Articles

Generation of articular chondrocytes from human pluripotent stem cells

© 2015 Nature America, Inc. All rights reserved.

April M Craft1,2,6, Jason S Rockel3, Yulia Nartiss1,2, Rita A Kandel4, Benjamin A Alman3,6 & Gordon M Keller1,2,5 The replacement of articular cartilage through transplantation of chondrogenic cells or preformed cartilage tissue represents a potential new avenue for the treatment of degenerative joint diseases. Although many studies have described differentiation of human pluripotent stem cells (hPSCs) to the chondrogenic lineage, the generation of chondrocytes able to produce stable articular cartilage in vivo has not been demonstrated. Here we show that activation of the TGFb pathway in hPSC-derived chondrogenic progenitors promotes the efficient development of articular chondrocytes that can form stable cartilage tissue in vitro and in vivo. In contrast, chondrocytes specified by BMP4 signaling display characteristics of hypertrophy and give rise to cartilage tissues that initiate the endochondral ossification process in vivo. These findings provide a simple serum-free and efficient approach for the routine generation of hPSC-derived articular chondrocytes for modeling diseases of the joint and developing cell therapy approaches to treat them. The hyaline articular cartilage that lines our synovial joints is generated from and maintained throughout adult life by a subpopulation of chondrocytes known as articular chondrocytes1–3. These chondrocytes are distinct from growth plate chondrocytes, which function to produce cartilage scaffolds for new bone formation. In debilitating joint diseases such as osteoarthritis, degeneration of articular cartilage leads to inappropriate activation of signaling pathways that can result in hypertrophy and apoptosis of articular chondrocytes and the appearance of osteophytes (bone spurs). As damaged articular cartilage has little capacity to regenerate itself, replacement therapies are the only means of treating patients with advanced osteoarthritis. Replacement of articular chondrocytes or articular cartilage with cells or tissues generated in vitro offers an alternative strategy to whole joint replacement, the current method of choice. Although adult mesenchymal stem cells can generate chondrocytes in vitro, most evidence indicates that these cells undergo premature hypertrophy after transplantation in vivo, characteristic of a growth plate chondrocyte fate4–6. hPSCs may provide an alternative source of chondrocytes as advances in our understanding of developmental biology in vitro have led to efficient strategies for producing a range of different cell types, including those of the chondrogenic lineage7–11. However, to our knowledge none of these studies has demonstrated the generation of articular chondrocytes. To derive this subpopulation of chondrocytes from hPSCs, we sought to recapitulate in vitro the sequence of events that lead to their development in early fetal life. Lineage tracing studies in the early embryo have provided compelling evidence that the two types of chondrocytes have different developmental origins and that articular chondrocytes derive from synovial

joint progenitors known as interzone cells, which are specified in regions of condensing chondrogenic mesenchyme12. These interzone progenitors also contribute to the meniscus and ligaments, but not to growth plate chondrocytes. The development of interzone progenitors is regulated in part by TGFβ signaling13–15, as joints do not develop in TGFβR2 conditional knockout mice. In this study we show that sustained TGFβ signaling in hPSC-derived mesenchymal progenitors specifies a population that initially expresses markers indicative of interzone cells and subsequently of articular chondrocyte development. The TGFβ-treated cells form stable articular cartilage–like tissue in vitro and in immunodeficient mice. In contrast, BMP4 promotes the development of chondrocytes that generate cartilage tissue composed of hypertrophic chondrocytes capable of initiating endochondral ossification in vivo. These findings demonstrate that, through an appropriately staged protocol, it is possible to efficiently and reproducibly generate articular chondrocyte–like cells that can be used to test the feasibility of chondrocyte and/or tissue transplantation for the repair of damaged articular cartilage and to develop models of cartilage degeneration and disease for drug screening. RESULTS Generation of chondrogenic mesoderm Our protocol was designed to recapitulate the key events that regulate cartilage formation during embryonic development (Fig. 1a). A primitive streak/early mesoderm population, characterized by the expression of CD56 (NCAM)16, PDGFRα17,18 and KDR18, was induced during the first four days of culture of the HES2 human embryonic cell (hESC) line (stage 1) by treatment of embryoid bodies

1McEwen

Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada. 2Princess Margaret Cancer Centre, Toronto, Ontario, Canada. and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada. 4Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. 5Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. 6Present addresses: Orthopedic Research Laboratories, Department of Orthopedic Surgery, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA (A.M.C.) and Division of Orthopedic Surgery, Duke University Medical Center, Durham, North Carolina, USA (B.A.A.). Correspondence should be addressed to A.M.C. ([email protected]) or G.M.K. ([email protected]). 3Developmental

Received 8 November 2014; accepted 25 March 2015; published online 11 May 2015; doi:10.1038/nbt.3210

