cells may or may not be attached to a solid surface or substrate.

[0026] The terms "contacting" or "culturing with" are intended to include incubating components and / or cells / tissues together in vitro (e.g., adding a compound to cells in culture), and the "contacting" or "culturing with" steps may be carried out in any suitable manner. For example, cells may be treated in adherent culture, suspension culture, or 3D culture, and components may be added substantially simultaneously in time (e.g., together in a cocktail) or sequentially (e.g., within an hour, a day, or more of addition of the first component). Cells may also be contacted with additional agents, such as growth factors or other differentiation agents or environments, to stabilize the cells or to further differentiate the cells, and may include culturing the cells under conditions known in the art.

[0027] The term "serum-free" refers to the absence of serum in a solution, e.g., in a medium used to culture a given cell population. For example, a serum-free medium or environment may contain less than 4, 3, 2, or 1% serum. In a preferred embodiment, a serum-free composition contains no serum or only trace amounts of serum derived from the isolation of components added to a restricted medium (containing 0% added serum).

[0028] The term "BMP inhibitor" means any inhibitor of BMP signaling, including, for example, type 1 BMP receptor inhibitors, BMP ligands, and / or soluble BMP receptors, such as dorsomorphin (DM), noggin, chordin, LDN-193189, soluble BMPR1a and / or soluble BMPR1b.

[0029] The term "Nodal agonist" as used herein means any molecule that activates Nodal signaling, e.g., "Nodal" (e.g., human Nodal, such as gene number 4338) or "Activin" in hepatocyte lineage cells.

[0030] The term "agonist" refers, for example, to an activator of a pathway or signaling molecule. An agonist of a molecule can retain substantially the same biological activity as the molecule (e.g., Nodal), or a subset of the biological activities of the molecule. For example, a Nodal agonist refers to a molecule that selectively activates Nodal signaling.

[0031] The term "inhibitor" refers to, for example, a selective inhibitor of a pathway or signaling molecule. An inhibitor or antagonist of a molecule (e.g., a BMP4 inhibitor) can inhibit one or more activities of the naturally occurring form of the molecule. For example, a BMP4 inhibitor is a molecule that selectively activates BMP4 signaling.

[0032] The term "selective inhibitor" refers to an inhibitor that inhibits a selective entity or pathway at least 1.5x, 2x, 3x, 4x, or 10x more efficiently than a related molecule.

[0033] The term "specific" refers to the process involved in directing cells to a specific cell fate before the cell type is committed and before any bias toward a specific fate can be reversed or converted to another fate. Specification induces a state in which the fate of a cell cannot be changed under typical conditions. Specification is the first step of differentiation, but can also refer to the differentiation of cells derived from the first step in subsequent steps or stages.

[0034] The term "stem cell" refers to an undifferentiated cell that can proliferate, self-renew, and give rise to many more progenitor or precursor cells that have the ability to generate a large number of mother cells that can differentiate or give rise to differentiable daughter cells. Daughter cells can, for example, be induced to proliferate and produce progeny cells that subsequently differentiate into one or more mature cell types, while at the same time retaining one or more cells of the parent's developmental potential. The term "stem cell" includes embryonic stem cells and pluripotent stem cells.

[0035] The term "embryonic stem cell" refers to the pluripotent stem cells of the inner cell mass of blastocysts (see, for example, U.S. Patent Nos. 5,843,780 and 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, U.S. Patent Nos. 5,945,577, 5,994,619 and 6,235,970).

[0036] The term "pluripotent stem cells" refers to cells that have the ability to differentiate under different conditions into two or more differentiated cell types, for example, into cell types that have characteristics of the three germ cell layers. Pluripotent cells are characterized by their ability to differentiate into two or more cell types, for example, using a nude mouse teratoma formation assay. Pluripotency is also evident by the expression of embryonic stem (ES) cell markers. Pluripotent stem cells include induced pluripotent stem cells (iPSCs) and embryonic stem cells. In one embodiment, the pluripotent stem cells are derived from somatic cells. In one embodiment, the pluripotent stem cells are derived from human somatic cells.

[0037] The terms "iPSC" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells artificially derived (e.g., by induced or complete reversal) from non-pluripotent cells, typically from adult somatic cells, by inducing expression of one or more genes, including, but not limited to, SOX2 (gene number 6657), KLF4 (gene number 9314), cMYC (gene number 4609), NANOG (gene number 79923), POU4F1 / OCT4 (gene number 5460) in combination with LIN28 / LIN28A (gene number 79727). This expression can be induced, for example, by forced gene expression or using small molecules, small RNAs, non-integrating gene expression vectors or proteins.

[0038] The term "chondrocyte-like cells" refers to chondrocytes, as well as cells that are cytochemically similar and behave as chondrocytes, expressing chondrocyte markers including, for example, Sox9 and collagen 2. Chondrocytes may be articular cartilage-like chondrocytes or precursors, or chondrocytes capable of hypertrophy (optionally referred to as growth plate chondrocyte (GPC)-like cells) or precursors thereof.

[0039] The term "cartilage-like tissue" refers to cartilage tissue, as well as tissue that is histologically similar, expresses cartilage markers such as collagen 2 and aggrecan, and behaves as cartilage, including articular cartilage tissue and / or growth plate cartilage-like tissue.

[0040] The term "articular chondrocyte-like cells and / or cartilage tissue" refers to an optionally enriched or mixed population comprising articular chondrocytes and / or articular chondrocyte-like cells, including, for example, cartilage-like tissue comprising articular chondrocyte-like cells.

[0041] The term "hypertrophic chondrocyte-like cells and / or cartilage tissue" or "GPC-like cells and / or cartilage tissue" refers to an optionally enriched or mixed population comprising hypertrophic chondrocytes and / or hypertrophic chondrocyte-like cells (e.g., chondrocytes within the growth plate of a developing bone), including, for example, cartilage-like tissue comprising hypertrophic chondrocyte-like cells.

[0042] The term "articular cartilage-like tissue" or "cartilage containing non-hypertrophic chondrocyte-like cells" resembles histologically, expresses articular cartilage markers such as lubricin (PRG4) and / or CILP2, and behaves as articular cartilage, e.g., articular cartilage is maintained as stable cartilage in vivo.

[0043] The term "growth plate cartilage-like tissue" as used herein refers to cartilage tissue that is histologically similar to, expresses cartilage markers found in, and behaves as, growth plate cartilage, including COL10A1, RUNX2, SP7, and / or ALPL, growth plate cartilage, e.g., functions in vivo to provide a scaffold upon which new bone is formed.

[0044] The term "isolated population," as used herein with respect to an isolated population of cells, refers to a population of cells that has been removed and isolated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells are isolated or enriched.

[0045] The term "substantially pure" refers to a population of cells that, with respect to a particular cell population, is at least about 65%, preferably at least about 75%, at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the entire cell population.

[0046] The terms "enrich" or "enriched" are used interchangeably herein and mean that the yield (proportion) of one type of cell is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% over the proportion of that type of cell in the starting culture or preparation. Enrichment and partial purification can be used interchangeably.

[0047] The population of cells can be enriched using different methods, for example, based on a marker, for example, based on a cell surface marker (eg, FACS sorting, etc.).

[0048] The term "subject" as used herein includes all members of the animal kingdom, including mammals, including, for example, primates, such as humans, monkeys or apes, domestic pets, farm animals, and laboratory animals.

[0049] The terms "treat," "treating," "treatment," and the like, when applied to an isolated cell, include subjecting the cell to any type of process or condition, or performing any type of operation or procedure on the cell. When applied to a subject, the term refers to providing the subject with medical or surgical care, therapy, or management.

[0050] The term "treatment" as applied to a subject refers to an approach aimed at obtaining beneficial or desired results, including clinical results, and includes, for example, medical procedures and applications, including pharmaceutical interventions, surgery, radiation therapy, and natural therapies, as well as experimental treatments to treat joint / bone disorders. Beneficial or desired clinical results may include, but are not limited to, relief or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, a stable (i.e., not worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total).

[0051] The terms "administering," "implanting," and "transplanting" are used interchangeably in the context of delivering the cells, tissues, and / or products described herein to a subject by a method or route that results in the introduced cells being at least partially localized at a desired site. Cells may be implanted directly into a joint, or alternatively may be administered by any suitable route that results in at least a portion of the transplanted cells or components of the cells being delivered viable to the desired location in the subject.

[0052] "Pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication that is commensurate with a reasonable benefit / risk ratio.

[0053] "Biocompatible" and "biocompatible" as used herein generally refer to a material that is generally non-toxic to the recipient and does not cause any significant adverse effects in the recipient, along with any metabolic or degradation products thereof. Generally speaking, a biocompatible material is one that does not induce significant inflammatory, immune or toxic responses when administered to an individual.

[0054] The terms "reduce," "inhibit," "alleviate," or "decrease" are used in comparison to a control with no other treatment or a treatment with a known degree of effectiveness. Those skilled in the art will readily identify appropriate controls to use in each experiment. For example, a reduction in a response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

[0055] The term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to treat, inhibit or alleviate one or more symptoms of the disease state being treated, or otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary according to various factors, such as the subject dependent variables (e.g., size / type of injury, age, joint health, immune system health, etc.), the disease or disorder, and the treatment being administered. The effective amount may be compared to a control. Such controls are known in the art and discussed herein, and may be, for example, the state of the subject before or without administration of the drug or drug combination, or, in the case of a drug combination, the effect of the combination may be compared to the effect of administration of only one of the drugs.

[0056] "Excipient" is used herein to include a compound that is not a therapeutically or biologically active compound. As such, the excipient should be pharma- ceutically or biologically acceptable or relevant, e.g., the excipient should be generally non-toxic to a subject. "Excipient" includes a single such compound, and is intended to include a plurality of compounds.

[0057] Throughout the description and claims of this specification, the word "comprise" and variations of this word, such as "comprising" and "comprises," mean "including but not limited to" and are not intended to exclude, for example, other additives, components, integers or steps.

[0058] "Optionally" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or exist, and that the description includes instances where the event, circumstance, or material occurs or exists as well as instances where it does not occur or exist.

[0059] Ranges may be expressed herein as "about" one particular value and / or to "about" another particular value. When such ranges are expressed, and what is specifically contemplated and considered to be disclosed is the range from one particular value and / or to the other particular value, unless the context specifically dictates otherwise. Similarly, when values ​​are expressed as approximations, using the antecedent "about," it is understood that the particular value forms another specifically contemplated embodiment that should be considered to be disclosed, unless the context specifically dictates otherwise. It is further understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint, unless the context specifically dictates otherwise. It should also be understood that all individual values ​​and subranges of values ​​included within an expressly disclosed range should be considered to be specifically contemplated and disclosed, unless the context specifically dictates otherwise. Finally, all ranges should be understood to refer to both the ranges recited as ranges and the ranges recited as a collection of individual numbers from the first endpoint (inclusive) to the second endpoint (inclusive). In the latter case, it should be understood that any of the individual numbers may be selected as one form of the quantity, value or characteristic to which the range refers. Thus, a range describes a set of numbers or values, from a first endpoint (inclusive) to a second endpoint (inclusive), from which a single member of the set (i.e., the singular number) may be selected as the quantity, value or characteristic to which the range refers. The foregoing applies regardless of whether some or all of these embodiments are expressly disclosed in a particular instance.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions, although certain useful methods, devices and materials are as described. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by prior invention. No admission is made that any reference constitutes prior art. The discussion of references describes what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. Although a number of publications are referenced herein, it is clearly understood that such references do not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0061] II. Composition Disclosed herein are human pluripotent stem cell (hPSC) derived articular chondrocytes having desirable characteristics for use in cartilage tissue engineering. Also provided herein are formulations containing the chondrocytes produced by the method and one or more excipients. Also provided are agents for use in chemical differentiation of hPSC cells into functional chondrocytes.

[0062] Kind Code: A1 Cell culture media compositions are provided that are supplemented with small molecules / proteins that can be used to drive chemical differentiation of human pluripotent stem cells (hPSCs) into articular chondrocytes.

[0063] A. Cell Culture Media Disclosed are cell culture medium compositions containing combinations of compounds that can be used to induce partial or complete chemical differentiation of human pluripotent stem cells (hPSCs) into articular chondrocytes. The required combination of small molecules / proteins can vary depending on the stage of chondrogenesis.

[0064] The cell culture medium comprises a basal medium supplemented with small molecule factors / proteins as disclosed herein. In one embodiment, optionally in steps 3 and / or 4, the medium is serum-free and comprises a basal medium of high glucose DMEM, optionally supplemented with dexamethasone, ascorbic acid, insulin, transferrin, selenium and proline.

[0065] As used herein, basal medium refers to a mixture of salts that provide cells with water and certain bulky inorganic ions essential for normal cell metabolism, maintain intracellular and extracellular osmotic balance, provide carbohydrates as an energy source, and provide a buffer system to maintain the medium within a physiological pH range. Examples of basal medium include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, Ham's F-10, Ham's F-12, alpha Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (O-MEM), and Iscove's Modified Dulbecco's Medium (IMDM), STEM PRO®, STEM PRO-34®, and mixtures thereof.

[0066] 1. Stage I Cell Culture Media First, in the induction of hPSC cells to form primitive streak mesoderm, the required combination of active signals includes (A) an FGF agonist, (B) a BMP4 agonist, and (C) a TGFβ agonist, and optionally a Wnt agonist, at levels effective to induce induction of hPSC cells to form primitive streak mesoderm. These signals can be produced endogenously by the hPSC-derived cells, or they can be induced by supplementation with molecules including (A) an FGF agonist, (B) a BMP4 agonist, and (C) a TGFβ agonist, and optionally a Wnt agonist, in amounts effective to induce induction of hPSC cells to form primitive streak mesoderm.

[0067] (a) FGF agonist In a preferred embodiment, the FGF agonist is a fibroblast growth factor agonist. The FGF agonist may be a molecule such as a cytokine, for example an FGF. "FGF," as used herein, refers to any fibroblast growth factor, optionally including bFGF, FGF2, FGF4, FGF9, and / or optionally FGF19, 21, 3, 5, 6, 8a, 16-18, 20, and / or 23, such as human FGF1 (Gene No. 2246), FGF2 (also known as bFGF; Gene No. 2247), FGF3 (Gene No. 2248), FGF4 (Gene No. 2249), FGF5 (Gene No. 2250), FGF6 (Gene No. 2251), FGF7 (Gene No. 2252), FGF8 (Gene No. 2253), FGF9 (Gene No. 2254), and FGF10 (Gene No. 2255), optionally including active conjugates and fragments thereof, including naturally occurring active conjugates and fragments. In some embodiments, the FGF is βFGF, FGF2, FGF4 and / or FGF9. As used herein, "active conjugates and fragments of FGF" include conjugates and fragments of fibroblast growth factors that bind to and activate FGF receptors, and optionally activate FGF signaling. In some forms, the FGF agonist is a molecule that activates the FGF signaling pathway, i.e., binds to and activates the FGF receptor. Preferably, the FGF agonist is FGF2 or FGF. Preferably, the FGF agonist is at any concentration between about 0.1 ng / ml and about 20 ng / ml, optionally about 5 ng / ml.

[0068] (b) BMP4 agonist In some embodiments, the BMP4 agonist is a bone morphogenetic protein 4 agonist, including any BMP or GDF that activates the receptor for BMP4. The term "BMP4" (e.g., gene number 652), as used herein, refers to bone morphogenetic protein 4, e.g., human BMP4, and to active conjugates and fragments thereof, optionally including naturally occurring active conjugates and fragments, that can activate, e.g., BMP4 receptor signaling. BMP4 agonists include, but are not limited to, GDF5, GDF6, GDF7, BMP4, BMP2, BMP6, BMP7, and / or BMP10. Preferably, the BMP4 agonist BMP4 is at any concentration from about 0.1 ng / ml to about 100 ng / ml, optionally about 3 ng / ml.

[0069] (c) TGFβ agonists TGFβ agonist refers to any molecule that activates TGFβ receptor. TGFβ receptor is a single-pass serine / threonine kinase receptor. Three types of TGFβ receptor include type I, II and III, namely TGFβ receptor 1, TGFβ receptor 2 and TGFβ receptor 3. Preferably, the TGFβ agonist is TGFβ3 or activin A. Examples of activin A include gene number 3624 (human activin) and include, but are not limited to, active conjugates and fragments thereof, optionally including naturally occurring active conjugates and fragments, and active conjugates and fragments thereof, optionally including naturally occurring active conjugates and fragments, capable of activating, for example, nodal signaling. Preferably, the TGFβ agonist is activin A at any concentration from about 0.1 ng / ml to about 100 ng / ml, optionally about 2 ng / ml.

[0070] (d) Wnt agonists Useful Wnt (Wingless and Int-1) agonists are molecules that activate Wnt / beta-catenin receptor signaling in chondrocyte lineage cells, including, for example, Wnt3a, as well as GSK3 selective inhibitors such as CHIR99021 (STEMOLECULE® CHIR99021 Stemgent), 6-bromoindirubin-3'-oxime (BIO) (Cayman Chemical (cat: 13123)) or STEMOLECULE® BIO (cat: 04003) from Stemgent. CHIR99021 is a selective inhibitor of GSK3. Contemplated GSK3 selective inhibitors are, for example, selective inhibitors of GSK-3α / β in the Wnt signaling pathway. Wnt3a, as used herein, refers to the wingless MMTV integration site family member factor 3A (e.g., gene number 89780), e.g., human Wnt3a, and to active conjugates and fragments thereof, including naturally occurring active conjugates and fragments. Other Wnt agonists include, but are not limited to, SB216763, TWS119, CHIR98014, tideglusib, SB415286, LY2090314, CHIR-98014, AZD1080, TDZD-8, and wnt3a.

[0071] 2. Phase II Cell Culture Media Second, to induce the formation of paraxial mesoderm from primitive streak mesoderm, the required combination of molecules includes an effective amount of (A) a BMP4 inhibitor and / or (B) an FGF agonist. Optionally, a Wnt inhibitor and a TGFβ inhibitor are used in effective amounts.

[0072] (a) BMP4 inhibitor A preferred BMP4 inhibitor is dorsomorphin, used at a concentration ranging from about 0.5 μM to optionally about 6 μM, preferably at a concentration of 4 μM. Other molecules that inhibit BMP4 signaling are known in the art and include, but are not limited to, LDN193189 dihydrochloride.

[0073] (b) FGF agonist Useful FGF agonists are the same as those disclosed above for Stage I medium. A preferred FGF agonist useful for supplementing Stage II medium is basic FGF / FGF2, used at a concentration ranging from about 1 ng / mL to about 100 ng / mL, about 10 to about 50 ng / ml, preferably about 20, 30 or 40 ng / ml, with intermediate values ​​being contemplated.

