CARTILAGE TISSUE

Abstract

The invention relates to a method for producing cartilage from pluripotent stem cells (PSCs), the method comprising providing chondrocytes by: 1) providing pluripotent stem cells (PSCs); 2) inducing differentiation of the PSCs into a primitive streak/mesendoderm by culturing the PSCs in hypoxic conditions, in a (mesendodermic) culture media comprising: i) a Wingless/Integrated (WNT) family member, ii) an Activin family member, and iii) a Fibroblast Growth Factor (FGF) family member; 3) inducing differentiation of the primitive streak/mesendoderm into a mesoderm by culturing the primitive streak/mesendoderm in hypoxic conditions, in a (mesodermic) culture media comprising: i) a FGF family member, ii) a bone morphogenetic protein (BMP) family member, iii) Follistatin, and iv) a Neurotrophin (NT); and 4) inducing differentiation of the mesoderm into chondrocytes by culturing the mesoderm in hypoxic conditions, in a (chondroinductive) culture media comprising: i) a FGF family member, ii) a BMP family member, iii) a Neurotrophin, and iv) a Growth/Differentiation Factor (GDF) family member; and forming a pellet of the chondrocytes and culturing the pellet of the chondrocytes in a culture media under hypoxic conditions to produce the cartilage. The invention further relates to methods of chondrocyte production, synthetically produced cartilage, and use in therapy.

Claims

1. A method for producing cartilage from pluripotent stem cells (PSCs), the method comprising providing chondrocytes by: 1) providing pluripotent stem cells (PSCs); 2) inducing differentiation of the PSCs into a primitive streak/mesendoderm by culturing the PSCs in hypoxic conditions, in a (mesendodermic) culture media comprising: i) a Wingless/Integrated (WNT) family member, ii) an Activin family member, and iii) a Fibroblast Growth Factor (FGF) family member; 3) inducing differentiation of the primitive streak/mesendoderm into a mesoderm by culturing the primitive streak/mesendoderm in hypoxic conditions, in a (mesodermic) culture media comprising: i) a FGF family member, ii) a bone morphogenetic protein (BMP) family member, iii) Follistatin, and iv) a Neurotrophin (NT); and 4) inducing differentiation of the mesoderm into chondrocytes by culturing the mesoderm in hypoxic conditions, in a (chondroinductive) culture media comprising: i) a FGF family member, ii) a BMP family member, iii) a Neurotrophin, and iv) a Growth/Differentiation Factor (GDF) family member; and forming a pellet of the chondrocytes and culturing the pellet of the chondrocytes in a culture media under hypoxic conditions to produce the cartilage.

2. The method according to claim 1, wherein the pellet of a chondrocytes is cultured in culture media under hypoxic conditions on a substrate.

3. The method according to claim 2, wherein the substrate comprises or consists of a porous membrane or cartilage.

4. A method for producing chondrocytes, the method comprising: 1) providing pluripotent stem cells (PSCs); 2) inducing differentiation of the PSCs into a primitive streak/mesendoderm by culturing the PSCs in hypoxic conditions, in a (mesendodermic) culture media comprising: i) a Wingless/Integrated (WNT) family member, ii) an Activin family member, and iii) a Fibroblast Growth Factor (FGF) family member; 3) inducing differentiation of the primitive streak/mesendoderm into a mesoderm by culturing the primitive streak/mesendoderm in hypoxic conditions, in a (mesodermic) culture media comprising: i) a FGF family member, ii) a bone morphogenetic protein (BMP) family member, iii) Follistatin, and iv) a Neurotrophin (NT); and 4) inducing differentiation of the mesoderm into chondrocytes by culturing the mesoderm in hypoxic conditions, in a (chondroinductive) culture media comprising: i) a FGF family member, ii) a BMP family member, iii) a Neurotrophin, and iv) a Growth/Differentiation Factor (GDF) family member.

5. The method according to any one of the preceding claims, wherein the chondroinductive culture media further comprises a TGF-β subfamily member, such as TGF-β.sub.3.

6. The method according to any preceding claim, further comprising a step of deriving a homogeneous population of the chondrocytes by selecting for or filtering the chondrocytes.