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Figure 1  Generation of paraxial mesoderm and chondrocyte progenitors from hPSCs. (a) Schematic representation of the protocol used for chondrogenic differentiation of hPSCs. The protocol consists of four stages: (1) induction of a primitive streak-like mesoderm population from days 1–4 as embryoid bodies; (2) specification to a paraxial mesoderm fate in monolayer culture by treatment with DM, days 4–6 and bFGF, days 4–15; (3) generation of chondrocyte progenitors in a high-density micromass culture in the presence of TGFβ3; and (4) specification to the articular chondrocyte or hypertrophic chondrocyte fate. (b) Flow cytometric analyses showing the proportion of CD56 +PDGFRα+ and KDR+PDGFRα+ cells in day 4 embryoid bodies induced with the combination of activin A (2 ng/ml), BMP4 (3 ng/ml) and bFGF (5 ng/ml). (c–h) qRT-PCR–based expression analyses of the indicated genes in day 6 (c,d) and day 15 (e–h) populations. Day 4 embryoid body–derived cells were treated for 48 h with no additional factors (mock), DM (4 µM) or bFGF (10 ng/ml), as indicated. Box plots depict the copy number of mRNA relative to TBP (n = 4). Error bars, mean ± s.d. (i) Flow cytometric analysis showing the proportion of CD73 +, CD105+ and PDGFRβ+ cells in the day-15 populations. Representative contour plots are shown. (j) qRT-PCR–based expression analyses of SOX9 and COL2A1 in micromass cultures after 1 week (n = 3). (k) Intracellular flow cytometric analysis of the proportion of cardiac troponin T+ cardiomyocytes in micromass cultures (1 week) generated from day-15 populations specified as indicated (n = 9). Significance compared to DM+FGF was calculated by Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars, mean ± s.d.

with the combination of activin A, BMP and basic (b)FGF (FGF2; Fig. 1b). At day 4, the embryoid bodies were dissociated and the cells cultured as a monolayer in the presence of the small-molecule inhibitor of type I BMP receptors, dorsomorphin (DM), bFGF or both (DM+FGF, stage 2) for 48 h to specify a paraxial mesoderm fate. Stage-specific inhibition of the BMP pathway is known to be required to specify this mesoderm subpopulation9,17,19. Next, the cells 

were maintained in bFGF for 9 days to promote the development of mesenchymal-like progenitors, then harvested and cultured in highdensity micromasses to initiate chondrogenesis. Within 2 days of monolayer culture, both nontreated (mock) and FGF-treated cells upregulated the lateral plate mesoderm transcription factor HAND1, whereas treatment with DM in the presence or absence of FGF resulted in the upregulation of the paraxial advance online publication  nature biotechnology

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Figure 2  TGFβ3 and BMP4 specify distinct TGFβ3 BMP4 TGFβ3 BMP4 populations of chondrocytes and cartilage TGFβ3 BMP4 tissues. (a) Photomicrographs of chondrocytes generated after 4 weeks of culture in TGFβ3 or BMP4. Both populations were generated in micromass cultures initiated in the presence of FSC-A FSC-A FSC-A TGFβ3 (10 ng/ml) with cells from 15-day-old TGFβ3 BMP4 monolayers. Ten days later, TGFβ3 was replaced 0 0.20027599 0.5298697 with BMP4 (50 ng/ml) in some of the cultures. hESC-derived Primary Fetal chondrocyte-derived (b) Flow cytometric analysis of forward light micromass human micromass (FSC-A; cell size) and side light scatter (SSC-A; cells TGFβ3 TGFβ3 BMP4 BMP4 cell granularity) parameters of hESC-derived chondrocytes following 5 weeks of micromass Weeks 0 1 2 3 4 6 8 1012 2 3 4 6 8 1012 1 4 6 8 12 4 6 8 12 culture. (c–e) Histological analyses (toluidine SOX9 COL2A1 blue) staining of cartilage tissues derived from ACAN hESCs after 8 weeks (c), hiPSCs after RUNX2 SP7 12 weeks (d) or primary fetal chondrocytes after ALP COL10A1 6 weeks (e) of culture in the presence of TGFβ3 GLI1 PTHLH or BMP4 as indicated. (f) Heat map showing PRG4 CILP2 expression levels of genes in hESC-derived COL1A1 COL22A1 chondrocytes (stage 3 and 4 of differentiation, CCND2 n ≥ 4 biological replicates) and primary fetal GDF5 ERG chondrocytes cultured as micromass (n = 6 from WNT9A SOSTDC1 two independently isolated fetal chondrocyte DCX populations), in the presence of TGFβ3 or BMP4 for the indicated period of time. Normalized values represent copy number mRNA relative to TBP and are compared to primary fetal chondrocytes (aged 16–19 weeks, n = 4), primary healthy adult articular chondrocytes (n = 2), and iliac crest (growth plate–like) chondrocytes isolated from a pediatric source (n = 1). Gray bars indicate no data were obtained due to unavailability of primary tissue sample. Graphs showing levels of expression are shown in Supplementary Figures 5 and 7. AC, articular chondrocytes. Scale bars for a 100 µm; c–e 200 µm. Fetal chondrocytederived, 6 weeks