[0074] (c) Wnt inhibitors / antagonists Useful Wnt antagonists or Wnt inhibitors refer to molecules that inhibit Wnt / beta-catenin receptor signaling in chondrocyte lineage cells and include, for example, IWP2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide; Sigma), Dickkopf-related protein 1 (DKK1; R&D Systems), Wnt-C59 (4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide) and / or XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one; Sigma). A preferred Wnt inhibitor is IWP2, used at a concentration ranging from about 0.5 μM to about 4 μM, preferably at a concentration of 2 μM.

[0075] (d) TGFβ inhibitors A preferred TGFβ inhibitor is an inhibitor of the type I activin receptor-like kinase (ALK) receptor SB431542. SB431542 is used at a concentration ranging from about 0.5 μM to optionally about 10 μM, preferably at a concentration of 5.4 μM. Other exemplary molecules that can inhibit the ALK receptor include GW788388 and A-83-01.

[0076] 3. Stage III Cell Culture Medium Third, to convert paraxial mesoderm cells into chondrocyte progenitors, the cocktail contains an effective amount of one or a combination of: (1) an FGF agonist, (2) a TGFβ agonist, and (3) a cyclic AMP agonist.

[0077] (a) TGFβ agonists Useful TGFβ agonists are the same as those disclosed above for Stage I medium. A preferred TGFβ agonist useful for supplementing Stage III medium is TGFβ3, used at a concentration ranging from about 1 ng / ml to about 50 ng / ml, optionally at a concentration of about 10 ng / ml.

[0078] (b) FGF agonist Useful FGF agonists are the same as those disclosed above for Stage I medium. A preferred TGF agonist useful for supplementing Stage III medium is FGF, used at a concentration ranging from 1 ng / ml to 100 ng / ml, preferably at a concentration of 10 ng / ml.

[0079] (c) Cyclic AMP agonists In a preferred embodiment, a molecule that improves or enhances chondrocyte differentiation is included. Preferably, the molecule that improves or enhances differentiation is a cyclic AMP (cAMP) agonist. Non-limiting examples of cyclic AMP agonists include prostaglandin E2 (PGE2), dibutyryl cyclic AMP (dbcAMP), 8-Br-cAMP, genistein, forskolin (FSK), colforsin, and rolipram. Preferably, the cyclic AMP agonist used to improve the differentiation of chondrocyte precursors to chondrocytes is forskolin. A preferred cAMP agonist is forskolin, used at a concentration ranging from 0.5 μM to about 100 μM, optionally at a concentration of about 30 μM.

[0080] 4. Stage IV Cell Culture Media In some embodiments, a cocktail of media containing TGFβ agonists is used to generate articular cartilage tissue from chondrocytes.

[0081] (a) TGFβ agonists Useful TGFβ agonists are the same as those disclosed above for Stage I medium. A preferred TGFβ agonist useful for supplementing Stage III medium is TGFβ3, used at a concentration ranging from about 1 ng / ml to about 50 ng / ml, preferably at a concentration of about 10 ng / ml.

[0082] B. Chemically Derived Chondrocytes and Preparations 1. Chemically Induced Chondrocytes Articular chondrocytes are preferably derived from human pluripotent stem cells (hPSCs). However, articular chondrocytes can be derived from PSCs of animals, including, but not limited to, dogs, horses, pigs, primates, such as monkeys and chimpanzees. PSCs can be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). ESCs are "true" ESCs derived from the inner cell mass of an embryo, which are ESCs produced by somatic cell nuclear transfer.

[0083] In some forms, the induced articular chondrocytes express one or more markers associated with a general cartilaginous phenotype, such as ACAN, SOX9, COL2A1, ACAN, COL9A1, SOX5, and SOX6. Additional markers specific for articular chondrocytes include PRG4, COL1A1, CILP2, COL22A1, COL15A1, FGF18, COMP, PTHLH, FGF1, ERG, COL6A1, UCMA, LECT1 / CNMD, CHI3L1, CHI3L2.

[0084] A population of articular chondrocytes can be isolated from stem cell-derived or progenitor cell-derived paraxial mesoderm cell cultures by selecting cells expressing one or more markers associated with a cartilaginous phenotype, such as ACAN, SOX9, COL2A1, COL9A1, SOX5, PRG4, and SOX6. This process ensures that no other cell types are present in the population of articular chondrocytes.

[0085] In some embodiments, the population of hPSC-derived articular chondrocytes is in a quiescent superficial zone or intermediate zone-like state with less than 10% continuing to differentiate / proliferate. In some embodiments, greater than about 20% of the cells are in a quiescent superficial zone or similar state (5-40%). In some embodiments, greater than about 60% of the cells are in a quiescent intermediate zone-like state (30-95%).

[0086] In some embodiments, the pluripotent cells and / or their progeny are autologous. In some embodiments, the pluripotent cells and / or their progeny are allogeneic. In some embodiments, the pluripotent cells and / or their progeny are syngeneic. In some embodiments, the pluripotent cells and / or their progeny are xenogeneic.

[0087] a. Structure of hPSC-derived articular chondrocytes Chondrocytes are specialized mesenchymal cells that occupy approximately 1-10% of the total tissue volume of articular cartilage. Chondrocytes have different morphologies due to their different locations in the cartilage. Superficial zone chondrocytes are located in the superficial layer of the cartilage tissue. They are distributed individually with small volumes and ovoid shapes. Their long axis is parallel to the surface of the cartilage. Developmentally, cells present in the superficial zone of cartilage can give rise to chondrocytes in the deep zone. Chondrocytes in the intermediate zone of articular cartilage, which is just below the superficial zone, are more or less uniformly distributed within the extracellular matrix. Under the electron microscope, chondrocytes have numerous rough endoplasmic reticulum, a well-developed Golgi complex and a small number of mitochondria in the cytoplasm. Chondrocytes are embedded in the cartilage stroma. They are located in small cavities called chondrocyte lacunae.

[0088] Adult articular cartilage has a highly organized structure with four zones: superficial (tangential), transitional (central / intermediate), deep (basal) and mineralized zones. The chondrocyte phenotype, density and cell morphology differ in the four zones. In some embodiments, the induced articular chondrocytes in the superficial zone make up 5-25% of the articular cartilage volume. In some embodiments, the induced articular chondrocytes in the transitional / intermediate zone make up 75-95% of the articular cartilage volume. In some embodiments, the induced articular chondrocytes in the deep zone make up <5% of the articular cartilage volume. No mineralized chondrocytes are present in the induced articular cartilage tissue.

[0089] Typically, naturally occurring chondrocytes in the superficial zone of articular cartilage are elongated and flat. The transition zone typically contains more round chondrocytes, while the deep zone contains spherical chondrocytes. In some embodiments, 5-40% of the induced articular chondrocytes are elongated and flat. In some embodiments, 30-95% of the induced articular chondrocytes are round. In some embodiments, <5% of the induced articular chondrocytes are spherical.

[0090] b. Function of hPSC-derived articular chondrocytes Articular chondrocytes produce and maintain a cartilaginous matrix composed primarily of collagen and proteoglycans. In some embodiments, articular chondrocytes are metabolically active cells that synthesize and turnover extracellular matrix (ECM) components such as collagen, glycoproteins, proteoglycans, and hyaluronan. In some forms, 70-100% of induced articular chondrocytes express ECM components such as collagen and proteoglycans.

[0091] In some forms, the induced articular chondrocytes maintain cartilage homeostasis by producing enzymes, growth factors and inflammatory mediators. The functions of the induced articular chondrocytes are similar to those of chondrocytes in developing human articular cartilage. These functions are significantly higher than chondrocytes induced from PSCs using other methods, as they produce ECM and produce cartilage tissue similar to native cartilage. In a preferred embodiment, the hPSC-derived articular chondrocyte tissue has an abundance of sulfated glycosaminoglycans (sGAGs), more preferably at levels comparable to the range in human articular cartilage.

[0092] Articular chondrocytes remodel and degrade ECM components by synthesizing and secreting proteinases required for tissue remodeling. Proteinases involved in cleavage of collagens and proteoglycans include matrix metalloproteinases (MMPs) and disintegrin metalloproteinases with thrombospondin motifs (ADAMTS), respectively, and to a lesser extent other types of enzymes, such as elastases and cathepsins.

[0093] i. Preparation In some embodiments, the formulation comprises articular chondrocytes provided in cell culture medium. In some forms, the number of cells in the formulation is about 1 million to 100 million cells, about 5 million to 50 million cells, preferably about 5 million cells.

[0094] In some embodiments, the preparation comprises articular chondrocytes provided in a cryopreserved state. In some forms, the number of cells in the preparation is about 1 million to 30 million cells, preferably about 10 million cells.

[0095] Media for cell storage are known in the art, such as CRYO-GOLD™ (cryopreservation medium) and CROSSTOR® (cryopreservation freezing medium), designed to mitigate temperature-induced molecular cellular stress responses upon freezing and thawing. All CROSSTOR® products are preformulated with UPS grade DMSO, a permanent solute cryoprotectant that helps mitigate damage from intracellular ice crystal formation. CROSSTOR® comes in several packages and is preformulated with DMSO at final concentrations of 2%, 5% and 10%. Preferred media for cell storage contain 5-10% DMSO, such as CRYOSTOR® CS10 (a proprietary serum-free, animal component-free, defined cryopreservation medium containing 10% dimethyl sulfoxide (DMSO)). In addition, cryoprotectants / cryoprotectant additives that can be included in the cell composition (for cryopreservation) are known in the art and include ethylene glycol (EG), antioxidants such as taurine, metformin, and gamma aminobutyric acid (GABA). The cells are frozen in "freezing medium", e.g., in cell culture medium containing 15-20% fetal bovine serum (FBS) and 7-10% DMSO, with or without 5-10% glycerol, at a concentration of, e.g., about 1-10×10 6 The cells can be suspended at a concentration of 1000000000 cells / ml. The cells are dispensed into glass or plastic vials, which are then sealed and transferred to the freezing chamber of a programmable or passive freezer. The optimal rate of freezing can be determined empirically. For example, a freezing program that gives a temperature change of -1°C / min due to the heat of fusion may be used. Once the vials containing the cells reach -80°C, they are transferred to liquid nitrogen storage.

[0096] In one preferred embodiment, the cryopreservation medium comprises about 50% FBS, about 10% DMSO, and about 40% IMDM (Iscove's Modified Dulbecco's Medium).

[0097] c. Articular chondrocytes and ECM In some embodiments, the method is used to produce a cartilage repair implant comprising an extracellular matrix (ECM) and a population of articular chondrocytes as disclosed herein. In some embodiments, the articular chondrocytes are suspended in a medium or biomaterial composition injection, the formulation being 5-100 million cells per milliliter, preferably 12.5 million cells. The injection material may contain biomaterials such as collagen, polyglycolic acid (pga), polylactic acid, alginates (e.g., calcium salts), polyethylene oxide, fibrin glue, polylactic acid-polyglycolic acid copolymers, proteoglycans, glycosaminoglycans, natural biomaterials such as Matrigel, chondrocyte-derived extracellular matrix that has been partially or completely destroyed by enzymes, or synthetic components such as RAD-16I (PuraMatrix).

[0098] In some embodiments, the preparation contains a population of articular chondrocytes surrounded by extracellular matrix (ECM). In some forms, the articular chondrocytes constitute 2-10% of the cartilage tissue volume. In some forms, the number of cells in the preparation is about 400,000-100 million cells, preferably about 5 million cells. In some forms, the ECM constitutes 90% of the cartilage tissue volume.

[0099] In some embodiments, the preparation of articular chondrocytes with ECM is an organized structure with two zones: the superficial (tangential) zone and the transitional (central / intermediate) zone. In some forms, the phenotype, density and cell morphology of the articular chondrocytes differ in the two zones. In some embodiments, the superficial zone comprises 5-25% of the articular cartilage volume. In some embodiments, the transitional zone is the thickest layer and comprises 75-95% of the articular cartilage volume. In some embodiments, the cartilage contains additional zones of cartilage: deep zone chondrocytes and calcified chondrocytes. The induced deep zone may comprise 10-30% of the articular cartilage volume.

[0100] In some embodiments, the superficial zone of the formulation is composed of collagen fibers oriented parallel to the articular surface. In some forms, the articular chondrocytes in the superficial layer are elongated and flat. In some embodiments, the articular chondrocytes in the superficial layer of articular cartilage are densely packed and aligned parallel to the articular surface. In some forms, the superficial layer of articular cartilage expresses proteoglycans and other ECM genes specific to the superficial layer of cartilage (e.g., PRG4 / lubricin, COL1A1).

[0101] In some embodiments, the intermediate zone of the formulation contains collagen fibers. In some forms, the collagen fibers are thicker, less organized, and typically oriented obliquely to the articular surface. In some forms, the repertoire of proteoglycans and other ECM genes expressed in the intermediate zone of the formulation is different from that in the superficial zone (e.g., more COL2A1, CNMD). In some forms, the collagen fibers are oriented perpendicular to the articular surface. In some forms, the morphology of articular chondrocytes in the intermediate zone is rounder than the flat chondrocytes in the superficial zone.

[0102] In some embodiments, the articular cartilage tissue has biomechanical properties similar to natural cartilage tissue. The biomechanical properties are measured by indentation assay and unconfined compression using three to four stress / relaxation phases (0-5%, 5-10%, 10-15%, 15-20%). The equilibrium elastic modulus (E) of the micromass-derived articular cartilage tissue ranges from 60 to 2000 kPa, preferably 200 to 600 kPa. An example of the elastic modulus (E) of the biomaterial-encapsulated cartilage tissue against indentation is 1.68 to 1.91 MPa.

[0103] In some embodiments, the ECM of articular cartilage is a hyperhydrated tissue. The percentage of water in the ECM can range from about 60% to about 95% of the total wet weight of the ECM. The ECM also contains macromolecular proteins, namely type II collagen, and the large, highly negatively charged proteoglycan aggrecan. In some embodiments, several other classes of molecules also comprise part of the ECM, including lipids, phospholipids, proteins, and glycoproteins. Type II collagen is the major constituent protein of the ECM and is the major fibrous collagen of articular cartilage, constituting 90% to 95% of the total collagen and 10% of the wet weight of articular cartilage. Types IX and XI collagens are also present.

[0104] III. Methods of Producing Chondrocytes and Cartilage Compositions Methods for generating chondrocytes and cartilage tissue involve inducing differentiation of human pluripotent stem cell (hPSC)-derived progenitors into chondrocytes, thereby providing chondrocytes with desirable characteristics for use in cartilage tissue engineering. Conventional methods for generating chondrocytes from hPSCs are described in detail in U.S. Pat. Nos. 9,993,504 and 10,736,923, which disclose a three-step process for obtaining articular chondrocytes from hPSCs. In contrast, the methods of the present disclosure, in some embodiments, use a four-step process from hPSCs to articular chondrocytes, using different combinations of chemical inducers to induce conversion of intermediate paraxial mesoderm seeded at low density (monolayer) into articular chondrocytes.

[0105] In some embodiments, the methods disclosed herein include that activation of the TGFβ pathway in hPSC-derived chondrocyte progenitors promotes efficient generation of articular chondrocytes capable of forming stable cartilage tissue in vitro and in vivo.

[0106] Typically, a method for differentiating paraxial mesoderm, chondrocyte progenitors, and cartilage tissue from human pluripotent stem cells (hPSCs) includes the steps of: (a) Inducing a primitive streak-like mesoderm population from hPSCs (Step I); (b) generating a paraxial mesoderm population from a primitive streak-like mesoderm population (stage II); (c) generating a chondrocyte precursor population from the paraxial mesoderm population via micromass (high cell density) culture (stage III); and (d) generating articular chondrocytes from the chondrocyte precursor population via micromass (high cell density) culture (Step IV); or In another embodiment, (e) Generating chondrocyte progenitors from the paraxial mesoderm population via low-density (monolayer) culture (phase III). Contains one or more of the following:

[0107] High cell density is used herein to refer to seeding about 200,000 cells to about 1,000,000 cells per surface area (2D) of about 0.2 cm to about 2 cm diameter, or with respect to micromass, at least about 100,000 cells per about 20 microliters of medium, or up to, for example, 2,000,000 cells per about 20 microliters of medium, allowing cells to adhere to a small surface area that allows for a micromass "spot." For membrane filters, the area depends on the commercially available membrane purchased and the volume used to determine the thickness of the biomaterial (if used), for example, approximately 400,000 cells to about 5,000,000 cells can be seeded in about 100 microliters to about 500 microliters of medium or biomaterial in, for example, a cylindrical membrane filter containing insert of about 0.5 cm to about 2 cm diameter to allow cells to adhere. In both micromass and membrane filter cultures, cells adhere at approximately 1-5 cell layers and tissues can be grown "thicker" after adhesion. Similar cell densities can be used to seed bone matrix or bone substitute scaffolds, such as calcium polyphosphate (CPP).

[0108] Cell cultures were grown at a cell density of 1 cm 2 20x10 3 ~1cm 2 20x10 4When the cell concentration varies from 0.1 to 100, it is referred to herein as a "monolayer culture." Above this cell concentration, the culture is defined as a high-density culture, which has very different characteristics than a monolayer.

[0109] The cells are generally subjected to adherent cell culture. The substrate for adherent culture can be any one or combination of tissue culture treated plastic, polyornithine, laminin, polylysine, purified collagen, gelatin, fibronectin, tenascin, vitronectin, entactin, heparin sulfate proteoglycan, poly glycolytic acid (PGA), polylactic acid (PLA) and polylactic-glycolic acid (PLGA). In one embodiment, the cells are seeded on MATRIGEL® coated plates. In another embodiment, the cells are seeded on fibronectin coated plates. The cells can be cultured in filter culture and micromass culture. In one embodiment, the cells are seeded on membrane filters, which are optionally placed in tissue culture dishes as part of a transwell system (e.g., MILLIPORE®, ALVATEX®). The substrate can also be a bone scaffold substitute such as CPP (calcium polyphosphate) or other pharma- ceutical available scaffolds.

[0110] Micromass culture is when a dense suspension of cells is allowed to adhere in a single cell layer to a small area of ​​the substrate (e.g., 200,000-500,000 cells adhere to a circular area of ​​the substrate with a diameter of 0.2-1 cm). Substrates of any shape or size can be used and can be prepared, for example, by 3D printing. The term "suspension" when used in the context of cell culture is the same as that used in the art. That is, a micromass culture suspension is a cell culture environment where the cells are not attached to a surface. Those skilled in the art are familiar with cell culture techniques and include, if necessary, the use of equipment such as flow hoods, incubators, and / or equipment used to keep the cells constantly moving, such as, but not limited to, a rotator platform, shakers.

[0111] A. Generation of a primitive streak-like mesoderm population (Stage I) Generally, any human pluripotent stem cell population can be used as the starting population, including induced pluripotent stem cell population. In one embodiment, the starting population is a human embryonic stem cell population (hESC) or induced pluripotent stem cell population (iPSC), optionally primary hESC and / or primary iPSC. Many human ESC lines are commercially available, for example, listed in the NIH HESC registry. In one embodiment, the human ESC population is HES2, H1, H9 or any hESC cell line available from the NIH ESC registry, or any human iPS cell line, such as any commercially available iPS cell line, for example, a cell line selected from those available from System Biosciences.