7. The method according to claim 3, wherein selecting for the chondrocytes comprising passaging and culturing the chondrocytes on a tissue culture plastic.

8. The method according to any one of claims 4-7, wherein the method further comprises the step of culturing the chondrocytes as a pellet, optionally wherein the chondrocytes are cultured as a pellet under hypoxic conditions.

9. The method according to any one of the preceding claims, wherein the PSCs are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

10. The method according to any one of the preceding claims, further comprising culturing the PSCs in a mesendodermic culture medium over a period of time sufficient to form primitive streak/mesendoderm.

11. The method according to any one of the preceding claims, further comprising culturing the PSCs in a mesendodermic culture media for 2 to 6 days, preferably 3 to 5 days, most preferably 4 days.

12. The method according to anyone of the preceding claims, wherein the step of inducing differentiation of PSCs into a primitive streak/mesendoderm comprises WNT3A, Activin A and FGF2.

13. The method according to any one of the preceding claims, further comprising culturing primitive streak/mesendoderm in a mesodermic culture medium over a period of time sufficient to form mesoderm.

14. The method according to any one of the preceding claims, comprising culturing the primitive streak/mesendoderm in a mesodermic culture media for 3 to 7 days, preferably 4 to 6 days, most preferably 5 days.

15. The method according to any one of the preceding claims, wherein the mesodermic culture media comprises FGF2, Follistatin, BMP4 and NT4.

16. The method according to any one of the preceding claims, wherein the step of inducing differentiation of mesoderm into chondrocytes comprises culturing mesoderm in a chondroinductive culture medium over a period of time sufficient to form chondrocytes.

17. The method according to any one of the preceding claims, wherein the step of inducing differentiation of a mesoderm into chondrocytes comprises culturing the mesoderm in a chondroinductive culture media for about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days or about 11 days in a chondroinductive culture media.

18. The method according to any one of claims 1 to 16, wherein the step of inducing differentiation of a mesoderm into chondrocytes comprises culturing the mesoderm in a chondroinductive media for 4 to 6 days, 4 to 7 days, 4 to 8 days, 4 to 9 days, 4 to 10 days or 4 to 11 days.

19. The method according to any one of the preceding claims, wherein the chondroinductive culture media of the step of inducing differentiation of a mesoderm into chondrocytes comprises FGF2, BMP4, NT4 and GDF5, optionally wherein the chondroinductive culture media further comprises a TGF β family member, such as TGF-β.sub.3.

20. A method of producing cartilage tissue comprising chondrocytes, the method comprising culturing a pellet of chondrocytes in a culture media under hypoxic conditions.

21. A method of producing cartilage tissue comprising chondrocytes, the method comprising culturing a pellet of chondrocytes on a substrate under hypoxic conditions.

22. The method according to claim 20 or 21, further comprising culturing a/the pellet of chondrocytes in a chondrogenic culture media comprising a TGF-β family member, such as TGF-β.sub.3.

23. The method according to claim 21, wherein the substrate is a cartilage extract or a porous membrane.

24. The method according to any one of claims 20 to 23, wherein the pellet of chondrocytes is cultured for at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 19 weeks or at least 26 weeks.

25. A method of repairing or replacing damaged cartilage tissue in a subject, the method comprising implanting in vitro derived cartilage tissue into the subject, wherein the in vitro derived cartilage tissue has been produced by the method according to any one of claims 1 to 3, or 5 to 24.

26. The method according to claim 25, wherein the implantation is within a defect of the subject's natural/native cartilage, or within a surgically prepared excision of the subject's natural/native cartilage.

27. A cartilage tissue comprising chondrocytes at a mean average density of approximately 5-20 chondrocytes/μm.sup.2.

28. A cartilage tissue produced by the method according to any one of claims 1 to 3 or 5 to 24, or a cartilage tissue according to claim 27, for use in therapy.

29. A cartilage tissue for use according to claim 28, wherein therapy comprises treating, restoring or repairing damaged cartilage tissue, or treating or preventing a cartilage disorder, optionally wherein the damage to the damaged cartilage tissue is caused by a cartilage disorder, such as be arthritis, osteoarthritis, chondritis, a partial thickness defect of native cartilage, or a full thickness defect of native cartilage.