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mesoderm–specific transcription factor TCF15 (PARAXIS) (Fig. 1c,d). Cells treated with both DM and FGF expressed higher levels of TCF15 (P = 0.011) and significantly lower levels of HAND1 (P = 0.033) than nontreated (mock) cells. By day 15, the population treated with DM+FGF upregulated the expression of paraxial mesoderm and somite-specific transcription factors MEOX1, PAX1 and NKX3.2, whereas those treated with only DM or FGF expressed these genes at significantly lower levels (P = 0.0008, 0.018, 0.013, respectively; Fig. 1e–g). The mock-treated population and the one treated with FGF alone upregulated the cardiac transcription factor NKX2.5 (Fig. 1h). Consistent with these gene expression patterns, the majority of the day-15 DM+FGF-treated cells co-expressed the surface markers CD73 (ecto-5′-nucleotidase), CD105 (endoglin) and PDGFRβ, patterns indicative of the emergence of mesenchymal-like cells (Fig. 1i). The population treated with FGF alone also contained CD73+CD105+ and CD105+PDGFRβ+ cells, but the proportion was lower than in the DM+FGF–treated population. A small number (5–10%) of endothelial cells expressing CD31 (PECAM) developed in all conditions. To promote chondrogenesis, we cultured day-15 mesoderm populations in micromasses in the presence of TGFβ3 (stage 3), conditions previously shown to promote the development of chondrocytes from limb bud mesenchyme (chick, mouse) and human mesenchymal stem cells20,21. After 1 week of micromass culture, DM+FGF–treated cells appeared to be undergoing chondrogenic differentiation as they expressed significantly higher levels of SOX9, a chondrogenic transcriptional regulator, and COL2A1, a major component of cartilage extracellular matrix (ECM), than the control population or populations treated with either DM or FGF alone (Fig. 1j). The control cells and FGF-treated cells generated troponin T (cTnT)-positive cardiomyocytes under these conditions, a fate consistent with their expression of NKX2.5 on day 15 (Fig. 1k). Taken together, these findings indicate that activation of bFGF signaling and concomitant inhibition of the BMP pathway are required for specification of paraxial/chondrogenic mesoderm. nature biotechnology  advance online publication

The generation of the CD73 +CD105+PDGFRβ+ population from CD56+KDR+PDGFRα+ primitive streak/early mesoderm induced from a second human embryonic cell (hESC) line (H7) and two human induced pluripotent stem cell (hiPSC) lines was also dependent on the combined effects of bFGF and DM (Supplementary Figs. 1–3; H7, iBJ and MSC-IPS1/Y2-1). Specification of articular and hypertrophic chondrocytes Previous studies have used either BMP agonists or sequential or concurrent activation of both the TGFβ and BMP pathways to generate chondrocytes from hPSCs8,9. As BMP signaling is known to induce hypertrophy in chondrocytes17,22,23, we investigated the effect of maintaining the micromass cultures in TGFβ3 without the addition of BMP4 and compared the resulting tissue to that generated by sequential TGFβ3 and BMP4 treatment. Although both micromass culture conditions promoted tissue development by 4 weeks, the morphology of the cells in the two types of tissue differed. Cells in the TGFβ3-induced tissue were small and fibroblastic, whereas those in the tissue induced with BMP4 were larger and arranged in a cobblestone pattern, similar to chondrocytes previously generated in culture 17 (Fig. 2a). Flow cytometric analysis confirmed the difference in size, as the BMP4-treated cells had higher forward light scatter than those cultured in TGFβ3 (Fig. 2b). Eight-week-old tissue generated under both conditions stained metachromatically with toluidine blue, indicative of a proteoglycan-rich, cartilage-like ECM. The differences in cell size detected at the early time point were also observed in the 8-week-old tissue (Fig. 2c). Similar differences in cell size and morphology were observed in micromass tissues generated from several additional hPSC lines and in 6-week-old tissues generated from fetal chondrocytes (Fig. 2d,e and Supplementary Figs. 3f,g, 4a,b, 5a). Flow cytometric analysis showed that the majority of cells in the day-15 population used to generate the micromass cultures expressed CD73, CD105 and PDGFRβ. To formally demonstrate that the hPSC-derived population which expressed these markers initiated 

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Figure 3  Comparison of hESC-derived cartilage tissues and the developing fetal femur. Sections of the human fetal femur at 19 weeks of gestation (left column) illustrate two anatomical locations, the developing articular surface and the growth plate containing the bone collar, hypertrophic chondrocytes and hematopoietic cells located near the primary ossification center. hESC-derived cartilage tissues derived in the presence of TGFβ3 or BMP4 were analyzed histologically after 12 weeks for the presence of extracellular matrix components (right column). (a,b) Metachromatic toluidine blue staining of both the fetal femur and hESC-derived tissues indicate the presence of proteoglycans and illustrate the size of chondrocytes present in each tissue. (c,d) Type II collagen was detected in the fetal articular cartilage and in the growth plate, as well as both hESC-derived cartilage tissues. (e–h) Both lubricin (e,f) and type I collagen (g,h) were present at the surface layers of the fetal articular cartilage and the hESC-derived cartilage tissue derived in the presence of TGFβ3, but absent from BMP4-treated tissue. Lubricin was not found in the growth plate; however, type I collagen was abundant in the bone collar near the fetal growth plate (g). (i,j) Type X collagen was detected only in the fetal growth plate regions containing hypertrophic chondrocytes. (k,l) Control. Scale bars (a–l), 100 µm.