[0112] In some embodiments, the pluripotent cell population is contacted with the primitive streak induction cocktail for about 1 to about 5 days. In one embodiment, the primitive streak induction cocktail comprises an activin agonist, such as activin A or nodal; a BMP4 agonist, such as BMP4, BMP2, BMP6, BMP7 and / or BMP10; and an FGF agonist, such as bFGF, FGF2, FGF4, FGF9 and / or optionally FGF19, 21, 3, 5, 6, 8a, 16-18, 20 and / or 23, preferably by culturing the cells in a cell culture medium supplemented with an effective amount of these factors. In some embodiments, the cell culture medium is also supplemented with a Wnt agonist. In some embodiments, stage I does not include a step that results in the formation of embryoid bodies. In another embodiment, the initial stage of differentiation involves inducing a primitive streak-like mesoderm population by contacting the pluripotent cells with a primitive streak-inducing cocktail comprising activin A, BMP4 and basic FGF on days 1-4 of differentiation. Typically, mesoderm is monitored by cell surface expression of CD56 and PDGFRα by flow cytometry on day 3. In some embodiments, contact with the primitive streak-inducing cocktail is shortened if the CD56+ / PDGDFRα+ population is generated earlier, e.g., on days 1-3.

[0113] Expression of brachyury is also induced at this stage, and gene expression is monitored from approximately days 2 to 3 and expression of cell surface markers PDGFRα and CD56 by day 4. In human PSCs, PS-like mesoderm induction is dependent on activin and Wnt signaling and is monitored by expression of brachyury and PDGFRα.

[0114] In some embodiments, CD56 is used to monitor, for example, human primitive streak cell formation. The appearance of cell surface markers, such as CD56 and PDGFRα, indicates that a primitive streak-like mesoderm population is generated. In some embodiments, the pluripotent cell population is induced with a primitive streak-inducing cocktail for 1 day, 2 days, 3 days, 4 days, or 5 days.

[0115] 1. Feeder-free monolayer culture In some embodiments, hPSCs are cultured on irradiated mouse fibroblasts / feeders (MEFs) and knockout serum based medium (KSR). In a preferred embodiment, hPSCs are cultured in feeder-free culture.

[0116] Maintaining undifferentiated human pluripotent stem cells (hPSCs) under xeno-free conditions requires the use of human feeder cells or extracellular matrix (ECM) coatings. However, human sources can introduce human pathogen contamination with viral or non-viral agents into the patient. Feeder-free culture systems are designed to keep the stem cells from differentiating while at the same time protecting them from direct contact with the feeder to prevent cross-contamination or introduction of non-human pathogens into the stem cells.

[0117] In some embodiments, the pluripotent cells are cultured as embryoid bodies. In other embodiments, the pluripotent cells are cultured as monolayers. Human embryonic stem cells (hESCs) are often co-cultured with mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multi-layered colonies of morphologically heterogeneous cells surrounded by flat mesenchymal cells. In contrast, hPSCs grown in feeder cell conditioned medium on Matrigel or other ECM coatings tend to grow instead as monolayers with uniform morphology.

[0118] 2. Addition of Wnt agonists Blocking Wnt signaling with an antagonist inhibits primitive streak formation. In some embodiments, a Wnt agonist is added to an hPSC line to enhance the development of a CD56+PDGFRa+ primitive streak-like population, for example, added on days 1-3.

[0119] In cell lines and starting populations where endogenous Wnt signaling is absent or low, the addition of a Wnt agonist improves the efficiency of primitive streak formation from PSCs. Thus, in a preferred embodiment, the primitive streak induction cocktail further comprises a Wnt agonist. Exemplary Wnt agonists are Wnt3a or GSK-3 selective inhibitors, such as CHIR-99021 (STEMOLECULE® CHIR99021 Stemgent), 6-bromoindirubin-3'-oxime (BIO) (Cayman Chemical (cat: 13123)) or STEMOLECULE® BIO (cat: 04003) from Stemgent.

[0120] Endogenous Wnt signaling is sufficient for primitive streak induction in some cell lines, e.g., HES2, H9, and thus, in other embodiments, no exogenous Wnt agonist is added to the hPSC lines to develop the primitive streak population.

[0121] B. Generation of the Paraxial Mesoderm Population In some embodiments, the method comprises culturing a primitive streak-like mesoderm population to generate a paraxial mesoderm population. In some embodiments, the primitive streak-like mesoderm population is a CD56+, PDGFRα+ primitive streak-like mesoderm population. In some embodiments, the primitive streak-like mesoderm population is a CD56+, PDGFRα+ primitive streak-like mesoderm population. (i) FGF agonists, (ii) BMP inhibitors such as noggin, LDN-193189 and / or dorsomorphin, and (iii) optionally a TGFβ inhibitor, optionally SB431542, and a Wnt inhibitor, optionally one or more of IWP2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide; Sigma), Dickkopf-related protein 1 (DKK1; R&D Systems), and / or XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one; Sigma). The cells are cultured in a paraxial mesoderm-specific cocktail containing

[0122] In some embodiments, the generation of paraxial mesoderm is generally characterized by the expression of transcription factors Meox1 and Nkx3.2. In preferred embodiments, the culture conditions induce the identification of a paraxial mesoderm population that expresses one or more of the cell surface molecules CD73, CD105, and / or PDGFRβ. In some embodiments, the paraxial mesoderm population expresses CD73+CD105+, or CD73+PDGFRβ, or CD73+CD105+PDGFRβ+.

[0123] In some embodiments, the primitive streak (PS)-like cells are induced to a paraxial fate in monolayer culture at this stage (e.g., day 3-15). In some embodiments, BMP signaling is inhibited in the primitive streak (PS)-like cells by the use of molecules such as dorsomorphin, and TGFβ signaling is inhibited by the use of small molecules such as SB431542. Human paraxial mesoderm requires the addition of FGF (e.g., bFGF), which is added to the culture medium, for example, on days 3-15 of monolayer culture. Data suggest that Wnt inhibition is better for later cartilage potential, and more tendon / ligament gene expression was observed without Wnt inhibition. Thus, in a preferred embodiment, a Wnt antagonist is also added. Human paraxial mesoderm is determined by the expression of cell surface markers including CD73, CD105, and PDGFRβ.

[0124] In one embodiment, human paraxial mesoderm is identified in monolayer cultures at days 3-14 of differentiation by the BMP inhibitor dorsomorphin (e.g., days 3-5) and FGF. Human paraxial mesoderm is characterized by cell surface markers CD73, CD105, PDGFRβ and / or Meox1 gene expression, and Nkx3.2 gene expression at day 14. Typically, expression of these markers begins at or about day 11 and is maximal at or about day 14.

[0125] 1. Timing of paraxial mesoderm specification In some embodiments, FGF treatment continues for 11 days to obtain "paraxial mesoderm" on day 14. In some embodiments, the culture to produce a paraxial mesoderm population is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 days. In preferred embodiments, the time required to produce a paraxial mesoderm population is about 1, 2, 3, 4, 5 days, or less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, less than 10 days.

[0126] C. Generation of chondrocyte precursor populations In some embodiments, the method includes culturing a paraxial mesoderm population to generate a chondrocyte precursor population. In some embodiments, the paraxial mesoderm population expressing CD73, CD105 and / or PDGFR-β is cultured at high cell density, optionally in serum-free or serum-containing medium. In other embodiments, the paraxial mesoderm population expressing CD73, CD105 and / or PDGFR-β is cultured at low cell density, e.g., in a monolayer, optionally in serum-free or serum-containing medium.

[0127] A paraxial mesoderm population expressing CD73+, CD105+ and / or PDGFRβ+ is cultured at high density in serum-free medium with a TGF-β agonist, such as TGFβ3, to generate a SOX9+, COL2A1+ chondrocyte / chondrocyte precursor population.

[0128] D. Generation of differentiated chondrocytes and cartilage tissue 1. Generation of differentiated chondrocytes by micromass culture of chondrocyte precursor cells In some embodiments, the method includes further culturing the chondrocyte precursor population to generate cartilage tissue. In this embodiment, the high cell density SOX9+, COL2A1+ chondrocyte precursor population is cultured with a TGFβ3 agonist for a time effective to produce articular-like non-hypertrophic chondrocytes and / or cartilage-like tissue. In other embodiments, the high cell density SOX9+, COL2A1+ chondrocyte precursor population is cultured with a BMP4 agonist for a time effective to produce hypertrophic chondrocyte-like cells and / or cartilage-like tissue.

[0129] In one embodiment, the paraxial mesoderm population is cultured as high density micromass in tissue culture medium containing a drug combination of a TGFβ agonist, an FGF agonist and a cyclic AMP agonist.

[0130] 2. Generation of differentiated chondrocytes by monolayer culture of paraxial mesoderm In some embodiments, the methods comprise generating chondrocyte precursors directly from a paraxial mesoderm population in low density monolayer culture, and subsequently generating chondrocytes from these chondrogenic precursors.

[0131] Methods for generating chondrocyte precursors from low density monolayers Experiments in the Examples below showed that forskolin increased the chondrogenic potential of monolayer cells treated with TGFβ agonists and FGF agonists, as well as cells cultured without TGFβ agonists or FGF agonists. Chondrocytes derived from monolayers with FGF treatment alone (no exogenous TGFβ) or with FGF with exogenous TGFβ with or without forskolin were suitable for use in cartilage tissue engineering. Advantages of deriving chondrocytes in monolayer culture over micromass culture include, but are not limited to, (1) higher cell yield, (2) greater chondrocyte viability, and (3) reduced time to generate articular cartilage (e.g., approximately 3 weeks to generate articular cartilage from monolayer-derived chondrocytes compared to at least 6-12 weeks to generate articular cartilage tissue in direct micromass culture). Thus, in some embodiments, cartilage derived from a monolayer of paraxial mesoderm cells has 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100% more viable cells, preferably chondrocytes expressing one or more of SOX9, COL2A1, ACAN and PRG4, than that derived from a high density culture of paraxial mesoderm cells, provided it has the same number of paraxial mesoderm cells as the starting population (Figures 16D-16F).

[0132] Thus, in some embodiments, paraxial mesoderm populations expressing CD73+, CD105+ and / or PDGFRβ+ are cultured with one or more of an FGF agonist, a cAMP agonist and a TGFβ agonist. Exemplary FGF agonists include basic FGF or FGF2. Additional FGFs include FGF4, FGF9, FGF19, 21, 3, 5, 6, 8a, 16-18, 20 and 23. Exemplary cAMP agonists include forskolin, 8-bromo-cAMP and colforsin. Exemplary TGFβ agonists include TGFβ1, TGFβ2, TGFβ3. In one embodiment, paraxial mesoderm populations expressing CD73+, CD105+ and / or PDGFRβ+ are cultured with an FGF agonist, a forskolin and a TGFβ agonist. In another embodiment, a paraxial mesoderm population expressing CD73+, CD105+ and / or PDGFRβ+ is cultured with forskolin alone, without an FGF agonist or a TGFβ agonist. In a further embodiment, a paraxial mesoderm population expressing CD73+, CD105+ and / or PDGFRβ+ is cultured with an FGF agonist alone, without a cAMP agonist or a TGFβ agonist.

[0133] In a preferred embodiment, chondrocytes derived from monolayers with any of the above conditions are further used for cartilage tissue engineering. Cartilage tissue of desired dimensions and composition can be subsequently produced via encapsulation in micromasses or biomaterials for implantation. For example, Figures 8A-8D and 9A-9F illustrate the results of experiments performed in exemplary embodiments. Figures 8A-D are bar graphs showing the results of qPCR experiments comparing the expression of genes associated with encapsulated cartilage tissue (COL2A1 and PRG4) grown from either monolayer-derived or micromass-derived chondrocytes. Figures 9A-9F show the qPCR results of the expression of cartilage-related genes in micromass tissue derived from monolayer-derived chondrocytes as disclosed herein. Chondrocyte progenitors derived from monolayers with either FGF only or TGFB+FGF+FSK can subsequently generate articular and growth plate cartilage in micromasses.

[0134] In a preferred embodiment, chondrocytes derived from a monolayer having any of the above conditions provide articular chondrocyte tissue with abundant sulfated glycosaminoglycans (sGAGs) and / or other components of the extracellular matrix, more preferably at levels comparable to the range within human articular cartilage.

[0135] IV. Methods for Cultivating and Expanding Chondrocytes In Vitro Improved methods of continuous generation and expansion of chondrocytes in vitro are also disclosed. In some embodiments, micromass-derived chondrocytes expanded in serum-containing or serum-free medium retained the ability to generate cartilage in new micromasses or to generate cartilage when encapsulated in biomaterials after passaging. In other embodiments, micromass-derived chondrocytes expanded and passaged for up to three passages in 2% serum medium, in serum-free medium with TGFβ and FGF, or in serum-containing or serum-free medium, in serum-free medium with FGF, in serum-free medium with TGFβ, FGF and FSK retained the ability to generate cartilage in new micromasses or to generate cartilage when encapsulated in biomaterials after passaging. In some embodiments, micromasses are cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks before continuous reseeding and / or expansion.

[0136] In some embodiments, the micromass-derived chondrocytes are reseeded and / or expanded for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 passages before generating cartilage tissue suitable for transplantation. In preferred embodiments, the cells after reseeding and / or expansion are responsive to TGFβ in articular cartilage and / or BMP in growth plate cartilage for all passages. In further preferred embodiments, the micromass-derived chondrocytes are reseeded and / or expanded in serum-free medium. In some embodiments, the micromass-derived chondrocytes are reseeded in a monolayer or in a new micromass (high density cell culture). In some embodiments, the micromass-derived chondrocytes after reseeding and / or expansion produce new cartilage tissue of smaller size compared to those that were reseeded and / or expanded in a monolayer with less or no reseeding.

[0137] Methods: Monolayer- and micromass-derived chondrocytes are enzymatically isolated, typically collagenase-mediated, from the enclosing extracellular matrix prior to passaging or cryopreservation. Approximately 500,000-5 million cells are seeded per well of gelatin, type II collagen or fibronectin-coated 6-well plates or in 10 cm dishes in culture conditions of DMED (and ITS / proline / dexamethasone) + 2% FBS + ascorbic acid or serum-free medium (SFD) supplemented with or without a combination of FGF, TGFB, FSK. Cells were cultured until confluent (2-3 cell doublings) (approximately 2-5 days), then trypsinized and replated at similar densities.

[0138] V. Methods of Encapsulating Chondrocytes for Tissue Engineering An example of a cartilage tissue graft / construct was created by encapsulating hPSC-derived articular chondrocytes in RAD16-I, a self-assembling peptide scaffold commercially known as Puramatrix (Corning), at a final concentration of 0.01%-0.5%, preferably 0.15%, of RAD16-I. The biomaterial is gradually equilibrated with culture medium over a period of minutes to 2 hours and polymerized by increasing pH.

[0139] Puramatrix has repeating units of hydrophilic-hydrophobic amino acids. In ionic or neutral environments, it spontaneously self-assembles in an antiparallel beta-sheet configuration, generating an intertwined network of nanofibers (pores 50-200 nm). Non-covalent interactions allow cell migration. Stiffness can be controlled by varying the concentration (100 Pa-6 kPa).

[0140] In some forms, hPSC-derived articular chondrocytes can also be encapsulated in a biomaterial composed of a combination of hyaluronic acid, chondroitin sulfate and collagen (mainly collagen I) and can similarly successfully give rise to cartilage tissue.

[0141] In some embodiments, the chondrocytes are encapsulated in a hydrogel as a tissue engineered scaffold. The hydrogel can be polymerized using light, UV radiation, redox agents (e.g., sodium thiosulfate in combination with sodium persulfate), changes in pH, or using some other suitable polymerization initiator, e.g., a divalent cation such as calcium.

[0142] The polymerization agent may include monomers, macromers, oligomers, polymers or mixtures thereof. The polymer composition may consist of only covalently crosslinkable polymers or a blend of covalently and ionically crosslinkable or hydrophilic polymers.

[0143] Suitable hydrophilic polymers include synthetic polymers such as poly(ethylene glycol), poly(ethylene oxide), partially or fully hydrolyzed poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers (poloxamer and meroxapol), poloxamine, carboxymethylcellulose and hydroxyalkylated celluloses such as hydroxyethylcellulose and methylhydroxypropylcellulose, and natural polymers such as polypeptides, polysaccharides or carbohydrates such as FICOLL™, polysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin or alginate, and proteins such as gelatin, collagen, albumin, or ovalbumin, or polymers or blends thereof. "Cellulose" includes cellulose and derivatives of the types described above, and "dextran" includes dextran and similar derivatives thereof.

[0144] Examples of materials that can be used to form hydrogels include modified alginates. Alginates are carbohydrate polymers isolated from seaweed that can be crosslinked to form hydrogels upon exposure to divalent cations such as calcium. Alginates can be ionically crosslinked at room temperature in water in the presence of divalent cations to form hydrogel matrices. Modified alginate derivatives can be synthesized that have improved ability to form hydrogels. The use of alginate as a starting material is advantageous because it is available from more than one source and is available with good purity and characteristics. The term "modified alginate" refers to chemically modified alginate with modified hydrogel properties. Naturally occurring alginate can be chemically modified to produce alginate polymer derivatives that degrade more rapidly. For example, alginate can be chemically cleaved to produce smaller blocks of gellable oligosaccharide blocks, and linear copolymers can be formed with different preselected moieties, for example, lactic acid or epsilon caprolactone. The resulting polymer contains alginate blocks that allow for ionically catalyzed gelation, and oligoester blocks that result in more rapid degradation depending on the synthetic design. Alternatively, alginate polymers may be used where the ratio of mannuronic acid to guluronic acid does not result in a film gel, and are derivatized with hydrophobic water-labile chains, for example oligomers of epsilon-caprolactone. The hydrophobic interactions induce gelation until degraded in the body.

[0145] In addition, polysaccharides that gel upon exposure to monovalent cathines, including bacterial polysaccharides such as gellan gum and plant polysaccharides such as carrageenan, can be crosslinked to form hydrogels using methods similar to those available for crosslinking alginates, described above. Polysaccharides that gel in the presence of monovalent cations will form hydrogels when exposed to solutions containing, for example, physiological levels of sodium. Hydrogel precursor solutions can also be osmolalized with anions, for example, with mannitol, and then injected to form gels.