Description

[0180] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example only, to the accompanying drawings, in which:—

[0181] FIG. 1 shows immunocytochemistry of hESCs demonstrating OCT4, SOX2, NANOG and TRA-1-60 expression.

[0182] FIG. 2 shows the protocol and growth factors used to induce differentiation of hESCs into primitive streak/mesendoderm, followed by mesoderm and then chondrocytes.

[0183] FIG. 3 shows immunocytochemistry of hESC-derived chondrocytes demonstrating SOX9 expression and the absence of OCT4.

[0184] FIG. 4 shows immunocytochemistry of hESC-derived chondrocytes demonstrating Type II collagen expression in the extracellular matrix.

[0185] FIG. 5 shows a Western Blot for proteins (OCT4, SOX2, NANOG, SOX9, Type II collagen and β-actin (control)) present in hESCs (stage 0) and hESC-derived chondrocytes (stage 3).

[0186] FIG. 6 shows bar charts which quantify the change in expression of OCT4, SOX2, NANOG, SOX9 and Type II collagen in hESCs (at stage 0) and hESC-derived chondrocytes (stage 3). For OCT4, SOX2 and NANOG, data were normalised to β-ACTIN and stage 3 was compared to stage 0, which was set to 1. For SOX9 and Type II collagen, data were normalised to β-ACTIN and stage 0 was compared to stage 3, which was set to 1. Values represent mean±SD. Asterisks indicate a statistically significant difference. *p≤0.05, **p≤0.01, ***p≤0.001 (Student's t-test);

[0187] FIG. 7 is a schematic of the protocol used for pellet culture of hESC-derived chondrocytes.

[0188] FIG. 8 shows photographs of hESC-derived cartilage pellets at 3 weeks, 4 weeks, 5 weeks, 16 weeks and 19 weeks.

[0189] FIG. 9 shows immunohistochemistry of hESC-derived cartilage pellets at 3 weeks, 4 weeks and 5 weeks of culture demonstrating SOX9 and Type II collagen and the absence of Type I collagen and Safranin O staining.

[0190] FIG. 10 shows Safranin O staining in hESC-derived cartilage pellet at 13 weeks (FIG. 10A), 16 weeks (FIG. 10B) and 19 weeks (FIG. 10C) of culture.

[0191] FIG. 11A shows the Young's moduli of human articular cartilage and 4-week hESC-derived cartilagepellets. Values represent mean±SEM. FIG. 11B shows the Young's moduli of human articular cartilage, 4-week and 19-week hESC-derived cartilage constructs (pellets). No significant differences were observed between human articular cartilage and the hESC-derived cartilage constructs. Values represent mean±SEM.

[0192] FIG. 12A shows a piece of full thickness, native human articular cartilage containing a partial thickness defect. FIG. 12B shows a schematic of the organotypic culture model.

[0193] FIG. 13 shows integration of the hESC-derived cartilage with the native human articular cartilage within a partial thickness defect.

[0194] FIG. 14 shows a photograph of hESC-derived cartilage cultured on a piece of native human articular cartilage.

[0195] FIG. 15 shows Safranin O staining of the co-cultured hESC-derived cartilage and native human articular cartilage.

[0196] FIG. 16 shows photographs of hESC-derived cartilage (approximately 4.5 mm diameter) cultured on a Polyethylene Terephthalate (PET) membrane.

[0197] FIG. 17 shows Safranin O staining (FIG. 17A) and Aggrecan immunostaining (FIG. 17B) of hESC-derived cartilage cultured on a PET membrane. Scale bars represent 500 μm, 100 μm and 50 μm.

[0198] FIG. 18 shows Western Blots for proteins (OCT4, SOX2, NANOG, SOX9, Type II collagen and β-ACTIN (control)) present in hiPSCs (stage 0) and hiPSC-derived chondrocytes (stage 3).