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the observed chondrogenesis, we isolated the day-15 CD73+CD105+ and CD73−CD105− and CD73+PDGFRβ+ and CD73−PDGFRβ− fractions and cultured the cells in micromass format (Supplementary Fig. 6a). Both the CD73+CD105+ and CD73+PDGFRβ+ k fractions generated chondrocytes within 2 weeks of culture (Supplementary Fig. 6b) and visible tissue by 5 weeks (Supplementary Fig. 6c). In contrast, neither of the negative fractions displayed any chondrogenic potential. These findings show that mesenchymal-like progenitors with chondrogenic potential generated from hPSC-derived paraxial mesoderm express CD73, CD105 and PDGFRβ. The tissue generated under the two conditions was next analyzed by qRT-PCR (Fig. 2f and Supplementary Fig. 7) and immunohistochemistry (Fig. 3). SOX9, COL2A1 and aggrecan (ACAN), genes expressed by both articular and hypertrophic chondrocytes, were upregulated by 2 weeks of culture in both TGFβ3- and BMP4-treated tissues. The levels of expression were similar to those found in primary human fetal articular chondrocytes, healthy adult articular chondrocytes and iliac crest (hypertrophic) chondrocytes. Expression of genes associated with hypertrophic chondrocytes, including RUNX2, SP7, alkaline phosphatase (ALP/ALPL) and COL10A1, was significantly higher in the 8- to 12-week-old, BMP4-treated tissue than in the tissue maintained in TGFβ3. GLI1, a transcription factor downstream of the hedgehog signaling pathway, which is active in growth plate chondrocytes in vivo24, was also expressed at higher levels in BMP4-treated than in the TGFβ3-treated chondrocytes. The reverse pattern was observed for genes known to be expressed by articular chondrocytes within the superficial zone of mature articular

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cartilage, including lubricin (PRG4), cartilage intermediate layer protein 2 (CILP2)25, COL1A1, COL22A1 (ref. 26) and parathyroidrelated protein (PTHLH), as well as for genes expressed in joint interzone cells, the progenitors of articular chondrocytes12, such as GDF5 (ref. 12), WNT9A1,3, ERG27, SOSTDC1 (ref. 28) and DCX29. The TGFβ3-treated chondrocytes expressed higher levels of cyclin D2 (CCND2) than those cultured in the presence of BMP4, indicative of a higher level of proliferative activity. Analysis of total cell numbers supports this interpretation as the 4- to 10-week-old tissue initiated with TGFβ3 contained significantly higher (P = 0.0020) numbers of cells than comparable aged tissue induced with BMP4 (Supplementary Fig. 8a). The BMP4-induced tissue had a larger proportion of annexin V– positive cells, an indication of apoptosis and a hallmark of terminal hypertrophic chondrocyte differentiation (Supplementary Fig. 8b). Based on these numbers, we estimate that we were able to generate ~10.86 ± 0.85 articular chondrocytes for each day-15 mesoderm cell plated in micromass culture. Our protocol gave rise to ~4.44 ± 1.20 day-15 mesoderm cells per input hESC, providing an overall yield of 48.22 ± 15.79 articular chondrocytes per input hESC. advance online publication  nature biotechnology

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Similar expression patterns were observed in cartilage tissues derived from H7 hESCs, hiPSCs and primary fetal chondrocytes (Fig. 2f and Supplementary Figs. 3–5,9). Several exceptions were, however, detected in the primary fetal chondrocyte–derived tissue, including the patterns of SOSTDC1 and DCX, which appear to be the reverse of that in the hPSC-derived population, and of COL10A1, which was upregulated in the presence of both TGFβ3 and BMP4. These differences may be due to differences in the developmental stage of the cells at the initiation of the micromass culture, and/or contamination of the fetal cell population with chondrocytes from both the future articular and growth plate sites. To further investigate the regulation of the hPSC-derived articular chondrocyte–like population, we treated micromass cultures with other TGFβ family member ligands, including TGFβ1 and TGFβ2 or GDF5, which we have previously shown to promote an articular chondrocyte fate from mouse PSC-derived paraxial mesoderm17. Both TGFβ1 and TGFβ2 induced expression of COL2A1, PRG4 and CILP2 to levels similar to that of TGFβ3 in 12-week-old tissue, indicating that both can specify the articular chondrocyte fate (Supplementary Fig. 10). In contrast, GDF5 alone or together with inhibitors of the BMP and Hedgehog pathways (G1aC), a combination that promoted optimal articular chondrocyte development in the mouse ESC model, did not induce significant upregulation of PRG4 or CILP2, despite the ability of these factors to support the development of cartilage tissues that expressed SOX9 and COL2A1 (Supplementary Fig. 11). We next compared the expression and distribution of ECM proteins in the hPSC-derived cartilage tissues to those found at the future articular surface and growth plate of the developing human fetal femur at ~19 weeks of gestation (Fig. 3). Type II collagen protein was present in the fetal femur and in hPSC-derived tissues generated under both conditions, as expected (Fig. 3c,d). In contrast, lubricin was detected in TGFβ3- but not in the BMP4-treated micromass tissue and was found preferentially in the flattened cells that line the top of the tissue structure, a pattern similar to the staining of the articular surface of the fetal femur (Fig. 3e,f). Type I collagen was present at the bone collar region of the fetal femur, consistent with its known expression in bone, as well as at the developing articular surface (Fig. 3g). Type I collagen was also expressed at the surface of TGFβ3-induced BMP4