[0146] Polysaccharides that are very viscous liquids or that are thixotropic and form gels over time due to slow structure development are also useful. For example, hyaluronic acid may be utilized that forms injectable gels with a consistency similar to hair gel. Modified hyaluronic acid derivatives are particularly useful. The term "hyaluronic acid" refers to natural hyaluronic acid and chemically modified hyaluronic acid. Modified hyaluronic acid can be designed and synthesized by preselected chemical modification to adjust the rate and extent of crosslinking and biodegradation. For example, modified hyaluronic acid can be designed and synthesized to be esterified with relatively hydrophobic groups such as propionic acid or benzilic acid to make the polymer more hydrophobic and gel-forming, or grafted with amines to promote electrostatic self-assembly. Thus, modified hyaluronic acid can be synthesized that is injectable in that it flows under stress but maintains a gel-like structure when not under stress.

[0147] Other materials that may be utilized include proteins such as fibrin, collagen, and gelatin. Other polymer hydrogel precursors include polyethylene oxide-polypropylene glycol block copolymers, such as PLURONICS® or TETRONICS™, which are crosslinked by hydrogen bonding and / or temperature changes as described in Steinleitner et al., Obstetrics & Gynecology, 77:48-52 (1991) and Steinleitner et al., Fertility and Sterility, 57:305-308 (1992). Polymer mixtures may also be utilized. For example, a mixture of polyethylene oxide and polyacrylic acid may be utilized, which gels upon mixing through hydrogen bonding. In one embodiment, a mixture of a 5% w / w solution of polyacrylic acid and a 5% w / w mixture of polyethylene oxide (polyethylene glycol, polyoxyethylene) 100,000 may be combined to form a gel over time, for example, rapidly within a few seconds.

[0148] Water-soluble polymers with charged side groups can be crosslinked by reacting the polymer with an aqueous solution containing ions of the opposite charge, either cations if the polymer has acidic side groups, or anions if the polymer has basic side groups. Examples of cations for crosslinking polymers with acidic side groups to form hydrogels are monovalent cations, such as sodium, divalent cations, such as calcium, and polyvalent cations, such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, as well as di-, tri-, or tetrafunctional organic cations, such as alkylammonium salts. Aqueous solutions of salts of these cations are added to the polymer to form soft, highly swellable hydrogels and membranes. The higher the concentration or valence of the cation, the greater the degree of polymer crosslinking. In addition, the polymer can be crosslinked enzymatically, such as fibrin with thrombin.

[0149] Suitable ionically crosslinkable groups include phenols, amines, imines, amides, carboxylic acids, sulfonic acids, and phosphate groups. Aliphatic hydroxyl groups are not considered to be reactive groups with respect to the chemistry disclosed herein. Negatively charged groups, such as carboxylate, sulfonate, and phosphate ions, can be crosslinked with cations, such as calcium ions. Crosslinking of alginate with calcium ions is an example of this type of ionic crosslinking. Positively charged groups, such as ammonium ions, can be crosslinked with negatively charged ions, such as carboxylate, sulfonate, and phosphate ions. Preferably, the negatively charged ions contain two or more carboxylate, sulfonate, or phosphate groups.

[0150] Preferred anions for crosslinking the polymer to form hydrogels are mono-, di- or trivalent anions such as low molecular weight dicarboxylic acids, e.g., terephthalic acid, sulfate and carbonate. Aqueous solutions of the salts of these anions are added to the polymer to form soft, highly swellable hydrogels and membranes as described for the cations.

[0151] A variety of polycations can be used to complex and thereby stabilize the polymer hydrogel to a semipermeable surface membrane. Examples of materials that can be used include polymers with basic reactive groups, such as amine or imine groups, with preferred molecular weights of 3,000 to 100,000, such as polyethyleneimine and polylysine. These are commercially available. One polycation is poly(L-lysine), and examples of synthetic polyamines are polyethyleneimine, poly(vinylamine) and poly(allylamine). There are also natural polycations, such as the polysaccharide, chitosan.

[0152] Polycations that can be used to form semipermeable membranes by reaction with basic surface groups of polymer hydrogels include polymers and copolymers of acrylic acid, methacrylic acid and other derivatives of acrylic acid, polymers with pendant SO3H groups, such as sulfonated polystyrene, and polystyrene with carboxylic acid groups. These polymers can be modified to contain active species polymerizable groups and / or ionically crosslinkable groups. Methods for modifying hydrophilic polymers to contain these groups are known to those skilled in the art.

[0153] The polymers may be inherently biodegradable, but preferably have low biodegradability (for predictability of dissolution), but low enough molecular weight to allow excretion. The maximum molecular weight that allows excretion in humans (or other species for which use is intended) varies with the type of polymer, but is often less than about 20,000 daltons. Also usable because of their inherent biodegradability, but less preferred for general use, are water-soluble natural polymers and synthetic equivalents or derivatives, including polypeptides, polynucleotides, and degradable polysaccharides.

[0154] The polymer may be a single block having a molecular weight of at least 600, preferably 2000 or more, more preferably at least 3000. Alternatively, the polymer may contain two or more water-soluble blocks linked by other groups, or may be a water-soluble block. Such linking groups may include biodegradable linkages, polymerizable linkages, or both. For example, unsaturated dicarboxylic acids such as maleic acid, fumaric acid, or aconitic acid may be esterified with hydrophilic polymers containing hydroxyl groups, such as polyethylene glycol, or amidated with hydrophilic polymers containing amine groups, such as poloxamers.

[0155] Covalently crosslinkable hydrogel precursors are also useful. For example, water-soluble polyamines such as chitosan can be crosslinked with water-soluble diisocyanates such as polyethylene glycol diisocyanate. Isothiocyanates react with amines to form chemically crosslinked gels. Aldehyde reactions with amines, for example with polyethylene glycol dialdehyde, can also be utilized. Hydroxylated water-soluble polymers can also be utilized.

[0156] Alternatively, polymers containing substituents that are crosslinked by radical reaction upon contact with a radical initiator may be utilized. For example, polymers containing ethylenically unsaturated groups that can be photochemically crosslinked may be utilized. In this embodiment, water-soluble macromers are provided that contain at least one water-soluble region, a biodegradable region, and at least two free-radically polymerizable regions. The macromers are polymerized by exposing the polymerizable regions to free radicals generated, for example, by photosensitive chemicals or light. An example of these macromers is PEG-oligolactyl acrylate, in which the acrylate groups are polymerized using a radical initiation system such as eosin dye, or by brief exposure to ultraviolet or visible light. In addition, water-soluble polymers containing cinnamoyl groups that can be photochemically crosslinked may be utilized, as disclosed in Matsuda et al., ASAID Trans., 38:154-157 (1992).

[0157] Generally, the polymers are at least partially soluble in aqueous solutions such as water, buffered salt solutions or aqueous alcohol solutions. Methods for the synthesis of the other polymers described above are known to those skilled in the art. See, for example, Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, edited by E. Goethals (Pergamen Press, Elmsford, NY (1980). Many polymers, such as poly(acrylic acid), are commercially available. Naturally occurring and synthetic polymers may be modified using chemical reactions available in the art, for example, as described in March, "Advanced Organic Chemistry", 4th Edition, 1992, Wiley-Interscience Publication, New York.

[0158] Preferably, the hydrophilic polymers containing active species or crosslinkable groups contain at least 1.02 polymerizable or malleable groups on average, and more preferably each contains an average of 2 or more polymerizable or crosslinkable groups. Since each polymerizable group polymerizes into a chain, crosslinked hydrogels can be produced using only slightly more than one reactive group per polymer (i.e., an average of about 1.02 polymerizable groups). However, higher percentages are also preferred, and excellent gels can be obtained in polymer mixtures in which most or all molecules have two or more reactive double bonds. Poloxamine, an example of a hydrophilic polymer, has four arms and can therefore be easily modified to contain four polymerizable groups.

[0159] The hydrogel solution is prepared, for example, by mixing 10% weight / volume (w / v) of the polymerizable polymer in a suitable solvent, sterile phosphate buffered saline (PBS), with the pH adjusted to about 7.4. In some embodiments, the polymer is either a photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) or a photopolymerizable poly(ethylene oxide) diacrylate (PEODA).

[0160] Optionally, various additives such as 100 U / ml penicillin and 100 μg / ml streptomycin can be included in the hydrogel solution to inhibit microbial contamination. However, these are not the only bioactive additives that can be included in the hydrogel solution. For example, bioactive additives can include growth factors, cell differentiation factors, other cell mediators, nutrients, antibiotics, anti-inflammatory drugs, and other pharmaceuticals, alone or in combination. Without being limited thereto, some suitable cell growth factors include heparin-binding growth factors (HBGFs), transforming growth factors (TGFα or TGFβ), alpha fibroblast growth factors (FGFs), epidermal growth factors (EGFs), vascular endothelial growth factors (VEGFs), various angiogenic factors, nerve growth factors (NGFs), and muscle morphogenetic growth factors, depending on the cell type to be encapsulated in either the same or adjacent hydrogel layers of the hydrogel.

[0161] In addition, the hydrogel solution optionally contains a suitable non-toxic polymerization initiator thoroughly mixed to a final concentration of 0.05% w / v. If PEGDA or PEODA is selected as the polymer, a polymerization initiator is preferably added and the photoinitiator Igracure 2959 (commercially available from Ciba Specialty Chemicals Corp., Tarrytown, NY) is selected, although other suitable photoinitiators may also be used.

[0162] Exemplary photopolymerizable polymers are PEGDA and PEODA. Suitable hydrophilic polymers include synthetic polymers such as partially or completely hydrolyzed poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers (poloxamer and meroxapol), poloxamine, carboxymethylcellulose and hydroxyalkylated cellulose such as hydroxyethylcellulose and methylhydroxypropylcellulose, and natural polymers such as polypeptides, polysaccharides or carbohydrates such as Ficoll® polysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin or alginate, and proteins such as gelatin, collagen, albumin or ovalbumin, or polymers or blends thereof. The term "cellulose" includes cellulose and derivatives of the types described above, and "dextran" includes dextran and similar derivatives thereof.

[0163] An exemplary photoinitiator is Igracure 2959. Other photoinitiators include HPK, available from Polysciences. In addition, various dyes and amine catalysts are known to form active species when exposed to external radiation. Specifically, light absorption by the dye causes the dye to enter a triplet state, which then reacts with the amine to form active species and initiate polymerization. Typically, polymerization can be initiated by irradiation with light of wavelengths of about 200-700 nm, most preferably 320 nm or longer, most preferably about 365-514 nm, in the ultraviolet or visible region.

[0164] A number of dyes can be used in the photopolymerization, including erythrosine, phloxime, rose bengal, thonine, camphorquinone, ethyl eosine, eosine, methylene blue, riboflavin, 2,2-dimethyl-2-phenylacetophenone, 2-methoxy-2-phenylacetophenone, 2,2-dimethoxy-2-phenylacetophenone, other acetophenone derivatives, and camphorquinone. Suitable catalysts include amines, such as N-methyldiethanolamine, N,N-dimethylbenzylamine, triethanolamine, triethylamine, dibenzylamine, N-benzylethanolamine, N-isopropylbenzylamine. Triethanolamine is a preferred catalyst for one of these dyes. Photopolymerization of these polymer solutions is based on the discovery that combinations of polymer and photoinitiator (at a noncytotoxic concentration of less than 0.1% by weight, more preferably 0.05-0.01% by weight of initiator) will crosslink when exposed to light equivalent to 1-3 mWatts / cm2.

[0165] Photopolymers are preferred for making hydrogels because of the convenience of controlling polymerization using external radiation provided by a surgical scope, but the invention can be practiced using other polymeric materials and polymerization initiators. Examples of other materials that can be used to form hydrogels include (a) modified alginates, (b) polysaccharides that gel upon exposure to monovalent cations (e.g., gellan gum and carrageenan), (c) polysaccharides that are very viscous liquids or that are thixotropic and form gels over time due to slow evolution of structure (e.g., hyaluronic acid), and (d) polymeric hydrogel precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins).

[0166] VI. Methods for cryopreserving chondrocytes Methods for cryopreserving chondrocytes with high viability are known to those skilled in the art. In some embodiments, as described in Example 7, hPSC-derived chondrocytes retain the ability to generate articular cartilage after freeze-thaw cycles following cryopreservation. As described herein, a cryopreservation solution is used for cryopreservation of live chondrocytes and subsequent generation of articular cartilage after thawing.

[0167] Cryopreservation solution refers to any solution or medium in which biological material (e.g., chondrocytes) is immersed prior to cryopreservation. Typically, cryopreservation solutions contain a balanced salt solution, such as phosphate buffered saline, and at least one cryoprotectant. A cryoprotectant is a substance that reduces damage suffered by cells or tissues during freezing and / or thawing. Most cryoprotectants are composed of intracellular cryoprotectants (e.g., DMSO, glycerol, ethylene glycol, polyethylene glycol, 1,2-propanediol, formamide) and / or extracellular cryoprotectants (e.g., sugars, proteins, carbohydrates, such as hydroxyethyl starch, dextran, etc.). Some optional cryopreservation solutions are glycosaminoglycan-free.

[0168] Cryoprotectants or cryoprotective agents (CPAs) necessary to prevent any freezing damage to cells are well known in the art (see, e.g., Fuller, Cryo. Lett. 2004;25:375-388, the contents of which are incorporated herein by reference). DMSO is the most common cryoprotectant used for cryopreservation of MSCs. Thus, in some embodiments, chondrocytes are frozen in one or more cryopreservatives including DMSO.

[0169] In some embodiments, the biomass-derived chondrocytes or monolayer-derived chondrocytes are frozen, for example, in a cryopreservation solution, ie, in the presence of a cryoprotectant.

[0170] In some embodiments, after being frozen and stored in a cryoprotectant for a period of time and then thawed, the micromass-derived chondrocytes or monolayer-derived chondrocytes retain high viability, e.g., 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% viable cells of the total number of frozen cells. In preferred embodiments, after being frozen and stored in a cryoprotectant for a period of time and then thawed, the biomass-derived chondrocytes or monolayer-derived chondrocytes retain their cartilage potential and are capable of generating new cartilage tissue.

[0171] Prior to cryopreservation, monolayer- and micromass-derived chondrocytes must be enzymatically isolated from the encapsulating extracellular matrix, typically mediated by collagenase. In some embodiments, chondrocytes are resuspended in cryopreservation medium composed of 50% serum, 40% IMDM, and 10% DMSO at a cell density ranging from 1 million to 50 million cells per milliliter, preferably about 5 million to 10 million cells per milliliter. In another embodiment, chondrocytes are resuspended in a commercially available cryopreservation medium, Cryostor-CS10. In some embodiments, thawed cells are immediately cultured into biomaterials or high-density micromasses to produce cartilage tissue. In another embodiment, thawed cells are seeded into monolayers and expanded 1, 2, or 3 times before encapsulation into biomaterials or cultured into high-density micromasses to produce cartilage tissue. Cells expanded in monolayers acquire a less chondrogenic phenotype, which is restored by high-density micromass seeding or encapsulation.

[0172] VII. Methods for Using Cartilage Tissue The method provides chondrocytes and / or cartilage tissue suitable for transplantation.

[0173] The methods can be used to treat or prevent one or more diseases or disorders in a subject in need thereof. In some embodiments, the subject has osteoarthritis, osteochondritis dissecans, polychondritis, other cartilage diseases, or injuries or damage affecting cartilage. Thus, in some embodiments, the disclosed compositions and methods of use thereof can improve one or more symptoms and / or treat osteoarthritis, osteochondritis dissecans, polychondritis, other cartilage diseases, or injuries or damage affecting cartilage.

[0174] Cartilage (hyaline or articular cartilage) is a 1-5 mm thin tissue that coats bony surfaces within joints and forms other lubricated, tough surfaces. The cartilage provides extremely low friction joints that ideally last a lifetime. Cartilage can be damaged by acute injury or degeneration over time. For example, osteoarthritis (OA) is a joint disorder that results in thinning of cartilage and progressive joint damage. Localized lesions of articular cartilage can progress to disabling widespread cartilage destruction and arthritis. In some embodiments, the method ameliorates one or more symptoms of cartilage damage, injury, and / or loss.

[0175] The chondrocytes and the cartilage tissue prepared therefrom can in principle be applied to any site in need of cartilage repair.Preferably, the disclosed cartilage tissue, such as articular cartilage tissue, which may be in the form of cartilage (osteochondral) autografts prepared from the chondrocytes, is a fully functional cartilage tissue suitable for implantation into defects, more preferably for integration into surrounding cartilage tissue.The disclosed cartilage tissue is particularly suitable for in vivo implantation.

[0176] In some embodiments, chondrocytes and cartilage tissue are administered or implanted to promote resurfacing, repair and / or regeneration of cartilage. In preferred embodiments, the percentage of repaired and / or regenerated cartilage is about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% of the volume of the initially damaged wasted cartilage.

[0177] Methods for repairing cartilage damage or defects using cartilage tissue or tissue grafts are described. In some embodiments, the methods and compositions are effective for repairing and / or regenerating cartilage surfaces, such as articular cartilage. In further embodiments, cartilage tissue is transplanted to repair articular cartilage at the surfaces of the femoral, tibial and / or patellar joints to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more than 300% of the damaged or wasted cartilage present at the time of treatment, measured by cartilage volume.

[0178] The invention will be further understood by reference to the following non-limiting examples. EXAMPLES

[0179] Example 1: Serial reseeding and expansion / reseeding of 12-week-old micromass-derived chondrocytes Materials and Methods Expansion by monolayer culture Chondrocytes within articular cartilage tissue (12-week-old micromass tissue) were dissociated from their matrix and either immediately reseeded into micromass cultures or briefly expanded in monolayer before reseeding into micromass cultures. For monolayer expansion, approximately 2 million cells were seeded per well of a gelatin-coated 10 cm dish in culture conditions of DMEM (and ITS / Proline / Dex) + 2% FBS + ascorbic acid. Expanded cells were cultured until confluent (approximately 3 days) and then trypsinized and reseeded into additional monolayer expansions or seeded into micromasses after the first passage.

[0180] result Two successive reseedings were performed without expansion in monolayer (Figures 1A-1C). The resulting tissues were cartilaginous but could be slightly reduced in size with each passage. They respond appropriately to TGFβ (in articular cartilage) and BMP (growth plate cartilage) across all passages. n=3 (or more) for each time point.

[0181] Twelve-week-old TGFβ-treated chondrocytes in micromasses (P0) were dissociated and the cells were either directly reseeded into new micromasses (E1), or expanded once in monolayer before reseeding into micromasses (E2), or expanded twice in monolayer before reseeding into micromasses (E3) (Figures 2A-2C). Histology confirms that these cells retain chondrogenic potential and respond to the two treatments (TGFB in articular cartilage and BMP4 in growth plate cartilage). qPCR results suggest that COL10A1 is restored at each passage after BMP4 treatment (as expected). PRG4 levels also return favorably after passaging. In total, n=3-6 tissues were analyzed per time point. Expansion medium contained 2% serum.