[0199] FIG. 19 shows bar charts which quantify the change in expression of OCT4, SOX2, NANOG, SOX9 and Type II collagen in hiPSCs (stage 0) and hiPSC-derived chondrocytes (stage 3). For OCT4, SOX2 and NANOG, data were normalised to β-ACTIN and stage 3 was compared to stage 0, which was set to 1. For SOX9 and Type II collagen, data were normalised to β-ACTIN and stage 0 was compared to stage 3, which was set to 1. Values represent mean±SD. Asterisks indicate a statistically significant difference. *p≤10.05, ***p≤0.001 (Student's t-test).

[0200] FIG. 20 shows immunocytochemistry of hiPSC-derived chondrocytes demonstrating robust SOX9 and Type II collagen expression, and the sporadic expression of OCT4. Scale bars for SOX9 and Type II collagen represent 200 μm and 50 μm; scale bar for OCT4 represents 100 μm.

[0201] FIG. 21 shows immunocytochemistry of hiPSC-derived chondrocytes following culture on tissue culture plastic demonstrating robust SOX9 and Type II collagen expression, and the absence of OCT4 expression. Scale bars represent 50 μm.

EXAMPLES

[0202] Materials and Methods

[0203] ESC Culture and Differentiation

[0204] HUES7 hESCs were cultured under hypoxic conditions (5% O.sub.2, 5% CO.sub.2, and balanced nitrogen) as previously described in Christensen et al., 2015 (Scientific Reports 5 (17500): 1 to 14). A directed differentiation protocol based on that developed by Oldershaw et al. (2010. Nat Biotechnol. November; 28(11):1187-94. doi: 10.1038/nbt.1683) was used to generate hESC-derived chondrocytes. The method used directs cells through the developmental stages of primitive-streak/mesendoerm (day 4), to mesoderm (day 9), and ultimately to chondrocytes (day 14) using the temporal addition of specific growth factor cocktails containing varying concentrations of the following factors: Activin A, WNT3A, FGF2, BMP4, Follistatin, NT4, GDF5, and TGFβ.sub.3. Here the differentiation protocol differs by the inclusion of TGF-β.sub.3 (10 ng/ml) from day 9 onwards, culture on Matrigel (Corning) coated tissue culture plates, and continuous culture under hypoxic conditions (5% O.sub.2 saturation). Cells were also passaged as colonies using collagenase dissociation rather than the trypsinization method described in Oldershaw et al. (2010. Nat Biotechnol. November; 28(11):1187-94. doi: 10.1038/nbt.1683) that used single cell culture. HUES7 hESCs were allowed to reach ˜70% confluency before initiation of differentiation.

[0205] Cartilage Generation Via Pellet Culture hESC-derived chondrocytes were dissociated and resuspended in chondrogenic media (α-MEM (Lonza) supplemented with 10 ng/ml TGF-β.sub.3 (Peprotech), 10 nM dexamethasone (Sigma), 100 μM ascorbate-2-phosphate (Sigma), 0.35 mM L-Proline (Sigma) and 1×ITS supplement (Gibco)) containing 3×10.sup.5 cells per 1 ml medium in a sterile universal tube. The cell suspension was centrifuged at 400 g for 5 minutes. Pellets were resuspended in 1 ml fresh chondrogenic media, and centrifuged as above. Pellets were cultured in a humidified incubator at 5% O.sub.2, 5% CO.sub.2, and balanced nitrogen for either 3, 4, 5, 13, 16 or 19 weeks.

[0206] Organotypic Cartilage Defect Culture

[0207] Near full-thickness articular cartilage pieces (1×1 cm.sup.2, 2 mm thick) were dissected from healthy non load-bearing regions of human femoral heads collected with approval of Southampton and South West Hampshire Research Ethics Committee (Ref. 210/01). A partial thickness defect (˜2×2 mm.sup.2, 1 mm deep) was created in each articular cartilage piece with a sterile drill bit, taking extreme care to avoid full penetration of the cartilage. A single 4-week hESC-derived chondrocyte pellet (the neocartilage graft) was implanted into each defect and the neocartilage graft-host cartilage construct was then placed on a Millipore filter insert and cultured in chondrogenic medium at the air-liquid interface in a humidified atmosphere at 37° C., 5% CO.sub.2 and 5% O.sub.2 for 16 weeks. Pieces of articular cartilage with empty defects cultured for 16 weeks served as controls. The samples were harvested, fixed in 4% paraformaldehyde (PFA) overnight at 4° C. and processed for histological analysis according to Li et al., 2014 (Lab on a Chip 14: 4475-4485).