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Figure 4  hPSC-derived chondrocytes generate cartilage tissue in vivo. Cells (500,000) from micromass tissues (aged 8–12 weeks) generated in the presence of either TGFβ3 or BMP4 were injected subcutaneously into immunodeficient mice. Grafts were harvested and analyzed histologically after 4 weeks. (a–d) Sections were stained with toluidine blue (a,c) and safranin O (d) to indicate the presence of proteoglycans and von Kossa (b) to identify areas of mineralization, of which there are none at this time. (e–h) Types II, X and I collagen and lubricin were detected immunohistochemically. Type II collagen was detected throughout grafts derived from both chondrocyte populations. Type I collagen and lubricin were detected at the periphery of grafts derived from TGFβ3treated chondrocytes (arrowheads, upper left corners). Low levels of type X and type I collagen were present throughout the grafts derived from BMP4-treated chondrocytes. Scale bars (a,b), 500 µm; (c–h), 100 µm.

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tissue (Fig. 3h). These expression patterns may be indicative of a fetal-like stage of articular chondrocyte development30. Type X collagen was detected at the periphery of hypertrophic chondrocytes in the fetal growth plate, but was not found at the articular surface or in either hPSC-derived tissues at this time point (Fig. 3i,j). Taken together, these findings provide strong support for the interpretation that sustained TGFβ3 signaling promotes the development of articular chondrocytes that can generate articular cartilage–like tissue, whereas BMP4 signaling induces the differentiation of hypertrophic growth plate–like chondrocytes that do not express markers found in fetal articular cartilage. To further characterize the potential of the two types of chondrocytes, we injected cells from dissociated 8- to 12-week-old tissue subcutaneously into NSG (nonobese diabetic/severe combined immune deficient/ IL2 receptor gamma chain deficient) mice. Both populations generated proteoglycan-rich cartilage tissue that expressed type II collagen with no evidence of mineralization by 4 weeks after transplantation (Fig. 4a–e). Differences in size between TGFβ3- and BMP4-derived chondrocytes were evident in the in vivo–generated tissue at this early stage (Fig. 4c). Type I collagen and lubricin were detected in peripheral regions of grafts derived from TGFβ3-treated chondrocytes (Fig. 4g,h, arrowheads), similar to the staining pattern found in the tissue in vitro. Low levels of type X and type I collagen, but not lubricin, were present throughout the grafts derived from BMP4-treated chondrocytes (Fig. 4f–h). Distinct differences between the grafts were observed at 12 weeks after transplantation. Tissues derived from BMP4-treated chondrocytes contained areas of reduced proteoglycan content, calcification and/or mineralization and hypertrophy, as revealed by positive von Kossa and type X collagen staining, respectively (Fig. 5a–c,f). All grafts derived from BMP4-treated chondrocytes contained regions of calcified cartilage that displayed high levels of types I, II and X collagen (Fig. 5e–g). Lubricin was not detected in any of these grafts at this stage (Fig. 5h). One of the grafts displayed obvious progression toward endochondral ossification, noted by the presence of 

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Figure 5  hPSC-derived chondrocytes maintain articular and hypertrophic phenotypes in vivo. Cells (500,000) from micromass tissues (aged 8–12 weeks) generated in the presence of either TGFβ3 or BMP4 were injected subcutaneously into immunodeficient mice. Grafts were harvested and analyzed histologically after 12 weeks. Two representative grafts derived from BMP4-treated chondrocytes are shown, one depicting areas of calcified cartilage (n = 5) and the other depicting areas undergoing endochondral ossification (n = 1). (a–d) Sections were stained with toluidine blue (a,c; purple staining) and safranin O (d, orange staining) to indicate the presence of proteoglycans. Positive von Kossa (b, brown/ black staining) identified areas of calcified cartilage and/or mineralization, both present in grafts derived from BMP4-treated chondrocytes only. (e–h) Types II, X and I collagen and lubricin were detected immunohistochemically. Type II collagen was detected in cartilaginous regions of grafts derived from both chondrocyte populations. Type I collagen and low levels of lubricin were detected at the periphery of grafts derived from TGFβ3-treated chondrocytes, whereas type X collagen was absent. Both type X and type I collagen were detected in the calcified cartilage and mineralized regions of grafts derived from BMP4-treated chondrocytes, whereas lubricin was absent. Scale bars (a,b), 500 µm; (c–h), 100 µm.