[0182] The phenotype of 12-week-old micromass-derived chondrocytes is the same as those expanded in monolayer (Figure 2A). Cells were expanded in monolayer as described, resulting in an approximately 2- to 3-fold increase in yield. Cells expanded in monolayer acquire a mesenchymal stem cell-like phenotype, and the chondrogenic phenotype is restored by high-density micromass seeding or encapsulation.

[0183] Example 2: Expansion / Reseeding of 6-Week-Old Micromass-Derived Chondrocytes Materials and Methods Chondrocytes within articular cartilage tissue (6-week-old micromass tissue) were dissociated from their matrix and expanded in monolayers before reseeding into micromass cultures or encapsulation in biomaterials for cartilage tissue formation. For monolayer expansion, approximately 500,000 to 2 million cells were seeded per well of a 6-well dish coated with gelatin, collagen, or fibronectin in culture conditions of DMEM (and ITS / proline / Dex) + 2% FBS + ascorbic acid, or serum-free culture medium supplemented with TGFβ and FGF, or serum-free medium with TGFβ, FGF, and FSK. Expanded cells were cultured until confluent (approximately 3-5 days) and then trypsinized and reseeded into additional monolayer expansions, or seeded into micromass, or encapsulated in biomaterials (data not shown).

[0184] result Micromass-derived chondrocytes (6 weeks old) when expanded in 2% serum medium retained the ability to produce cartilage in new micromasses or when encapsulated in biomaterial after passaging.

[0185] Micromass-derived chondrocytes, when expanded and passaged for up to three passages in 2% serum medium, in serum-free medium with TGFβ and FGF, or in serum-free medium with TGFβ, FGF and FSK, retained the ability to produce cartilage in new micromass or when encapsulated in biomaterial after passaging.

[0186] These studies show that: 1. Micromass-derived chondrocytes can be expanded and retain chondrogenic potential (e.g., after 6 and 12 weeks). This is confirmed following expansion in both serum-containing and serum-free media. 2. Micromass‐derived chondrocytes can be serially passaged in micromasses for 12 weeks each (up to 3 passages) and retain their chondrogenic potential.

[0187] [Example 3] Induction of chondrocytes by monolayer culture Materials and Methods 100,000-200,000 paraxial mesoderm cells were seeded per well of a 24-well plate in culture conditions of serum-free differentiation medium supplemented with or without FGF and / or TGFB and / or FSK. The cells were cultured in the medium for 1, 2, 3 or 4 weeks and up to 6 weeks.

[0188] result Monolayers are initiated by seeding cells at low density into tissue culture vessels. This differs from micromasses, which are initially seeded in "spots" at very high density. Monolayer cultures require days or weeks to become confluent and chondrogenic induction can occur. It has been observed that more confluent or dense areas form chondrocytes first, a phenomenon known in chondrocyte culture. The basal medium for monolayers differs in composition of nutrients and supplements, in addition to differing growth factors or molecules.

[0189] Forskolin (FSK) improved cartilage production in the short term (1 day) and long term (2 weeks) in monolayers. Gene expression of COL2A1 in monolayers after 2, 3, 4 days or 2 weeks in the presence of TGFB and FGF is shown in Figures 3A and 3B. SCX expression is reduced by FSK in the presence of TGFB and FGF. SCX expression in monolayers after 2, 3, 4 days or 2 weeks is shown in Figure 3C.

[0190] Example 4. Phenotype and gene expression patterns of monolayer-derived chondrocytes from different compositions Materials and Methods Two starting populations (day 14 paraxial mesoderm / meso): 420 = 4 μM of the BMP inhibitor dorsomorphin and 20 ng / ml FGF at stage 2; 420 mesoderm shows a tendency towards tendon and little chondrogenic potential (generally in most experiments). 420i mesoderm = 420+I treated with the Wnt inhibitor IWP2 from day 3-5 of differentiation / stage 2 increased chondrogenic potential. Five experiments were performed and qPCR tests were performed on the cells after 4 weeks, data were pooled and analyzed.

[0191] In the second study, 420 and 420i paraxial mesoderm cells were seeded into micromass cultures supplemented with TGFβ and qPCR was performed after 12 weeks of micromass expansion.

[0192] In the third study, 420 and 420i paraxial mesoderm cells were seeded into serum-free monolayer cultures with no additional factors and qPCR was performed after 4 weeks of culture. A second group of 420 and 420i paraxial mesoderm cells was seeded into serum-free monolayer cultures supplemented with either FGF alone or a mixture of FGF, TGFβ and forskolin (FSK). qPCR was performed after 4 weeks of culture. Each condition had a sample size of 3.

[0193] result The 4 week old chondrogenic monolayer phenotype is chondrogenic in all 420i mesodermal starting populations.

[0194] Chondrocytes derived from monolayers after 4 weeks in different media compositions were analyzed for gene expression including COL2A1 (cartilage gene), SCX (tendon gene) and MKX (tendon gene) (Figures 4A-4C). qPCR data show that 420i has higher cartilage gene expression and only 420 has high tendon / ligament gene expression (SCX and Mohawk or MKX expression). Only 420 has higher tendon / ligament gene expression (Mohawk or MKX expression) in 12 week old TGFβ treated micromasses (Figure 4D). 420i induced mesoderm also demonstrated higher chondrogenic potential in monolayer cultures after 4 weeks, showing higher COL2A1 expression (cartilage related gene) in 420i cultures and higher SCX expression (tendon gene) in 420 cultures when cultured in media not supplemented with any additional factors (Figures 5A and 5B). In medium supplemented with FGF, 420-induced mesoderm demonstrated higher chondrogenic potential in monolayer culture after 4 weeks, with higher COL2A1 expression (cartilage-related gene) in 420i cultures and higher SCX expression (tendon gene) in 420 cultures (Figures 5C and 5D).

[0195] These results demonstrate that paraxial mesoderm produced in monolayer culture with different compositions of medium can have different chondrogenic potential for therapeutic purposes.

[0196] [Example 5] The role of forskolin in inducing chondrogenesis in the absence of FGF or TGFB Materials and Methods 100,000-200,000 paraxial mesoderm cells were seeded per well of a 24-well plate in culture conditions of serum-free differentiation medium supplemented with or without FSK. The cells were cultured in the medium for 1, 2, 3 or 4 weeks.

[0197] result In the absence of exogenous TGFβ / BMP agonists, endogenous TGFβ / BMP signaling must be present (usually in areas where cells are more confluent / higher density) for chondrogenesis to occur. In monolayers cultured without TGFβ and FGF, FSK (alone) induced cartilage. DMSO is the vehicle control for FSK (reconstituted in DMSO). Increase in chondrogenic gene expression (COL2A1) by FSK results in minimal changes or reductions in tendon genes (SCS and MKX) (Figures 6A-6C).

[0198] [Example 6] The effect of FSK is weakened by CBP inhibitor Materials and Methods 100,000-200,000 paraxial mesoderm cells were seeded per well of a 24-well plate in culture conditions of serum-free differentiation medium supplemented with TGFβ with or without FGF and / or FSK and / or CREB binding protein inhibitor (CBPi). Cells were cultured in medium for 1, 2, 3 or 4 weeks.

[0199] result The increased cartilage and / or tendon effects observed with FSK were attenuated by a CBP inhibitor, suggesting that the effects of FSK are mediated by PKA / CREB (Figures 7A-7C).

[0200] Example 7 hPSC-derived chondrocytes can be cryopreserved and retain their chondrogenic potential upon thawing.

[0201] Materials and Methods Micromass tissues (6 weeks old) were dissociated by collagenase treatment and resuspended in cryopreservation medium: 50% FBS + 10% DMSO + 40% IMDM. Approximately 5 million cells were aliquoted into each vial (ranges of 2-10 million cells per vial were tested) and immediately stored at -80°C for 2 days before being transferred to liquid nitrogen.

[0202] Frozen cells were thawed and reseeded into micromasses or encapsulated in biomaterials (RAD16-I / Puramatrix) and analyzed for viability and chondrogenic potential after 6 weeks.

[0203] result Micromass-derived chondrocytes are viable following cryopreservation / thawing. The viability / yield of micromass-derived chondrocytes following thawing is summarized in Table 1 below.

[0204] [Table 1]

[0205] After the freeze-thaw cycle, the micromass-derived chondrocytes generated cartilage tissue. Histology showed that the cartilage after the thaw cycle appears to be of better quality (i.e., thicker tissue with more uniform toluidine blue staining) compared to cartilage generated from the original micromass before cryopreservation. Thus, the cryopreserved micromass-derived chondrocytes retain their cartilage potential and their ability to generate cartilage tissue after thawing.

[0206] Monolayer-derived chondrocytes can also be cryopreserved and / or passaged and retain similar chondrogenic potential as micromass-derived chondrocytes.

[0207] Example 8: Monolayer-derived chondrocytes encapsulated in biomaterials for tissue engineering of implants Materials and Methods Chondrocytes from monolayer cultures were dissociated from their original matrix or thawed from cryopreservation and encapsulated in RAD16-I / PURAMATRIX™ scaffolds using Millicell-CM inserts for 6 weeks. Each insert was loaded with 12,000 cells / μL of cell / RAD16-I solution and polymerized by pH equilibration. Tissue engineered constructs were cultured in serum-free medium containing ascorbic acid, dexamethasone, proline, insulin and transferrin cell culture medium supplement (ITS) and TGFβ for 6 weeks.

[0208] result Cartilage formation from hESC-derived chondrocytes derived from monolayer culture in the PURAMATRIX™ system was compared to micromass-derived chondrocytes using histology analysis. The constructs were cultured prior to toluidine blue staining analysis. Toluidine blue staining showed a proteoglycan-rich matrix (ideal for cartilage) and tissue thickness was uniform, ideal for implantation. TGFβ+FGF+FSK-induced monolayer-derived chondrocytes were successfully encapsulated for tissue engineering and are comparable to micromass-derived chondrocytes.

[0209] Monolayer-derived chondrocytes behaved similarly to micromass-derived chondrocytes in terms of COL2A1 and PRG4 expression when encapsulated in biomaterials for tissue engineering of explants for 6-8 weeks (Figures 8A-8D).

[0210] A more nuanced chondrogenic potential of monolayer-derived cells was observed: differentially induced monolayer-derived chondrocytes could produce unique cartilage tissue when seeded into micromass cultures.

[0211] Micromasses were seeded with monolayer-derived chondrocytes and treated with TGFβ to induce articular cartilage (i.e., to regenerate "superficial zone-like" chondrocytes expressing PRG4) or BMPs to induce growth plate cartilage (GPCs; i.e., to generate hypertrophic chondrocytes expressing COL10A1). Monolayer-derived chondrocytes were generated with the following compositions: FGF only, or TGF+FGF+FSK30.

[0212] Monolayer-derived chondrocytes are seeded onto TGFB-treated micromasses to induce an articular-like cartilage phenotype. Monolayer-derived chondrocytes are seeded onto BMP4-treated micromass cultures to induce a growth plate cartilage phenotype. Hypertrophic chondrocyte gene or GPC gene COL10A1 is more abundant in BMP micromasses despite monolayer treatment prior to micromass culture (Figure 9A). Articular cartilage gene (PRG4) is more abundant in TGFB micromasses as expected (Figure 9B), and more abundant in micromasses derived from FGF+TGFβ+FSK-treated cells. Chondrocyte progenitors derived from monolayers with either FGF only or TGFB+FGF+FSK can subsequently generate articular and growth plate cartilage in micromasses (Figures 9A-9F).

[0213] In summary, monolayer-derived chondrocytes can be subsequently influenced to become articular or growth plate chondrocytes.Addition of exogenous TGFβ agonists was not required for FGF-treated cells to become chondrocytes.

[0214] Example 9: Derivation and molecular characterization of human pluripotent stem cells (hESC / iPSC) capable of giving rise to both growth plate and articular cartilage A modified directed differentiation approach based on a previously described method (Craft et al., Nat Biotechnol., 2015, 33(6):638-645; PMID:25961409) was used to generate chondrocytes derived from human pluripotent stem cells (i.e., hESCs / iPSCs) that can give rise to both growth plate and articular cartilage. The aim of this study was dual. The objective was to determine the molecular architecture governing chondrocyte fate and differentiation by comprehensively probing the molecular signature of generated hESC-derived chondrocytes along with chondrocytes derived from developing mouse and human cartilage. The transcriptomes of hESC-derived articular chondrocytes (AC) and growth plate chondrocytes (GPC) were characterized by bulk RNA sequencing (RNA-seq). Findings showed: (1) lineage-specific gene expression whose directionality significantly overlaps with gene expression differences observed between human developing epiphyseal and growth plate cartilage; (2) the concomitant regulatory landscape of hESC-derived chondrocyte lineages and mouse chondrocytes isolated by cell sorting via rigorous ATAC sequencing (ATAC-seq) protocols (i.e., protocols previously described in Gui et al., Elife, 2017, Vol. 5(6):e29329, PMID: 29205154; Richard et al., 2020, Cell, Vol. 181(2):362-381, PMID: 32220312); (3) confirmation of putative gene regulatory networks specific to either growth plate or articular chondrocytes via incorporation of aggregate transcriptomic and epigenetic datasets; and (4) evidence supporting representative regulatory networks of RUNX2 in growth plate chondrocytes and RELA (aka transcription factor p65) in articular chondrocytes.

[0215] result Transcriptome profiles of hESC-derived chondrocytes and human fetal chondrocytes Our previous studies have shown that hESC-derived chondrocytes / tissues acquire characteristics reminiscent of developmental human articular or growth plate chondrocytes / cartilage after treatment with TGFβ3 (TGFβ) or BMP4 (BMP), respectively (PMID:25961409). Both cartilage tissues are similar in size and rich in proteoglycans, as confirmed by histological assessment. The superficial layer of hESC-derived articular cartilage is smooth and contains flat chondrocytes arranged parallel to the surface, while the deeper zone of this tissue contains chondrocytes that are relatively uniform in size and uniformly distributed within the extracellular matrix. Chondrocytes in BMP-treated cartilage matrix are relatively large or hypertrophied.

[0216] Previous studies have taken a candidate-based (qRT-PCR and immunohistochemistry) approach to establish that lineage-specific genes are specifically expressed in each respective tissue, such as PRG4 expression in the joint lineage and COL10A1 in the growth plate lineage (Craft et al., Nat Biotechnol., 2015, 33(6):638-645; PMID:25961409). Beyond these traditional chondrocyte markers and information from studies of other species, understanding of the transcriptional profiles corresponding to human joint and growth plate cartilage remains limited. To generate unbiased transcriptome profiles for each of these lineages, a series of RNA-sequencing experiments were performed on both chondrogenic lineages derived in parallel from seven independent differentiations of the H9 hESC lineage. Twelve-week-old chondrocyte / cartilage tissues (n=6 tissues in total) from four experiments were collected to facilitate robust comparisons between lineages. RNA-sequencing was also performed on chondrocytes isolated from developing human epiphyseal or growth plate cartilage to serve as in vivo standards for each cell type. Primary chondrocytes were isolated from the left and right distal femurs of embryonic day (E)67 fetal donor samples. Differentially expressed genes (DEGs) in vitro or in vivo between each chondrocyte lineage were studied.

[0217] The resulting transcriptomic signatures of each chondrogenic lineage were unique as shown by principal component analysis (Figure 10A). Principal component 1 (PC1) represents the differences observed between in vitro and in vivo samples (Figure 10A, squares vs. circles / triangles). Within each experimental group, joint and growth plate samples were separated along PC2 (Figure 10A, PC2). As we predicted from the candidate gene expression trends, hESC-derived articular chondrocytes (orange circles / triangles) clustered closest to fetal epiphyseal cartilage (red triangles), while hESC-derived growth plate cartilage (light blue circles / triangles) clustered closest to fetal growth plate cartilage (dark blue squares). The differences between articular and growth plate cartilage were more pronounced for hESC-derived chondrocytes than their in vivo counterparts, as indicated by the greater distances separated along the PC2 axis.

[0218] The top 40 genes with the highest degree of differential expression between hESC-derived articular and growth plate chondrocytes are UCMA, R3HDML, CHI3L1, GPR171, PRG4, LYPD1, COL15A1, TNMD, COMP, MEOX1, ADORA1, FAP, FGF1, DOC2B, CD79B, MMP3, GALNT16, GH1, PAX1, AIF1, IL17B, MC3R, TRIM53BP, CD24P4, SCNN1B, SP7, VN1R40P, SCLBA3, LRRC38, APOB, COL10A1, TRIM49D1, ALPL, CXCL3, PRB2, TRIM53AP, MEPE, HDC, PANX3, and IBSP (heatmap not shown). Most of these genes displayed similar differential expression to fetal epiphyseal and fetal growth plate chondrocytes (data not shown). When the data set was expanded to the top 200 differentially expressed genes, this trend persisted and reached statistical significance (p-value < 0.05) (Figure 10B, compare upper and lower graphs). Similar results were obtained when starting from the top differentially expressed genes between fetal epiphyseal and fetal growth plate chondrocytes (data not shown). Finally, gene set enrichment analysis (GSEA) was performed on the set of genes upregulated in hESC-derived articular or hESC-derived growth plate cartilage. In hESC-derived articular cartilage, enrichment was found in periods related to (1) ECM organization, (2) response to TGF stimulation, and (3) collagen processes (Table 2). In hESC-derived growth plate cartilage, enrichment was found in periods related to (1) ossification, (2) ECM organization, and (3) cartilage development (Table 3). Similar enrichment periods were obtained when the same analysis was performed on genes upregulated in fetal epiphyseal or fetal growth plate cartilage. Taken together, these data provide strong support for the notion that the two chondrogenic cell types derived from hESCs truly represent chemically induced articular and growth plate chondrocytes.

[0219] [Table 2]

[0220] [Table 3]

[0221] In addition to identifying differentially expressed genes in hESC-derived articular and growth plate chondrocytes after 12 weeks of differentiation, we also identified differentially expressed genes between the two lineages at earlier time points in a new set of experiments, i.e., 4 and 8 weeks of differentiation, and included corresponding cultures after 12 weeks. In 4-week hESC-derived articular cartilage, enrichment was found specifically for periods related to (1) chondrogenesis, (2) skeletal development, and (3) extracellular matrix (data not shown).

[0222] In 4-week hESC-derived growth plate cartilage, condensation was found particularly in the periods related to (1) chondrocyte differentiation, (2) extracellular matrix organization, and (3) collagen fibril organization (data not shown).

[0223] In 8-week hESC-derived articular cartilage, enrichment was found particularly in terms of (1) the extracellular matrix, (2) response to transforming growth factor, and (3) collagen fibril organization (data not shown).

[0224] In 8-week hESC-derived growth plate cartilage, condensation was found particularly in the periods related to (1) ossification, (2) extracellular matrix organization, and (3) bone mineralization (data not shown).

[0225] In 12 week replicate hESC-derived articular cartilage, enrichment was found particularly in periods related to (1) extracellular matrix organization, (2) collagen fibril organization, and (3) skeletal development (Tables 2 and 3).