[0208] Organotypic Co-Culture Model

[0209] Near full-thickness articular cartilage pieces (1×1 cm.sup.2, 2 mm thick) were dissected from healthy non load-bearing regions of human femoral heads. A single 4-week hESC-derived chondrocyte pellet was placed on top of the piece of articular cartilage and co-cultured in chondrogenic medium on a Millipore filter insert at the air-liquid interface in a humidified atmosphere at 37° C., 5% CO.sub.2 and 5% CO.sub.2 for 16 weeks. The sample was harvested, fixed in 4% PFA overnight at 4° C. and processed for histological analysis according to Li et al., 2014.

[0210] Safranin O staining was performed as described previously (Tare et al., 2005 Biochemical and Biophysical Research Communications 333: 609-621).

[0211] Immunocytochemistry

[0212] Samples were analysed for immunocytochemistry as previously described (Christensen et al., 2015). Primary antibodies against OCT4 (Santa Cruz) 1:100, SOX2 (Cell Signalling Technology) 1:200, NANOG (Abcam) 1:100, TRA-1-60 (Santa Cruz) 1:100, SOX9 (Millipore) 1:150, Type II Collagen (Calbiochem) 1:500 were used.

[0213] Immunohistochemistry

[0214] Samples were analysed for immunohistochemistry as previously described (Li et al., 2014). Primary antibodies against SOX9 (1:150), Collagen Type I (1:1000) and Collagen Type II (1:500) were used.

[0215] Western Blotting

[0216] Samples for Western blotting were analysed as previously described (Christensen et al., 2015). Primary antibodies against OCT4 (1:1000), SOX2 (1:3000), NANOG (1:500) and SOX9 (1:850), Type II Collagen (1:1000), β-actin (Sigma) 1:50,000 were used.

[0217] Mechanical Testing

[0218] A custom-built mechanical testing rig was used to compress samples between two flat metal plates. The device generated force and displacement readings used to determine the Young's elastic modulus (E) for each sample. For native cartilage, 5 mm.sup.2 samples of full thickness articular cartilage were harvested from the non-load bearing region of the femoral head. hESC-derived chondrocyte pellets were tested following 4 or 19 weeks culture.

Example 1—Characterisation of Human Embryonic Stem Cells (hESCs) by Immunocytochemistry

[0219] The hESCs, which would be used to create chondrocytes, were analysed. It was confirmed that they express the biomarkers OCT4, SOX2, NANOG and TRA-1-60. Thus they are deemed to be hESCs.

Example 2—Protocol for the Production of hESC-Derived Chondrocytes

[0220] In order to differentiate the pluripotent, hESCs into chondrocytes they were temporarily cultured in media comprising three different mixtures of growth factors (see FIG. 2).

[0221] The first mixture was used to create a mesendodermic culture media. It comprises WNT3A, Activin A and FGF2, and was used to differentiate the hESCs (stage 0) into primitive streak/mesendoderm (stage 1).

[0222] After 4 days of culture (day 0 to day 4), the first mixture of growth factors was replaced by a second mixture, which was used to create a mesodermic culture media. The second mixture comprises FGF2, BMP4, Follistatin and NT4, and was used to differentiate the primitive streak/mesendoderm (stage 1) into a mesoderm (stage 2).

[0223] After 5 days of culture (day 4 to day 9), the second mixture was replaced with a third mixture, which was used to create a chondroinductive culture media. The third mixture comprises FGF2, BMP4, NT4, GDF5 and TGF-β3, and was used to differentiate the mesoderm (stage 2) into chondrocytes (stage 3).