bone ossicles and hematopoietic cells (Fig. 5, BMP4 graft 2, Supplementary Fig. 12). The remaining grafts derived from BMP4-treated cells showed evidence of vascularization, demonstrated by the presence of CD31positive endothelial cells at the periphery. Low numbers of TRAP-positive cells were also detected in most of these grafts, indicative of the presence of bone-remodeling osteoclasts (Supplementary Fig. 13). Thus, 12-week grafts derived from BMP4-treated cells had initiated endochondral ossification, which likely would have progressed given additional time in vivo. In contrast, grafts from the TGFβ3-treated chondrocytes maintained a proteoglycan- and type II collagen–rich ECM (Fig. 5a,c–e) with no evidence of calcification or mineralization, hypertrophy or type X collagen expression (Fig. 5b–d,f). Type I collagen staining remained robust at the periphery of grafts derived from TGFβ3-treated chondrocytes; however, the relative abundance of lubricin at the protein level was lower in 12-week grafts compared to 4-week grafts (Fig. 5g,h). Furthermore, vascularization was not evident and TRAP+ cells were not detected in any of these grafts (Supplementary Fig. 13), indicating that this tissue persists as stable cartilage for up to 12 weeks in vivo. The findings from these transplantation studies demonstrate that the two chondrocyte populations are functionally distinct and provide additional evidence that the TGFβ3-treated cells represent articular chondrocytes as they generate and maintain stable cartilage for over 12 weeks in vivo. BMP4-graft 2

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Modeling cartilage disease using hPSC-derived tissues Access to an unlimited supply of hPSC-derived, articular-like cartilage provides an opportunity to analyze the effects of pro-inflammatory cytokines, 

such as interleukin-1β (IL1β), and other molecules known to play a role in the early stages of osteoarthritis31,32. Treatment of 10-week-old hPSC-derived articular chondrocytes with IL1β in the absence of TGFβ3 for 2 weeks (Fig. 6a) resulted in significant upregulation of expression of catabolic enzymes including matrix metallopeptidase 13 (MMP13, P = 0.0006) (Fig. 6b) and ADAMTS4 (P = 0.0016) and ADAMTS5 (P = 0.0001) (Fig. 6c,d). The upregulation of MMP13 and ADAMTS4 was observed only in the absence of TGFβ3, whereas ADAMTS5 was induced with or without TGFβ3 present. Notably, the removal of TGFβ3 alone resulted in a significant (P < 0.0001) loss of PRG4 and CILP2 expression (Fig. 6e,f), although levels were also downregulated when IL1β was added to tissues in the presence of TGFβ3. IL1β treatment led to a reduction in COL2A1 and ACAN expression (Fig. 6g,h), an increase in vascular endothelial growth factor (VEGF) expression (Fig. 6i) and a noticeable loss of proteoglycans in the tissue (Fig. 6j,k). The most striking effects were observed in the absence of TGFβ3. Together, these findings suggest that IL1β signaling can initiate a transition from an anabolic environment to a catabolic state in hPSCderived articular chondrocytes, similar to that observed in native cartilage during early osteoarthritis pathogenesis. The observation that some changes were detected only in the absence of TGFβ3 suggests that the initiating events in osteoarthritis likely involve a change in the endogenous levels of TGFβ signaling together with the activation of inflammatory signaling pathways. advance online publication  nature biotechnology

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a

Generation of chondrocyte progenitors

Specification of articular chondrocytes/cartilage Experimental Gene expression Stage 4 treatment(s) ± TGFβ3 and histology TGFβ3 ± IL1β 1.5 weeks 10 weeks 12 weeks Articular cartilage

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j TGFβ3 DISCUSSION Here we report a method to efficiently and reproducibly generate hPSC-derived articular chondrocytes that give rise to and maintain stable cartilage in vitro and in vivo. The differentiation protocol is based on our understanding of the developmental pathways that specify this population in the embryo. Lineage-tracing studies have provided compelling evidence that articular chondrocytes derive from interzone cells in regions of condensing chondrogenic mesenchyme12. Our observations that the hPSCderived articular chondrocytes express genes expressed in interzone progenitors (GDF5, WNT9A, ERG, SOSTDC1 and DCX) and develop in the presence of TGFβ indicate that chondrogenic specification in vitro recapitulates key aspects of chondrogenic lineage development in the embryo. The hPSC-derived articular chondrocytes were able to generate and maintain stable cartilage tissue in vivo when transplanted subcutaneously at a highly vascularized site. Transplantation to an ectopic site rather than the joint has limitations, including the inability to determine the levels of cartilage integration, its capacity to bear weight and long-term survival in the joint space. Additionally, the signaling environment is likely to differ from that found at the articular surface, which is avascular and expresses inhibitors of the prohypertrophic BMP signaling pathway. Thus, ectopic sites are likely to promote ossification of the transplanted tissue. Indeed, cartilage derived from adult human mesenchymal cells or mouse ESCs readily undergoes ossification when transplanted to ectopic sites33–35. Our BMP4-induced tissues also initiated the ossification process during the 12-week engraftment period. The failure to complete the process may be due to low levels of expression of pro-angiogenic genes such as VEGF in these chondrocytes at the time of transplantation. The lack of any evidence of ossification, including vascularization, in the tissue derived from the hPSC articular chondrocytes is a good indication that it differs from BMP4-induced tissue and does indeed represent articular cartilage. The ECM composition of TGFβ3-induced cartilage tissues shared many characteristics of the developing fetal articular cartilage, including surface localization of lubricin and type I collagen. Although the nature biotechnology  advance online publication

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Stage 3 Day 15 0 weeks mesoderm