[0226] In 12 week replicate hESC-derived growth plate cartilage, condensation was found particularly during periods related to (1) endochondral ossification, (2) endochondral bone morphogenesis, and (3) skeletal system morphogenesis (Tables 2 and 3).

[0227] A subset of differentially expressed genes (DEGs) identified by RNA-seq were validated using in situ hybridization (ISH), quantitative RT-PCR (qPCR) and immunohistochemistry (IHC) in additional cartilage tissue derived from hESCs, epiphyseal and growth plate chondrocytes from distal femurs and proximal tibias of (embryonic) E59-E72 stage fetal donor specimens, where the tissues are actively differentiating and developing fetal joints / growth plates (Figures 11A-11P). Some of the top DEGs, such as PRG4 and COMP, have been previously demonstrated in cartilage biology, while the observed expression of other DEGs extracted from the current dataset of cartilage, such as COL15A1 and EFHD1, were previously unknown in cartilage biology.

[0228] Spatial resolution of RNA or protein expression was used to confirm DEG expression in specific zones of cartilage. To localize mRNA expression in situ, RNAscope was performed on hESC-derived cartilage tissue and fetal knee joint and distal femoral growth plate sections using probes recognizing COL2A1, PRG4, TNMD, and COL10A1. Type II collagen, encoded by the gene COL2A1, is a major structural component of both articular and growth plate cartilage, and thus expression is observed in cartilaginous structures both in vitro and in vivo (micrographs not shown). PRG4 is expressed in the superficial layer of hESC-derived TGFβ-treated articular cartilage and is absent in BMP4-treated growth plate-like cartilage (micrographs not shown). Similarly, in vivo, PRG4 is expressed in fetal articular cartilage and in the superficial zone of intraarticular ligaments and menisci, and is absent in the growth plate. COL10A1 mRNA was detected in hESC-derived BMP4-treated growth plate cartilage but not in TGFβ-treated articular cartilage, consistent with the expression pattern found in fetal knees, where COL10A1 is expressed in hypertrophic chondrocytes of the growth plate but not in epiphyseal chondrocytes (micrographs not shown). Tenomodulin (TNMD) (PMID:15632070), a well-known marker for tendon fate, was a top DEG in the articular / epiphyseal lineage. Interestingly, TNMD expression was detected in the superficial layers of most of the hESC-derived articular cartilage and fetal knee epiphyseal cartilage. TNMD was also expressed in intra-articular ligaments in vivo, as expected.

[0229] Several DEGs were further validated using qPCR in hESC-derived chondrocytes (Figures 11A-11H) and in fetal chondrocytes from distal femur and proximal tibia at three developmental time points (E59 (Carnegie stage 23), E67, and E72; Figures 11I-11P). The receptor-ligand pairs FGFR3 and FGF18, and PTH1R and PTHLH, are known to be differentially expressed between articular cartilage (FGF18, PTHLH) and growth plate cartilage (FGFR3, PTH1R) (PMID:31290205, PMID:12960068, PMID:23060229, 15781473, PMID:27142453, PMID:8314082). As predicted from previous studies, the expression levels of FGF18 and PTHLH were significantly higher in hESC-derived articular chondrocytes, and the levels of FGFR3 and PTH1R were significantly higher in hESC-derived growth plate chondrocytes. A similar pattern was observed in fetal chondrocytes. Pannexin 3 (PANX3), the second most abundant DEG in the growth plate lineage, was previously found to promote chondrogenic differentiation of prehypertrophic and hypertrophic chondrocytes of the growth plate (PMID:20404334), and we found its expression to be significantly higher in both hESC-derived chondrocytes and fetal growth plate chondrocytes. Alkaline phosphatase (ALPL) is known to be expressed in both hypertrophic chondrocytes and osteoblasts, and plays a role in bone mineralization (PMID:11850436). As expected, ALPL was also highly expressed in hESC-derived chondrocytes and fetal growth plate chondrocytes compared to their respective articular / epiphyseal cartilage. In the joint lineage, chitinase 3-like protein 1 (CHI3L1, also known as YKL-40) and mesenchymal homeobox 1 (MEOX1) were found to be top DEGs and their lineage-specific expression was confirmed in vitro and in vivo. CHI3L1 expression has been previously described in cultured chondrocytes and osteoarthritic cartilage (PMID:11315922, PMID:15015934), while MEOX1 is well known for its role in somitogenesis and axial skeletal formation (PMID:19520072).

[0230] Using IHC, DEG-encoded proteins were detected in hESC-derived cartilage and in developing phalangeal (E70) and knee (E59) joints. Cartilage oligomeric matrix protein (COMP) (PMID:16340129, PMID:24558358, PMID:16542502), a non-collagenous extracellular matrix protein expressed in cartilage, ligaments, and tendons, was more highly expressed in hESC-derived articular cartilage but not differentially expressed between fetal epiphysis and fetal growth plate, consistent with the overlap of chondrocytes being similar between these two samples (fetal cartilaginous elements are continuous structures and anatomical locations were approximate). COMP was detected in both the matrix and cells in hESC-derived articular cartilage tissue, but was found only intracellularly in hESC-derived growth plate tissue. In both metacarpophalangeal and knee joints, COMP was detected in the matrix of both epiphysis and growth plate cartilage, consistent with fetal RNA-seq data. COMP is also detected in ligament and perichondrium tissue. SP7 (also known as Osterix) (PMID:11792318, PMID:21075078), a transcription factor essential for growth plate chondrocyte and osteoblast differentiation, was significantly higher in hESC-derived and fetal growth plate cartilage. At the protein level, it was localized to the nuclei of hypertrophic chondrocytes in hESC-derived and fetal growth plate cartilage, but was not detectable in epiphyseal chondrocytes at the articular surface.

[0231] The selected unbiased transcriptomic approach also revealed previously undescribed potential markers of articular and growth plate chondrocytes. Collagen XV (COL15A1) is a non-fibrous basement membrane-associated collagen protein that has previously been detected in the perichondrium around bone and mesenchymal stem cells undergoing osteogenic differentiation (PMID:11827796, PMID:19365806). Although not differentially expressed in fetal tissues, COL15A1 was significantly higher in hESC-derived articular cartilage compared to hESC-derived growth plate cartilage. We localized the highest levels of collagen XV to the matrix of the superficial zone of hESC-derived articular cartilage, and also detected it within cells in the deep zone of articular cartilage and within some cells within growth plate cartilage. Collagen XV is expressed in the matrix of the epiphysis of metacarpophalangeal joints and on the surface of knee articular cartilage, but is absent in the matrix around hypertrophic chondrocytes of the growth plate. These data suggest that COL15A1 expression may be specific to the superficial zone of articular cartilage. EF-hand domain-containing protein 1 (EFHD1) expression was significantly higher in both hESC-derived chondrocytes and fetal growth plate chondrocytes. EFHD1 is a calcium-binding protein localized to the inner mitochondrial membrane and has not been previously described in cartilage (PMID:33537316). EFHD1 protein was localized to the cytoplasm of BMP4-treated hypertrophic chondrocytes and hypertrophic cartilage of fetal growth plate, but not to articular or epiphyseal cartilage as expected. These data indicate that EFHD1 is specifically expressed in hypertrophic chondrocytes of the growth plate.

[0232] Taken together, these experiments highlight the utility of established in vitro differentiation methods to define chondrocyte lineages, confirm previously described and also undescribed markers, and further illustrate the strength of the described transcriptomic dataset in identifying potential regulators of articular and growth plate cartilage.

[0233] Classification of differences in gene expression and chromatin accessibility between hESC-derived articular chondrocytes and hESC-derived growth plate chondrocytes 277 differentially expressed transcription factors (TFs) were identified in at least one of the four cell types profiled, for which binding motifs were described. A subset of these (n=36) was highly expressed by both hESC-derived chondrocytes and fetal articular chondrocytes, while a distinct subset (n=23) was highly expressed by both hESC-derived chondrocytes and fetal growth plate chondrocytes (top 40 TFs shown in Figure 12A). To refine this list of potential chondrocyte lineage regulators, their interactions with the genome of growth plate or articular chondrocytes were investigated by performing ATAC-seq (Ludwig et al., Cell Reports, 2019, vol. 27, pp. 3228-3240, PMID: 31189107). ATAC sequencing (ATAC-seq) is a method to characterize chromatin accessibility on a genome-wide basis for the subset of hESC-derived chondrocytes used for transcriptome analysis, facilitating robust comparison of gene expression and chromatin accessibility. For in vivo comparisons, ATAC-seq data were generated from populations of mouse embryonic chondrocytes expressing Col2a1 or hypertrophic chondrocytes expressing Col10a1. Col2a1+ or Col10a1+ chondrocytes were isolated using cell sorting from E15.5 transgenic mice carrying fluorescent reporters driven by Col2a1 or Col10a1 regulatory elements. The genome-wide overlap of peaks found in the two types of human chondrocytes and mouse chondrocytes is summarized in Table 4. Col2a1+ chondrocytes can contain chondrocytes that also express Col10a1, and it is expected that Col10a1+ chondrocytes also express Col2a1, although this population is more restricted in the hypertrophic lineage.

[0234] [Table 4]

[0235] Profiling hESC-derived chondrocytes by ATAC-seq and calling significant reproducible open chromatin regions (i.e., peaks) revealed a total of 37,780 unique peaks corresponding to putative regulatory elements. These regions were classified based on differential accessibility to either growth plate chondrocytes or articular chondrocytes, and 12,154 regions were identified as more accessible in growth plate chondrocytes and 11,571 regions were identified as more accessible in articular chondrocytes. These differentially accessible regions (DARs) suggest cell type-specific regulatory activity and are the focus of subsequent analysis. Examples of DARs identified in growth plate chondrocytes and articular chondrocytes are shown for the IHH locus and FGF1 locus, respectively. Using the GREAT region-based association tool [PMID:20436461], we identified periods significantly associated with DARs from growth plate chondrocytes, including abnormalities in appendicular diaphysis and ECM organization. Similarly, terms associated with DAR from articular chondrocytes were also identified, including ECM organization, collagen metabolic processes and osteoarthritis. The top 20 DEGs for each lineage in this subset of hESC-derived tissues, their corresponding promoter accessibility and their respective cis-regulatory scores (CRS, see below) are listed below and are merely representative of the larger dataset: (1) In hESC-derived articular (TGFβ-correlated) chondrocytes, genes with high expression and promoter accessibility scores ranging from 0 to 2 include SERTAD4-AS1, PENK, ADORA1, FGF18, MCUB, FGF1, SNTB1, SSC5D, ADAMTSL2, CD70 CILP2, COMP, ANGPTL6, SERTAD4, FAP, COL22A1, GALNT16, COL15A1, LYPD1, and CHI3L1. Conversely, these genes also exhibit low expression and promoter accessibility in hESC-derived growth plate (BMP-correlated) chondrocytes.

[0236] (2) In hESC-derived growth plate (BMP-correlated) chondrocytes, DARs with high expression and promoter accessibility scores ranging from 0 to 2 include IRF6, S100P, COL10A1, LPAR3, IHH, LG14, MGST1, FST, SLC13A5, ADGRD1, FAM177B, FXYD3, TSPAN18, WDR86, VSTM2L, HOXB6, SPINK5, TOX2, and C5AR2. Conversely, these DARs also exhibit low expression and promoter accessibility in hESC-derived articular (TGFβ-correlated) chondrocytes. 57 TF-encoding genes were differentially expressed primarily in hESC-derived chondrocytes, 134 TF-encoding genes were differentially expressed primarily in fetal chondrocytes, and 86 genes encoding TFs overlapped between hESC-derived and fetal chondrocytes (subset shown in Figure 12A).

[0237] To begin to understand the mechanisms of differential transcriptional regulation in these two lineages, de novo motif analysis was used to identify transcription factor (TF) motifs that were over-represented in DARs specific to either articular chondrocytes or growth plate chondrocytes (Figures 12B and 12C). A small number of these TFs were also differentially expressed in the corresponding cell type, but the majority were not. The same two sets of DARs were further examined for enrichment of motifs belonging to TFs that were differentially expressed in the corresponding cell type. This resulted in a reduced set of TFs, some of which were also observed in the de novo motif analysis (Figures 12B and 12C). This approach confirmed that motif enrichment does not substantially correlate with sequence complexity. Analysis of sets of lineage-specific DARs of neighboring genes that exhibit lineage-specific expression revealed similar enrichment for motif occurrence of some of these TFs in both sets of regions, despite conditioning on lineage-specific expression of these factors (data not shown). For example, the top 10 highly expressed genes encoding TFs specific to the BMP lineage are IRF6, MAFA, EGR3, HOXB6, FOXA2, OSR2, RUNX3, RUNX2, TBX20, and MEF2C. These TF-encoding DEGs are downregulated in the TGFβ cell lineage. However, enrichment of binding motifs of these TFs (including those of MEF2C) in the DARs of BMP and TGFβ revealed non-specificity of the motifs only in the BMP lineage, with the exception of RUNX2 and RUNX3, whose motifs were significantly enriched in the DARs of BMP but not in the DARs of TGFB. Conversely, the top 10 highly expressed genes encoding TFs specific to the TGFβ cell lineage are POUF2, MSC, NFATC2, HIC1, EGR2, NFATC4, EBF2, PKNOX2, SOX15, and ERG (heatmap data not shown). These TF-encoding DEGs are downregulated in the BMP cell lineage. However, enrichment of TF-binding motifs (including those of POU2F2) in DARS was not specific to the TGFB lineage alone, thus demonstrating a lack of significant specificity of the TF for this lineage.This suggests that a very simple model of gene expression, in which upregulation of a given TF is associated with increased accessibility to elements to which the TF may bind and subsequently with increased expression of its putative targets, is not sufficient to explain the gene regulatory network information obtained by the described ATAC-seq / RNA-seq strategy.

[0238] Defining hypothetical gene regulatory networks and their divergences in cell lineage development ATAC-seq and RNA-seq datasets were integrated to better capture the regulatory behavior described in the sequencing dataset. The selected approach defined three metrics of expression and accessibility at a given locus: 1) gene expression, 2) proximal (promoter) accessibility, and 3) distal (enhancer) activity, defined as cis-regulatory scores. For the top 20 DEGs, generally good correspondence was observed between these three metrics. However, when the scope of this integration approach was extended to all DEGs, the correspondence became unclear, i.e., genes exhibiting lineage-specific expression (as assessed by RNA-seq analysis) demonstrated variable and overlapping promoter accessibility (as assessed by ATAC-seq analysis). Potentially, multiple regulatory principles may be at work. By modifying an analytical approach described in a previous study describing the cis-regulatory behavior of immunological genes in mice (Yoshida et al., Cell, 2019, 176(4)pp. 897-912, PMID:30686579), genes were classified into four distinct regulatory behaviors based on the proportion of expression variance explained by chromatin accessibility within their respective loci. Briefly, these include genes whose expression variability is best explained by (1) a combination of promoter accessibility and distal cis-regulatory accessibility ("combo-centric" cluster 2), (3) promoter accessibility alone ("promoter-centric" cluster 3), or (4) distal cis-regulatory accessibility alone ("enhancer-centric" cluster 4). In general, genes falling into clusters 2-4 exhibited greater fold change in expression between articular and growth plate chondrocytes compared to genes falling into the "unknown variation" classification (cluster 1; Figure 13A). Similarly, a large proportion of genes from clusters 2-4 (genes whose variation in expression can be attributed to the accessibility of promoters or enhancers or both) were differentially expressed compared to those from cluster 1 (genes whose variation cannot be attributed to differential accessibility of any putative regulatory elements; Figure 13A).Further analysis confirmed that gene sets isolated in this manner showed increased sharing of direction (i.e., sequence bias) for predicted parameters (e.g., "combo-centric" gene expression had greater correspondence to cis-regulatory bias metrics than "promoter-centric" gene expression).

[0239] Further analysis was performed to characterize TF motif enrichment in DARs of genes belonging to clusters 2-4 (i.e., combo-centric, promoter-centric, and enhancer-centric genes) with the following initial restrictions: (1) motifs were considered only for TFs that were differentially expressed between hESC-derived articular chondrocytes and growth plate chondrocytes (TGFβ=124, BMP=83); enrichment for each motif was considered only for DARs or promoters whose direction of accessibility (growth plate vs. joint) matched the direction of expression (growth plate vs. joint). For each motif demonstrating enrichment according to these criteria, further validation was performed to determine whether enrichment could be significantly detected in the set of DARs / promoters where the way of accessibility was opposite to the direction of expression. This approach yielded a small number of motifs that were enriched in either promoter or enhancer sequences from genes in clusters 2-4 and were biased towards articular chondrocytes or growth plate chondrocytes.

[0240] [Table 5]

[0241] Analysis was performed focusing on motifs most highly enriched in DARs from enhancer-centered (cluster 4) and combo-centered (cluster 2) genes, as these groups exhibited the strongest tendency for differential expression across lineages (Figure 13A). TFs whose motifs were specifically enriched in TGFβ-treated articular cartilage-specific DARs included ETV1, FLI1, RELA, RFX2, NFKB1, RFX1, and ATF7, which were either significantly depleted or not significantly enriched in BMP-specific growth plate cartilage-specific DARs (Figures 13B-13D). For example, RELA was one TF whose motifs were specifically enriched in TGFβ-treated articular cartilage-specific DARs (Figures 13B-13D) and significantly depleted in BMP-specific growth plate cartilage-specific DARs. TFs whose motifs were specifically enriched in BMP-specific growth plate cartilage-specific DARs and not significantly enriched in any TGFB-specific articular cartilage DARs included FOXF2, FOXO4, FOXA2, CEBPB, RUNX2, DLX5, and EMX2 (Figures 13E-13G). The RUNX2 motif was specifically enriched in BMP-specific DARs (Figures 13E-13G) and not significantly enriched in any TGFB-specific articular cartilage DARs. DARs containing RELA or RUNX2 motifs were specifically enriched in RELA or RUNX2 binding sites, respectively (see Methods). RELA, also known as p65, belongs to the NF-κB family of transcription factors that share a REL homology domain and can form transcriptionally active dimers with other family members. It is a transcriptional activator of SOX9, a master regulator of chondrocyte differentiation as well as of its early differentiation, and of anabolic factors such as SOX6 and COL2A1, late factor HIF-2α and catabolic gene ADAMTS5. It also plays a role in cartilage homeostasis and degradation in osteoarthritis. RUNX2, also known as CBFA1, PEBP2 or AML3, belongs to a class of transcription factors that contain Runt homology domains (PMID:8341710). RUNX2 has long been recognized as a "master" skeletogenic factor at the apex of the regulatory cascade that governs osteoblast differentiation (PMID:9182763;9182762;9182764).Since its initial discovery, the role of RUNX2 in skeletogenesis has been expanded to include regulating chondrocyte hypertrophy in growth plate cartilage (PMID:10213384;10072783;15107406). It also has a similar, but pathogenic, role in articular chondrocytes, which acquire the hallmark hypertrophy in joint diseases such as osteoarthritis (PMID:32913706;31189030;28539595).