Example 3—Characterisation of hESC-Derived Chondrocytes

[0224] In order to confirm that the hESCs have differentiated into chondrocytes, the stage 3 cells were analysed (by immunohistochemistry) to determine if they express the chondrogenic transcription factor, SOX9. It was found that the hESC-derived chondrocytes do express SOX9 but do not express, OCT4, which is a key pluripotency transcription factor and marker of hESCs (see FIG. 3). Moreover, the chondrocytes synthesise an extracellular matrix that is rich in Type II collagen, a cartilage specific collagen (see FIG. 4).

[0225] These results above were confirmed by Western Blot (see FIG. 5) and quantified in FIG. 6. The Western Blots also confirmed that the stage 3 cells have negligible expression of OCT4, SOX2 and NANOG proteins, while SOX9 and Type II collagen are significantly increased. β-ACTIN expression was used as a control. It is present in hESCs (stage 0) and hESC-derived chondrocytes (stage 3).

[0226] These results confirm that the stage 3 cells are no longer hESCs, and that they are in fact chondrocytes.

Example 4—Schematic Representation of the Method of Pellet Culture

[0227] A schematic representation of the protocol used to culture a mass of hESC-derived chondrocytes is provided in FIG. 7.

[0228] Step 1—provide a suspension of the stage 3 hESC-derived chondrocytes in a vessel with a pointed base.

[0229] Steps 2 and 3—centrifuge the chondrocytes so that they form a pellet in the pointed base of the relevant vessel.

[0230] Step 4—remove the media in which the cells were suspended in order leave behind the pellet of cells.

[0231] Step 5—culture the pellet of cells in chondrogenic medium, under hypoxic conditions for approximately 21 days.

Example 5—Photographs of hESC-Derived Cartilage Pellets

[0232] It has been found that 3D cartilage tissue is produced by a pellet of hESC-derived chondrocytes after at least three weeks in culture under hypoxic conditions. hESC-derived cartilage pellets cultured for 3 weeks, 4 weeks, 5 weeks have a diameter of approximately 1 mm, while pellets cultured for 16 weeks and 19 weeks have a diameter of approximately 3 mm (see FIG. 8).

Example 6—Characterisation of hESC-Derived Cartilage Pellets

[0233] hESC-derived cartilage pellets were analysed after 3 weeks, 4 weeks and 5 weeks of culture. The pellets express SOX9 and Type II collagen, which indicates the presence of cartilage (FIG. 9). However, the pellets do not express Type I collagen or Safranin O staining. FIG. 10 shows Safranin O staining of hESC-derived cartilage pellet at 13 weeks (FIG. 10A), 16 weeks (FIG. 10B) and 19 weeks (FIG. 10C) of culture. At 19 weeks the hESC-derived cartilage pellet displays robust Safranin O staining and typical hyaline cartilage morphology (chondrocytes in lacunae embedded in dense extracellular matrix).

Example 7—Measurement of Biomechanical Properties of Full Thickness Articular Human Cartilage, 4-Week and 19-Week hESC-Derived Cartilage Pellets (Constructs)

[0234] The Young's moduli were determined in human articular cartilage, 4-week and 19-week hESC-derived cartilage pellets (constructs). The average value for the Young's modulus of full thickness articular human cartilage is comparable to that of 4-week and 19-week hESC-derived cartilage pellets (see FIG. 11A and FIG. 11B).

Example 9—Organotypic Culture System to Model Repair of Partial Thickness Defect in Human Articular Cartilage

[0235] FIG. 12A shows a piece of full thickness, native human articular cartilage containing a partial thickness defect. The hESC-derived cartilage construct was placed in the partial thickness defect created in a piece of full thickness native human articular cartilage. This was then placed on a tissue culture insert and cultured at the air liquid interface (for 16 weeks), in chondrogenic medium (FIG. 12B). The hESC-derived cartilage integrated into the host native cartilage and contributed to the repair of the partial thickness defect (see FIG. 13).

Example 10—Scale Up of hESC-Derived Cartilage Generation

[0236] FIG. 14 shows an approximately 1 cm.sup.2 piece of hESC-derived cartilage generated by culturing a 1 mm 4-week hESC-derived cartilage pellet on full thickness native human articular cartilage in chondrogenic medium for 16 weeks.