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Figure 6  hPSC-derived articular-like cartilage responds to IL1β. (a) Schematic representation of the experimental plan used to test the effects of IL1β. Articular cartilage tissues generated in micromass cultures (10 week) were treated for 2 weeks with IL1β (10 ng/ml) in the absence or presence of TGFβ3, as indicated. Following treatment, the tissue was analyzed histologically or dissociated and analyzed for gene expression patterns. (b–i) qRT-PCR–based expression analyses of the indicated genes in the tissues cultured under different conditions (n = 3 biological replicates for TGFβ3 + IL1β condition, n = 7 for all other treatments). Box plots show the copy number of mRNA relative to TBP. Significance was calculated by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars, mean ± s.d. (j,k) Toluidine blue staining of tissue cultured in the presence of IL1β or TGFβ3. Scale bars, 200 µm.

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presence of type I collagen in the adult can be indicative of fibrocartilage, its expression in developing tissue is not inconsistent with the emergence of the articular population30,36. The presence of type I collagen in the hPSC-derived tissue could thus be regarded as recapitulating a developmental stage that occurs during cartilage formation in the embryo. A recent report9 described the generation of hPSC-derived tissue, which was characterized as nonhypertrophic based largely on the lack of type X collagen at day 24 of culture. We also did not detect type X collagen at the protein level in the BMP4-induced tissue in vitro, even after 12 weeks of culture. However, these cells did give rise to tissue containing hypertrophic cells that expressed type X collagen and initiated the ossification process in 12-week-old grafts in vivo. These findings indicate that it is difficult to fully characterize chondrocyte potential based on short-term in vitro assays. In summary, access to hPSC-derived articular chondrocytes and derivative tissue provides an opportunity to test the feasibility of cell- and tissue-based therapy for injured or degenerative joints. Our observation that these chondrocytes can generate and maintain a stable cartilage graft in an ectopic site in vivo for several months is a first step toward this goal. Validation that they can produce functional tissue in orthotopic sites in a large animal model is the next challenge. In addition to its therapeutic potential, hPSC-derived articular cartilage provides a good model for studying the early events that lead to osteoarthritis, such as the changes in gene expression patterns observed in the tissue after the removal of TGFβ3 and/or the addition of IL1β. With the availability of unlimited amounts of such tissue, it will be possible to test a wide range of factors and cytokines thought to participate in the initiation and progression of this disease and to screen for drugs that attenuate these responses. 

Articles Methods Methods and any associated references are available in the online version of the paper. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank D. Taylor for providing primary human chondrocyte samples, H. Whetstone for histological expertise and helpful discussions, and A. Grigoriadis, F. Beier and members of the Keller laboratory for critical reading of the manuscript. We thank T. Araki and B. Neel (Ontario Cancer Institute, Toronto) for the iBJ hiPSC line and M. Warman (Boston Children’s Hospital) and G. Jay (Lifespan Health System, RI) for the lubricin antibody. This work was supported by a grant from Canadian Institutes of Health Research (MOP 219710) to B.A.A. and G.M.K., and a generous contribution from the Krembil Foundation to A.M.C. and G.M.K.

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AUTHOR CONTRIBUTIONS A.M.C. jointly conceived the study, interpreted results and wrote the paper with G.M.K. A.M.C. also designed and performed experiments and analyzed data; J.S.R. analyzed data and discussed results; Y.N. performed experiments and analyzed data; B.A.A. and R.A.K. provided primary human samples, gave conceptual advice, discussed results and edited the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Archer, C.W., Dowthwaite, G.P. & Francis-West, P. Development of synovial joints. Birth Defects Res. C Embryo Today 69, 144–155 (2003). 2. Colnot, C. Cellular and molecular interactions regulating skeletogenesis. J. Cell. Biochem. 95, 688–697 (2005). 3. Pacifici, M. et al. Cellular and molecular mechanisms of synovial joint and articular cartilage formation. Ann. NY Acad. Sci. 1068, 74–86 (2006). 4. Pelttari, K. et al. Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis Rheum. 54, 3254–3266 (2006). 5. Pelttari, K., Steck, E. & Richter, W. The use of mesenchymal stem cells for chondrogenesis. Injury 39 (suppl. 1), S58–S65 (2008). 6. Steinert, A.F. et al. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res. Ther. 9, 213 (2007). 7. Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008). 8. Oldershaw, R.A. et al. Directed differentiation of human embryonic stem cells toward chondrocytes. Nat. Biotechnol. 28, 1187–1194 (2010). 9. Umeda, K. et al. Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells. Sci. Rep. 2, 455 (2012). 10. Hwang, N.S. et al. In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells. Proc. Natl. Acad. Sci. USA 105, 20641–20646 (2008). 11. Toh, W.S. et al. Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells. Biomaterials 31, 6968–6980 (2010). 12. Koyama, E. et al. A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis. Dev. Biol. 316, 62–73 (2008). 13. Spagnoli, A. et al. TGF-beta signaling is essential for joint morphogenesis. J. Cell Biol. 177, 1105–1117 (2007).