[0242] Analysis was performed on enhancer and promoter elements selected to be assigned to DEGs with putative binding sites for RUNX2 or RELA in hESC-derived chondrocytes and validated using CHIP-qPCR (Tables 6 and 7). To confirm candidates for validation, putative binding sites were cross-referenced with ATAC-seq data collected from E15.5 mouse Col2a1+ and Col10a1+ chondrocytes and with published ChIP-seq data in several cell types. Targets that met some or all of these criteria were selected, along with some genes previously described in chondrocyte biology and other genes with only binding sites with overlapping ChIP-seq peaks in other cell types.

[0243] Seven putative RELA target loci (Table 6) were selected for validation by ChIP-qPCR and included several genes known to be involved in articular cartilage identity and maintenance. These included PRG4 (lubricin), a functional marker of the superficial zone of articular cartilage; LOXL2 (lysyl oxidase-like 2), which induces anabolic gene expression and plays a potentially protective role against osteoarthritis (Alshenibr et al., Arthritis Res Ther., 2017, 19(1):179; PMID:28764769); DKK3 (Dickkopf-3), a non-canonical member of the Dkk family of Wnt antagonists that plays a role in articular cartilage maintenance (PMID:26687825); and TLR2 (Toll-like receptor 2), which mediates articular cartilage homeostasis (PMID:24237425).

[0244] [Table 6]

[0245] [Table 7]

[0246] In addition, targets not previously described for articular cartilage were selected for validation, including LTBP2, COL15A1, and GLIPR2. Representative binding regions with RELA motifs are the GLIPR2 promoter (overlapping with ChIP-seq data of RELA and overlapping with Col2a1+ mouse chondrocytes; data not shown) and the upstream enhancer of LOXL2 (overlapping with ChIP-seq data of RELA and histone acetylation peaks, data not shown). RELA and these putative target genes are expressed at significantly higher levels in hESC-derived chondrocytes (Figures 14A-14H). RELA, COL15A1, and LOXL2 were not differentially expressed between fetal epiphyseal chondrocytes and growth plate chondrocytes, whereas the remaining RELA targets were significantly more abundant in fetal epiphyseal chondrocytes (Table 6 and data not shown). Of note, COL15A1 is not a DEG in fetal chondrocytes, but its protein expression appears to be higher in the matrix of fetal epiphyseal compared to that of fetal growth plate.

[0247] Following ChIP-mediated pull-down of genomic regions bound by RELA in TGFB-treated articular cartilage, 6 of 7 loci were enriched at least 2-fold compared to the negative control in TGFB-treated articular cartilage (Table 6 and Figures 15A-15B), and 5 of 7 loci were enriched at least 5-fold. The bound region of the COL15A1 locus was only enriched 1.3-fold compared to the negative control in hESC-derived TGFB-treated articular cartilage, suggesting that RELA was not sufficiently bound to this locus in this sample or that COL15A1-expressing cells were not sufficiently enriched in these samples.

[0248] Ten putative RUNX2 targets (Table 7) were selected for validation by ChIP-qPCR and included genes known to be important for chondrocyte and growth plate biology, including ACAN (aggrecan), a proteoglycan essential for the extracellular matrix of both articular and growth plate cartilage (PMID:28804204, PMID:25446537); COL10A1 (type X collagen), a marker of hypertrophic chondrocytes important for endochondral bone formation (PMID:25321476); Included were WNT10B (PMID:17337262, PMID:15728361), a Wnt family ligand thought to play a role in chondrocyte differentiation and osteoblastogenesis; ATOH8 (Atonal homolog 8), a transcription factor important for chondrocyte proliferation and differentiation of the cartilaginous element of endochondral bone (PMID:31449527); and RXRA (Retinoid X Receptor Alpha), a retinoic acid receptor that plays a role in endochondral ossification (Sun et al., 2019, Osteoarthritis and Cartilage, vol. 27(1):S177-S178; PMID:11277079). Additionally, targets previously undescribed in cartilage biology were analyzed, based on previously described ChIP-seq interactions or homologies with ATAC-seq peaks from mouse chondrocytes, including C16ORF72, RCL1, GPR153, MAP4K3, and SCUBE1 (Table 7). Representative gene regulatory elements with RUNX2 motifs are the upstream ATOH enhancer (ChIP-seq data of RUNX2, overlapping with Col2a1+ mouse chondrocytes and histone acetylation marks, data not shown) and the upstream enhancer of ACAN (data not shown), which overlaps with a peak found in mouse Col2a1+ chondrocytes and is homologous to an enhancer identified in mouse chondrocytes (PMID: 29343853). RUNX2 and putative target DEGs are more highly expressed in hESC-derived growth plate cartilage, except for ACAN, which is expressed in both cartilage lineages (Figures 14I-14S). Similarly, RUNX2 and most of the putative target genes were more highly expressed in fetal growth plate chondrocytes compared to fetal epiphyseal chondrocytes, except for C16ORF72, which was expressed at similar levels.After ChIP-mediated pull-down of genomic regions bound by RUNX2 in BMP-treated growth plate cartilage, all 10 target loci selected for validation were enriched at least 2-fold compared to the negative control (Table 7 and FIG. 15C). Seven of the 10 loci were enriched at least 5-fold compared to the negative control, confirming the RUNX2 binding events at these gene regulatory elements.

[0249] As the majority of putative DARs predicted to carry motifs recognized by RELA and RUNX2 in hESC-derived articular cartilage and hESC-derived growth plate cartilage, respectively, were indeed enriched compared to negative control loci, these data sets are valuable for further exploring the molecular mechanisms underlying chondrocyte fate decisions.

[0250] Consideration Provided herein is a thorough characterization of both the transcriptome signature and gene regulatory landscape of human articular and growth plate chondrocytes. We also provide evidence that these hESC-derived lineages are molecularly similar to their in vivo counterparts through transcriptome profiling of human fetal epiphyseal and growth plate chondrocytes, and through epigenetic profiling of mouse embryonic chondrocytes isolated from either Col2a1 reporter mice (representing the majority of all mouse chondrocytes) or Col10a1 reporter mice (representing mouse growth plate chondrocytes). Specifically, we found a strong correlation between hESC-derived articular cartilage samples and fetal epiphyseal samples, as well as between hESC-derived growth plate cartilage samples and fetal growth plate samples.

[0251] We performed extensive experimental validation of the differentially expressed genes and confirmed the lineage-specific patterns in multiple independent hESC differentiation experiments and primary cell data sets. The receptor-ligand pairs fibroblast growth factor 18 (FGF18) and its receptor FGFR3 (PMID:31290205, 12960068, 23060229, 15781473) and parathyroid hormone-like hormone (PTHLH) and its receptor PTH1R (PMID:27142453, PMID:8314082) are known to be differentially expressed between articular and growth plate cartilage, respectively. We found that these expression patterns, as well as those of other known markers, were true in both hESC-derived and fetal chondrocytes. In addition to known markers, we identified novel genes that mark unique cartilage lineages, such as MEOX1 and CHI3L1 in the articular cartilage lineage and EFHD1 in the growth plate cartilage lineage. MEOX1 and CHI3L1, whose expression has been reported in axial skeleton and osteoarthritic cartilage, respectively, have not yet been confirmed in developing articular cartilage. EFHD1 was found to be strongly localized in hypertrophic cells of hESC-derived chondrocytes and fetal growth plate chondrocytes. EFHD1, whose role as a calcium sensor was previously investigated (PMID:26975899), may play a role in mediating cellular responses to calcium in hypertrophic chondrocytes (PMID:11404353). We surprisingly also found that the superficial zone of articular cartilage expresses tenomodulin (TNMD), which is co-expressed with PRG4. TNMD, closely related to chondromodulin 1 (CNMD), is known as a functional marker of tendon cells (PMID:15632070). Although there is conflicting evidence of TNMD expression in resting and proliferative chondrocytes of growth plate cartilage (PMID:11357195, 18239943), TNMD expression in the superficial zone of articular cartilage has not been described previously. This result seemed unexpected, as both cartilage and tendons / ligaments depend on TGFB signaling and may arise from a common developmental ancestor (PMID:19304887, PMID:18295755, PMID:27292641). This intriguing finding provides a rationale for further exploration of the developmental relationship between cartilage and adjacent connective tissues in the joint.Furthermore, these data reveal several other novel genes that remain to be explored in chondrocyte biology and highlight the potential utility of tissue- or zone-specific markers and the opportunity to investigate their function in cartilage development or maintenance in human cells and other models.

[0252] Despite the overall strong correspondence between hESC-derived and fetal chondrocytes, there were some notable exceptions, including genes whose expression patterns were the opposite of those seen in vitro. This was an expected result, as cartilage dissected from fetal samples is more heterogeneous than hESC-derived tissue, and there is overlap with chondrocytes in these samples due to the continuous structure and approximate anatomical boundaries of fetal cartilage. For example, dissected epiphyseal cartilage contains perichondrium, resting zone chondrocytes, proliferative chondrocytes, in addition to chondrocytes that participate in events related to secondary ossification centers, and chondrocytes that ultimately give rise to neonatal and adult articular cartilage. Similarly, growth plate cartilage contains proliferative, prehypertrophic, and hypertrophic chondrocytes, in addition to perichondrium cells, but our microdissection approach omitted osteoblasts and hematopoietic cells. Furthermore, it remains unclear whether hESC-derived articular and growth plate cartilage reflects developmental time compared to fetal cartilage. Some obvious differences in developmental stages are the presence of late growth plate marker gene expression, e.g., integrin-binding sialoprotein (IBSP) hESC-derived growth plate chondrocytes, which were absent in fetal growth plate chondrocytes, and the presence of a distinctive superficial zone of cartilage in hESC-derived articular cartilage, which was less developed in the fetal tissue used in this study (i.e., we showed that the surface of the epiphysis corresponded to the site of the future superficial zone of articular cartilage). Potentially, we confirmed that the more developed superficial zone of hESC-derived articular cartilage differentially expressed superficial zone-specific genes, e.g., COL15A1 (PMID:24043668), a non-fibrous basement membrane-associated collagen, in hESC-derived articular cartilage, but not in fetal epiphyseal cartilage. Future studies focused on transcriptome profiling of chondrocytes at the single cell level and / or over time during differentiation and time will address some of these long-standing questions.

[0253] Relying on the diverse properties of hESC-derived chondrocyte lineages and that these reflect true in vivo lineage properties, we also explored using this system to define putative gene regulatory networks (GRNs) that may govern lineage specificity and gene expression patterns in developing chondrocytes. We used ATAC-seq to probe the regulatory landscape of hESC-derived chondrocytes, as this approach gives insight into the potential binding of all factors using only one assay. By generating paired ATAC-seq and RNA-seq libraries of hESC-derived articular (TGFB-treated) and growth plate (BMP-treated) chondrocyte lineages, we were able to consider chromatin accessibility and adjacent gene expression at the locus-specific level of key lineage marker genes as well as across the genome more broadly. In addition, this assay also presented us with potential targets of candidate regulatory transcription factors (TFs).

[0254] As TFs typically have a major role in governing lineage-specific expression patterns, we identified a number of factors that demonstrate bias in expression across lineages and the occurrence of motifs enriched in putative lineage-biased regulatory elements. Finding that a simple model of GRNs is insufficient to explain the behavior observed in our epigenetic and expression datasets, we applied a locus-by-locus approach to the integration of ATAC-seq and RNA-seq genes to define sets of genes with distinct putative regulatory behaviors. We found that these groups exhibited distinct patterns of association between differential gene expression, chromatin accessibility data, and importantly, enrichment occurrence of lineage-biased TFs (Figure 4). Of note, our findings that grouped genes have different degrees of differential expression are consistent with previous studies that stratified immune genes based on regulatory behavior. We leveraged these findings to identify TFs exhibiting enrichment of DARs around DEGs to define either "enhancer-centric" or "combo-centric" enrichment except for certain lineages (Figure 5). Through this analysis, we identified, among others, RELA and RUNX2 as transcription factors that may play a role in lineage-specific regulatory networks, and used the results of our integrated ATAC-seq / RNA-seq analysis to prioritize candidate regulatory regions that may bind to regulate differential expression of lineage-specific genes. Surprisingly, when we performed ChIP-qPCR on these candidate regions, we found that the majority (16 out of 17 tested) were indeed bound to predicted TFs in the predicted lineages. These findings highlight that the simultaneous use of epigenetic and expression datasets generated in this study the definition of putative gene regulatory networks that may be active in developing chondrocyte populations, and the definition of key regulators that control these networks.

[0255] Our transcriptome profiling approach revealed several interesting TFs in both hESC-derived cartilage lineages, as well as some that were not identified as significantly differentially expressed in the smaller subset of samples for which we performed ATAC-seq. Many of these TFs and their associated family members showed similar expression patterns in fetal cartilage specimens, and some have been previously identified in the context of cartilage and joint biology, validating our data and the hESC model system studied here. Such TF families identified in TGFB-induced hESC-derived articular cartilage include ETS factors that contain a conserved ETS DNA-binding domain, including polyoma virus enhancer activator 3 (PEA3) family members (ETV1, ETV4, ETV5) and ETS-related gene (ERG) family members (ERG, FLI1, FEV) (PMID:23870508). Here, we specifically identified ETV1 and FLI1 as regulators of enhancer-centered and combo-centered genes in the hESC-derived articular cartilage lineage (Figure 5). PEA3 family members are significantly differentially expressed in both hESC-derived articular cartilage and fetal epiphyseal chondrocytes. These are FGF-responsive genes, and there is some evidence that loss of these proteins results in reduction and disorganization of humeral cartilage (PMID:26555052). ERG and FLI1 are differentially expressed in hESC-derived articular cartilage (but not significantly in fetal data), while FEV is differentially expressed in growth plate cartilage (not differentially expressed in fetuses). ERG has been well studied for its role in long-term maintenance of articular cartilage and, together with FLI1, upregulates articular cartilage genes such as PTHLH and PRG4 (PMID:17336282, https: / / doi.org / 10.1016 / j.joca.2013.02.063). The CREB family of TFs includes CREB5 and CREB3L1, both of which are differentially expressed in hESC-derived articular cartilage (CREB5 is differentially expressed in fetal epiphyseal cartilage, whereas CREB3L1 is not significant in fetal data).CREB5 is a known regulator of PRG4 expression in articular cartilage (PMID:33712729), which shares sequence homology with the ATF family of TFs, such as ATF7, and we highlight it as a regulator of combo-center genes in hESC-derived articular cartilage lineages (Figure 5). Nuclear factor of activated T cells (NFAT) family members NFATC2 and NFATC4 are both differentially expressed in hESC-derived articular cartilage (NFATC4 is also differentially expressed in fetal epiphysis). NFATC2 is also more highly expressed in superficial zone chondrocytes compared to deep zone chondrocytes in bovine cartilage, and NFAT family members play a role in chondrocyte gene expression and articular cartilage maintenance (PMID:24248346, PMID:12239209, PMID:24257415). The homeobox proteins MEOX1 and MEOX2, and the LIM homeobox protein LHX9, are also DEGs in both hESC-derived articular cartilage and fetal epiphyseal chondrocytes. MEOX1 and MEOX2 are essential for the development of all somitic compartments and for normal development of the head and neck joints (PMID:19520072). LHX9 is induced by FGF signaling and has previously been investigated for its role in osteosarcoma progression (PMID:31788020). Notably, the TFs and TF families we identified in these studies provide a rationale for further exploration of their individual and joint roles in articular cartilage development and stability.

[0256] In the growth plate lineage, the DLX family of TFs DLX2, DLX5, and DLX6 are highly expressed in growth plate cartilage (DLX5 and DLX6 are also expressed in fetal growth plates, while DLX2 was differentially expressed only in hESC-derived growth plates) and are known to be important regulators of cartilage differentiation during endochondral ossification (PMID:17051482). In particular, DLX5 has been shown to regulate the differentiation of immature proliferative chondrocytes into hypertrophic chondrocytes and into osteoblasts (PMID:12482714). Similarly, two RUNX family members, RUNX2 and RUNX3, are differentially expressed in both hESC-derived and fetal growth plate cartilage. RUNX2 is a TF important for chondrocyte maturation and osteoblast differentiation, and acts in concert with DLX5 and SP7 for correct skeletal development (https: / / doi.org / 10.1016 / S1348-8643(14)00032-9). RUNX3 acts redundantly with RUNX2 in chondrocyte maturation (PMID:15107406). Forkhead box (FOX) proteins are a superfamily of TFs, several members of which are differentially expressed in either articular cartilage or growth plate lineages. Within this large family, FOXA2, expressed in hESC-derived growth plate cartilage, is a key regulator of chondrocyte hypertrophic differentiation (PMID:22595668) and has been implicated in cartilage degradation and OA progression (www.oarsijournal.com / article / S1063-4584(19)30273-0 / fulltext). Myocyte enhancer factor 2c (MEF2C) is differentially expressed in both hESC-derived and fetal growth plate cartilage and activates a genetic program of hypertrophy during endochondral ossification (PMID:17336904). These TFs and other TFs identified in the studies herein can now be investigated for their biological roles in growth plate biology and chondrocyte function.

[0257] The data here are important resources for studying human joint and growth plate cartilage development. Thus, we have established and validated an in vitro human pluripotent stem cell chondrogenesis system as a robust and useful tool in investigating joint and growth plate cartilage lineages. This is particularly important for understanding how to specify and maintain articular cartilage, since the affected tissue after cartilage injury sometimes shows hypertrophic-like changes, even in regenerating tissues (PMID:22178514). From tissue-specific transcriptome data, we have identified several novel genes that mark two different tissues, and further identified and validated zone-specific markers of cartilage. Efforts to identify genes and networks that regulate cartilage development can be driven by this comprehensive study and analysis of the transcriptome and epigenetic signatures of joint and growth plate cartilage. We believe that this comparative perspective will greatly aid our understanding of cartilage development and joint disease biology.

[0258] Example 10: Comparison of micromasses derived from high density inoculations of paraxial mesoderm at stage III with those derived from low density inoculations (monolayers) The macroscopic morphology of micromasses derived from the use of different protocols was examined after 1.5, 3 and 6 weeks (data not shown). High density inoculation of paraxial mesoderm at stage III resulted in uneven and irregular micromasses and uneven toluidine blue staining, suggesting inefficient cartilage formation. Micromasses derived from monolayer-derived chondrocytes stained evenly with toluidine blue, suggesting uniform and efficient cartilage formation. These data clearly indicate that monolayer-derived micromasses are of higher quality than micromasses derived from high density inoculation of paraxial mesoderm at stage III.