Example 11—Safranin O Staining of the Co-Cultured hESC-Derived Cartilage and Native Human Articular Cartilage

[0237] Robust Safranin O staining is present in both the scaled-up hESC-derived cartilage and the native human articular cartilage. The hESC-derived cartilage exhibits typical hyaline cartilage morphology comprising of chondrocytes in lacunae embedded in dense extracellular matrix (see FIG. 15).

Example 12—Photographs of hESC-Derived Cartilage Cultured on a Polyethylene Terephthalate (PET) Membrane

[0238] FIG. 16 shows an approximately 4.5 mm diameter hESC-derived cartilage construct generated by culturing a 4-week hESC-derived cartilage pellet on a PET membrane in chondrogenic medium for 16 weeks. Robust Safranin O staining (FIG. 17A) and Aggrecan expression (FIG. 17B) is present in the hESC-derived cartilage construct. The hESC-derived cartilage construct exhibits typical hyaline cartilage morphology comprising of chondrocytes in lacunae embedded in dense extracellular matrix (see FIGS. 17A and B).

Example 13—Characterisation of hiPSC-Derived Chondrocytes

[0239] Chondrocytes have been generated from human induced pluripotent stem cells (hiPSCs). The NIBSC-8 hiPSC cell line was used, but other hiPSC lines may be used. hiPSCs were cultured on a substrate, vitronectin, in Essential 8 medium under hypoxic conditions (5% O.sub.2, 5% CO.sub.2, and balanced nitrogen). hiPSCs were differentiated into hiPSC-derived chondrocytes on vitronectin-coated plates using a directed differentiation protocol based on that used to generate hESC-derived chondrocytes. The method used directs cells through the developmental stages of primitive-streak/mesendoerm (day 4), to mesoderm (day 9), and ultimately to chondrocytes (day 14) using the temporal addition of specific growth factor cocktails containing varying concentrations of the following factors: Activin A, WNT3A, FGF2, BMP4, Follistatin, NT4, GDF5, and TGFβ3.

[0240] In order to confirm that the hiPSCs have differentiated into chondrocytes, the stage 3 cells were analysed for OCT4, SOX2, NANOG, SOX9 and Type II collagen by Western blotting (FIG. 18). Quantification of the Western blots confirmed that the stage 3 cells have negligible expression of OCT4, SOX2 and NANOG proteins, while expression of SOX9 and Type II collagen increased significantly (FIG. 19). β-ACTIN expression was used as a control. It is present in hiPSCs (stage 0) and hiPSC-derived chondrocytes (stage 3).

[0241] Immunocytochemistry was used to investigate the expression of SOX9, Type II collagen and OCT4 in hiPSC-derived chondrocytes. Robust expression of SOX9 and Type II collagen was observed in the hiPSC-derived chondrocytes. However, sporadic expression of OCT4 was also observed (FIG. 20).

[0242] To eliminate OCT4 expression, hiPSC-derived chondrocytes on day 14 were passaged onto tissue culture plastic and cultured under hypoxic conditions in chondroinductive medium for a further 3 days. Immunocytochemistry of hiPSC-derived chondrocytes (day 17) demonstrated absence of OCT4 protein expression, while expression of SOX9 and Type II collagen persisted (FIG. 21). This demonstrates the generation of a robust, homogeneous population of hiPSC-derived chondrocytes.

[0243] PSC Growth Medium Examples

[0244] mTESR™ Comprises:

[0245] DMEM/F12; L-ascorbic acid; Selenium; Transferrin; NaHCO.sub.3; Insulin; FGF2; TGFB1; Albumin (BSA); Glutathione; L-glutamine; Defined lipids; Thiamine; Trace Elements B; Trace Elements C; Beta-mercaptoethanol; Pipecolic acid; LiCl; GABA; and H.sub.2O.

[0246] Essential 8™ Comprises:

[0247] DMEM/F12; L-ascorbic acid; Selenium; Transferrin; NaHCO.sub.3; Insulin; FGF2; and TGFB1.

[0248] All references described herein are incorporated by reference.