[0259] The 3-week-old micromasses using high density seeding of paraxial mesoderm at stage III are unable to retain their shape when repositioned in a Petri dish using forceps. In contrast, the 1.5-week-old monolayer-derived micromasses are able to retain their shape better than the 3-week-old micromasses derived from using high density seeding of paraxial mesoderm at stage III. These observations clearly show the difference in the structure of those micromasses derived from different methods. The micromasses derived from low density monolayer seeding of paraxial mesoderm at stage III provide higher quality, more extracellular matrix, and more uniform distribution of extracellular matrix (images not shown).

[0260] Four- and six-week-old micromasses derived from high-density inoculation of paraxial mesoderm at stage III often had uneven morphology, with less dense matrix and less area of ​​proteoglycan, indicating suboptimal chondrogenesis efficiency. Monolayer-derived micromasses after 4 and 6 weeks of age showed uniform shape and uniform staining of monolayer-derived micromasses, indicating highly efficient chondrogenesis (images not shown).

[0261] The superior cartilage-like quality of the monolayer-derived cartilage tissue was further confirmed by the expression levels of COL2A1 (a cartilage gene) after 6 weeks of micromass culture or after 6 weeks of encapsulation in RAD16-I biomaterial. COL2A1 expression is higher in tissue derived from monolayer-derived cells compared to tissue derived from high-density micromass culture of paraxial mesoderm cells (Figure 16A). PRG4 expression (an articular cartilage gene) was also expressed at similar levels in monolayer-derived micromasses and encapsulations compared to 6-week-old micromasses or encapsulations derived from the use of high-density inoculation of paraxial mesoderm at stage III (Figure 16B). In general, the levels of RRG4 are higher in micromasses compared to encapsulations in both populations (Figure 16B).

[0262] Gene expression graphs show that monolayer-derived micromasses undergo chondrogenesis earlier and more efficiently than initial protocol micromasses. Gene expression was compared in 1-4 week chondrogenic cultures in micromasses, monolayer cultures, and monolayer-derived micromasses to determine comparative levels of chondrogenic genes COL2A1, SOX9, PRG4, and ACAN. The data suggest that monolayer and micromass cultures initiated with monolayer-derived cells undergo chondrogenesis earlier and more efficiently, based on the upregulation of COL2A1, SOX9, and ACAN gene expression, and generate articular cartilage, based on the upregulation of PRG4. Figures 16C-F depict mRNA copy numbers after 1 week, 2 weeks, 3 weeks, or 4 weeks in paraxial mesoderm at stage III (day 14), micromass cultures derived from paraxial mesoderm seeded into micromass at stage III (micromass), monolayer cultures derived from paraxial mesoderm seeded into monolayer at stage III (monolayer), and monolayer-derived micromass cultures derived from monolayer cells seeded into micromass culture, as indicated.

[0263] 17A-C depict COL10A1 mRNA copy numbers normalized to TBP in micromass cultures at the indicated time points and treatment regimens. TGFβ3-treated articular cartilage tissue was cultured in TGFβ3-supplemented media for the indicated time periods (2, 4, 6, 8, 10, or 12 weeks) until triggered with BMP4 by switching media supplementation from TGFβ3 to BMP4 (e.g., BMP4 at week 2 (@) indicates cultures were treated with TGFβ3 for 2 weeks and then switched to BMP4 supplementation for an additional (+) 2, 6, 12, or 24 weeks). Micromass cultures treated with TGFβ for 8 weeks or less prior to the BMP4 treatment regimen were able to respond to BMP4 and upregulate COL10A1 mRNA, indicating that cells remain bipotent and can undergo growth plate cartilage differentiation. Micromass cultures treated with TGFβ for at least 10 weeks prior to BMP4 treatment did not show BMP4-induced upregulation of COL10A1 mRNA and are therefore resistant to BMP4-mediated growth plate differentiation. Data represent the average of four biological replicates, with error bars indicating standard error. Figure 17A shows that stability of hPSC-derived articular cartilage tissue was achieved after 8-10 weeks of TGFβ treatment, as it became resistant to BMP4 induction and growth plate chondrocyte differentiation. Figures 17B and 21C show that the cartilage lineage is stable for long periods in culture, as PRG4 gene expression (Figure 17B) remains significantly higher in the TGFβ-induced articular cartilage lineage, and COL10A1 expression (Figure 17C) is expressed only by BMP4-induced growth plate cartilage up to 24 weeks. Unique cartilage tissue does not express genes associated with other lineages even after 24 weeks of in vitro culture.

[0264] FIG. 18A shows an example of quantified amounts (μg per μg DNA content) of sulfated (s) glycosaminoglycans (GAGs) in micromass cultures cultured in the presence of TGFβ3 or BMP4 for the indicated time periods (weeks). sGAG content increases over time in both cartilaginous tissues. FIG. 18B depicts representative quantification of both sulfated GAGs and hydroxyproline (OH-Pro; an alternative biochemical quantification of collagen content) in TGFβ3-treated articular cartilage tissue cultured for 12 weeks. Values ​​were calculated as μg per μg DNA content per culture. Error bars represent the mean standard error. FIG. 18C depicts relative differential expression levels of representative collagen genes in articular (TGFβ) and growth plate (BMP) cartilage micromass tissues. Values ​​represent the mean (n=6) counts by RNA sequencing, and error bars indicate standard deviation. The graph depicts only genes that were found to be significantly differentially expressed, ie, it is non-inclusive to all collagen genes expressed in these tissues.

[0265] Collagen genes more highly expressed in articular cartilage: COL6A3; COL4A4; COL14A1; COL22A1; COL16A1; COL8A2; COL8A1; COL12A1; COL4A2; COL5A2; COL24A1; COL3A1; COL1A2; COL18A1; COL4A1; COL1A1; COL5A1; COL6A2; COL6A1; COL7A1; COL15A1; COL13A1; COL25A1.

[0266] Collagen genes highly expressed in growth plate cartilage: COL20A1; COL10A1; COL11A1; COL4A6; COL4A5; COL11A2; COL2A1; COL9A1; COL9A2; COL9A3.

[0267] A glycoprotein gene highly expressed by articular cartilage: SBSPON THBS4;TNFAIP6;SPON1;SRPX;LAMA1;COCH;COMP;FN1;AEBP1;MXRA5;MATN2;TGFBI;SNED1;LAMB3;ECM1;EFEMP2;LAMA 2;LAMB2;MFAP2;MFAP4;SRPX2;LTBP3;POSTN;CILP;PCOLCE;FBLN7;LTBP2;EMILIN1;VWA1;FBLN1;MGP;GAS6;CTHRC1; IGFBP7;FBLN5;FNDC1;ECM2;CRISPLD2;THBS3;LAMC3;HMCN1;LTBP4;CILP2;LTBP1;SVEP1;PXDN;THBS2;DPT;EDIL3;A BI3BP;IGFBP6;LAMC2;NELL1;MFAP5;LAMA3;SPARC;FBN2;FBN1;ELN;THBS1;EMILIN3;SMOC1;AGRN;NTN3;SSPO;OTOG; Glycoprotein genes highly expressed by growth plate cartilage: NID1;SLIT3;NTNG1;LGI4;PAPLN;EMILIN2;RSPO2;NTNG2;RSPO4;MATN4;MATN3;NPNT;SPP1;DMP1;SPARCL1;NELL2;VWDE;VWA5A;TSKU;IGFBP2;FGL2;TINAGL1;EMID1;TNC;CRISPLD1;SPON2;IGSF10;GLDN;LAMA4;MFAP3;MFGE8;PCOLCE2; Proteoglycans highly expressed in articular cartilage: LUM; FMOD; PODNL1; BGN; ASPN; HAPLN3; PRG4; PODN; OGN; Proteoglycans highly expressed by growth plate cartilage: IMPG2; CHAD; SPOCK3; CHADL; HAPLN1; DCN; HAPLN2; PRELP; ACAN; Secreted factor genes more highly expressed in articular cartilage: EGFL8;MEGF6;SFRP1;LEFTY2;WFIKKN1;AMH;FSTL1;BMP5;SCUBE3;GDF6;IL17D;PDGFC;GDF7;CHRDL1;GDF5;WIF1;NRTN;THPO;FGF2;ANGPTL6;INHBA;TGFB3;VEGFB;TNFSF12;FGF18;IL11;NGF;CLCF1;IL10;CX3CL1;VWC2;S100A6;FGF1; TNFSF10;PDGFA;WNT5B;ANGPTL1;ANGPTL7;SFRP5;EGFL6;ARTN;FGF9;CHRD;MEGF10;CBLN3;MST1;LIF;S100A3;S100A16;HBEGF;S 100A4;TNFSF9;CRLF1;WNT9A;INHBE;CTF1;IGF1;SFRP2;NRG1;S100B;NTF3;FGF11;ANGPTL4;FSTL3;TGFB1;VEGFC;HCFC1;S100A2; Secreted factor genes highly expressed by growth plate cartilage: TGFA;WNT16;WNT11;CXCL13;KITLG;WNT5A;BMP8B;MEGF11;ANGPTL5;WNT10B;ANGPTL2;S100A13;WNT3;WFIKKN2;S100P;ANGPT1;S100A1;SCUBE1;BMP4;HGF;NRG4;IHH;FGFBP2;FGF7;ISM1;EGF;BMP2;IL17B;CXCL14;FGF14;GDF15;FGF13;WNT2B;PDGFD;PIK3IP1;CHRDL2;FST;WNT4;CRHBP;S100Z;BMP6;HHIP;TGFB2;SCUBE2;ANGPT2;FRZB;MEGF9;CSF1; ECM-related genes expressed more highly in articular cartilage: ANXA1; PLXDC2; MUC1; PLXDC1; LGALS1; MUC20; ANXA6; COLEC12; PLXNA4; C1QTNF5; SEMA3C; SEMA3A; SEMA4C; CSPG5; C1QL1; GREM1; SEMA3F; SEMA4D; SDC1; C1QTNF2; CLEC11A; GPC2; SEMA4A; CLEC2D; SEMA6A; C1QTNF8; C1QL4; CLEC3A; C1QTNF1; ANXA3; SDC2; ECM-related genes highly expressed in growth plate cartilage: MUC5B; ANXA5; SEMA5A; LGALS3; SDC4; SEMA4G; PLXNA1; PLXNB3; SEMA3D; GPC1; GPC4; ANXA2; SEMA3E; CLEC3B; GPC6; C1QTNF7; SEMA5B; GPC5; SEMA6B; GPC3; PLXNB1; SDC3.

Claims

1. A method for chemically inducing the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes, (a) A step of inducing the formation of protostial mesoderm (PSM) from hPSCs in a first cell culture medium, (b) A step of inducing the formation of paraxial mesoderm (PM) from PSM in a second cell culture medium, and (c) A step of inducing the conversion of PM cells to chondrocyte precursors (CPCs) in a third cell culture medium supplemented with a TGFβ agonist, an FGF agonist and / or a cyclic AMP agonist, wherein (i) The PM cells are cultured in a third cell culture medium at a low cell density, or (ii) The step of culturing paraxial mesoderm cells at a high cell density in a third cell culture medium, and (d) A step of inducing chondrocyte formation from CPCs, comprising culturing the CPCs in a fourth cell culture medium. Includes, Herein, if step (c) includes culturing at a high cell density, the cells are cultured in a cell culture medium supplemented with a TGFβ agonist, an FGF agonist, and / or a cyclic AMP agonist.

2. The method according to claim 1, wherein one or more of an FGF agonist, a BMP4 agonist, and a TGFβ agonist are supplied to a first cell culture medium in an effective amount, and the hPSCs are cultured in the first cell culture medium for a time effective to generate PSM cells expressing the cell surface markers CD56 and PDGFRα.

3. The method according to claim 2, wherein the FGF agonist is selected from the group consisting of FGF, bFGF, FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20, and FGF23.

4. The method according to claim 2, wherein the BMP4 agonist is selected from the group consisting of BMP4, GDF5, GDF6, GDF7, BMP4, BMP2, BMP6, BMP7, and BMP10.

5. The method according to claim 2, wherein the TGFβ agonist is selected from the group consisting of TGFβ1, TGFβ, TGFβ3, and activin A.

6. The method according to claim 2, wherein the first cell culture medium comprises a Wnt agonist, and the Wnt agonist is selected from the group consisting of CHIR99021, SB216763, TWS119, CHIR98014, Tideglusib, SB415286, LY2090314, CHIR-98014, AZD1080, TDZD-8, and wnt3a.

7. The method according to claim 2, wherein (a) the FGF agonist is FGF, (b) the BMP4 agonist is BMP4, and / or (c) the TGFβ agonist is activin A.

8. The method according to claim 1, wherein step (a) preferably results in the formation of an embryoid body.

9. The method according to claim 1, wherein step (a) preferably omits the step of causing the formation of an embryoid body.

10. The method according to any one of claims 1 to 9, wherein a second cell culture medium is supplemented with an effective amount of a BMP4 inhibitor and / or an FGF agonist, and the PSM cells are cultured in the second cell culture medium for a time sufficient to generate the formation of paraxial mesoderm cells expressing D73, CD105 and / or PDGFRβ.

11. The method according to claim 10, wherein the BMP4 inhibitor is selected from the group consisting of dorsomorphine and LDN193189 dihydrochloride.

12. The method according to claim 10, wherein the FGF agonist is selected from the group consisting of FGF, bFGF, FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20, and FGF23.

13. The method according to claim 10, wherein the second cell culture medium comprises a Wnt antagonist, and the Wnt antagonist is selected from the group consisting of IWP2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide), Dickkopf-related protein 1 (DKK1), Wnt-C59 (4-(2-methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide) and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one).

14. The method according to claim 10, wherein the second cell culture medium comprises a TGFβ inhibitor, and the TGFβ inhibitor is selected from the group consisting of SB431542, GW788388, and A-83-01.

15. The method according to any one of claims 1 to 9, wherein an effective amount of a combination of agents selected from the group consisting of a TGFβ agonist, an FGF agonist, and a cyclic AMP agonist is supplied to a third cell culture medium, and PM cells are cultured in the third cell culture medium for an effective time to generate chondrocytes expressing one or more of SOX9, COL2A1, and PRG4.

16. The method according to claim 15, wherein the TGFβ agonist is selected from the group consisting of TGFβ1, TGFβ, TGFβ3, and activin A.

17. The method according to claim 15, wherein the FGF agonist is selected from the group consisting of FGF, bFGF, FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20, and FGF23.

18. The method according to claim 15, wherein the cyclic AMP agonist is selected from the group consisting of prostaglandin E2, dbcAMP, 8-bromo-cAMP, genistein, forskolin, colforsin, and rolipram.

19. The paraxial mesoderm cells in step (b) are 1 cm including the values ​​at both ends. ２ Approximately 20 x 10 ３ ~1 cm ２ 20 x 10 ４ The method according to claim 1, wherein the cells are cultured at a low cell density.

20. The method according to claim 15, wherein the number of chondrocytes expressing one or more of SOX9, COL2A1, and PRG4 is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more when induced from paraxial mesoderm cells cultured at a low cell density in step (b), provided that the same number of paraxial mesoderm cells are used as the starting population.

21. The method according to claim 15, further comprising encapsulating cartilage cells in a biomaterial.

22. The method according to claim 21, wherein the TGFβ agonist is selected from the group consisting of TGFβ1, TGFβ, TGFβ3, and activin A.

23. The method according to claim 21, wherein chondrocytes are encapsulated in a hydrogel as a tissue modification scaffold.

24. The method according to claim 15, further comprising the step of freezing chondrocytes or cartilage tissue in a salt solution and one or more cryoprotective substances.

25. The method according to claim 24, further comprising the step of thawing frozen chondrocytes or their cartilage tissue for further culture and / or in vivo tissue transplantation.

26. Chondrocytes or cartilage tissue thereof, prepared by the method described in claim 1.

27. Chondrocytes or cartilage tissue according to claim 26, prepared for in vivo transplantation.

28. A pharmaceutical composition for use in a method of treating or preventing one or more diseases or disorders requiring cartilage repair in a subject requiring cartilage repair, comprising transplanting cartilage cells or cartilage tissue as described in claim 26 or 27.

29. The pharmaceutical composition according to claim 28, wherein the subject has osteoarthritis, osteochondritis dissecans, polychondritis, other cartilage diseases, or injuries or damage affecting cartilage.

30. The pharmaceutical composition according to claim 28 or 29, for treating or improving one or more symptoms related to osteoarthritis, osteochondritis dissecans, polychondritis, other cartilage diseases, or injuries or damage affecting cartilage.

31. A tissue culture medium for inducing the conversion of paraxial mesoderm cells to chondrocytes, comprising a combination of drugs: a TGFβ agonist, an FGF agonist, and a cyclic AMP agonist, wherein the drug combination is effective in inducing a greater number of chondrocytes expressing one or more of SOX9, COL2A1, and PRG4, and when induced from paraxial mesoderm cells cultured at a low cell density of approximately 20 × 10³ to 20 × 10⁴ per cm², the resulting tissue culture medium is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than when the paraxial mesoderm cells are cultured at a high cell density, provided that the same number of paraxial mesoderm cells are used as the starting population.

32. A tissue culture medium according to claim 31, in an amount effective for producing chondrocytes expressing one or more of SOX9, COL2A1, and PRG4.

33. The tissue culture medium according to claim 31 or 32, wherein the TGFβ agonist is selected from the group consisting of TGFβ1, TGFβ, TGFβ3, and activin A.

34. The tissue culture medium according to claim 33, wherein the TGFβ agonist is TGFβ3.

35. The tissue culture medium according to claim 33, wherein the concentration of the TGFβ agonist is approximately 1 ng / ml to approximately 50 ng / ml, approximately 5 ng / ml to approximately 20 ng / ml, or preferably approximately 10 ng / ml, including the values ​​at both ends.

36. The tissue culture medium according to claim 31, wherein the FGF agonist is selected from the group consisting of FGF, bFGF, FGF2, FGF4, FGF9, FGF19, FGF21, FGF3, FGF5, FGF6, FGF8a, FGF16, FGF17, FGF18, FGF20, and FGF23.

37. The tissue culture medium according to claim 36, wherein the FGF agonist is bFGF.

38. The tissue culture medium according to claim 36, wherein the concentration of the FGF agonist is approximately 1 ng / ml to 100 ng / ml, approximately 5 ng / ml to 50 ng / ml, or preferably approximately 10 ng / ml, including the values ​​at both ends.

39. The tissue culture medium according to claim 31, wherein the cyclic AMP agonist is selected from the group consisting of prostaglandin E2, dbcAMP, 8-bromo-cAMP, genistein, forskolin, colforsin, and rolipram.

40. The tissue culture medium according to claim 39, wherein the cyclic AMP agonist is forskolin.

41. The tissue culture medium according to claim 40, wherein the concentration of the cyclic AMP agonist, including the values ​​at both ends, is approximately 5 μM to approximately 100 μM, approximately 10 μM to approximately 50 μM, or approximately 30 μM.

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