14. Li, T. et al. Joint TGF-beta type II receptor-expressing cells: ontogeny and characterization as joint progenitors. Stem Cells Dev. 22, 1342–1359 (2013). 15. Serra, R. et al. Expression of a truncated, kinase-defective TGF-beta type II receptor in mouse skeletal tissue promotes terminal chondrocyte differentiation and osteoarthritis. J. Cell Biol. 139, 541–552 (1997). 16. Evseenko, D. et al. Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells. Proc. Natl. Acad. Sci. USA 107, 13742–13747 (2010). 17. Craft, A.M. et al. Specification of chondrocytes and cartilage tissues from embryonic stem cells. Development 140, 2597–2610 (2013). 18. Kattman, S.J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011). 19. Tanaka, M. et al. BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells. Stem Cell Res. (Amst.) 3, 126–141 (2009). 20. Roark, E.F. & Greer, K. Transforming growth factor-beta and bone morphogenetic protein-2 act by distinct mechanisms to promote chick limb cartilage differentiation in vitro. Dev. Dyn. 200, 103–116 (1994). 21. Mackay, A.M. et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. 4, 415–428 (1998). 22. Enomoto-Iwamoto, M. et al. Bone morphogenetic protein signaling is required for maintenance of differentiated phenotype, control of proliferation, and hypertrophy in chondrocytes. J. Cell Biol. 140, 409–418 (1998). 23. Volk, S.W., Luvalle, P., Leask, T. & Leboy, P.S.A. BMP responsive transcriptional region in the chicken type X collagen gene. J. Bone Miner. Res. 13, 1521–1529 (1998). 24. Kronenberg, H.M. & Chung, U. The parathyroid hormone-related protein and Indian hedgehog feedback loop in the growth plate. Novartis Foundation symposium 232, 144–152 discussion 152–147 (2001). 25. Bernardo, B.C. et al. Cartilage intermediate layer protein 2 (CILP-2) is expressed in articular and meniscal cartilage and down-regulated in experimental osteoarthritis. J. Biol. Chem. 286, 37758–37767 (2011). 26. Koch, M. et al. A novel marker of tissue junctions, collagen XXII. J. Biol. Chem. 279, 22514–22521 (2004). 27. Iwamoto, M. et al. Transcription factor ERG and joint and articular cartilage formation during mouse limb and spine skeletogenesis. Dev. Biol. 305, 40–51 (2007). 28. Guo, S. et al. Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling. Cell. Mol. Biol. Lett. 15, 153–176 (2010). 29. Zhang, Q. et al. Expression of doublecortin reveals articular chondrocyte lineage in mouse embryonic limbs. Genesis 49, 75–82 (2011). 30. Fukunaga, T. et al. Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression. Bone 33, 911–918 (2003). 31. Wang, X., Li, F., Fan, C., Wang, C. & Ruan, H. Effects and relationship of ERK1 and ERK2 in interleukin-1beta-induced alterations in MMP3, MMP13, type II collagen and aggrecan expression in human chondrocytes. Int. J. Mol. Med. 27, 583–589 (2011). 32. Goldring, M.B. et al. Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis. Eur. Cell. Mater. 21, 202–220 (2011). 33. Janicki, P., Kasten, P., Kleinschmidt, K., Luginbuehl, R. & Richter, W. Chondrogenic pre-induction of human mesenchymal stem cells on beta-TCP: enhanced bone quality by endochondral heterotopic bone formation. Acta Biomater. 6, 3292–3301 (2010). 34. Farrell, E. et al. In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells. BMC Musculoskelet. Disord. 12, 31 (2011). 35. Scotti, C. et al. Engineering of a functional bone organ through endochondral ossification. Proc. Natl. Acad. Sci. USA 110, 3997–4002 (2013). 36. Sasano, Y. et al. Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage. Anat. Embryol. (Berl.) 194, 247–252 (1996).

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ONLINE METHODS

hPSC maintenance and differentiation. hPSCs were maintained as described37. Two hESC lines (HES2 and H7) and two hiPSC lines (iBJ38 and MSC-IPS1/Y2-1 (refs. 18,39)) were used for this study. Cell lines were routinely tested for mycoplasma contamination and confirmed negative. Embryoid bodies were differentiated to the primitive streak/early mesoderm as previously described18,40. In brief, HES2 hESC embryoid bodies were generated from small aggregates by culture in StemPro-34 (Invitrogen) media containing BMP4 (0.5 ng/ml). hiPSC embryoid bodies were generated from single-cell suspensions by culture in HES media containing basic fibroblast growth factor (bFGF; 10 ng/ml) and Y-27632 (5 µM) on a rotator overnight. On day 1, embryoid bodies were harvested and resuspended in induction medium consisting of StemPro-34 supplemented with bFGF (FGF2; 5 ng/ml), activin A (2–3 ng/ml), BMP4 (1–3 ng/ml) and the GSK-3β inhibitor (CHIR99021, 1 µM), as indicated. On day 4 (day 3 for iPSC), the embryoid bodies were harvested from the induction medium, the cells dissociated with TrypLE (Invitrogen) and cultured as monolayers (500,000 cells per ml) in 96-well tissue culture plates (Falcon, Becton Dickinson) in StemPro-34 supplemented with the inhibitor of type I activin receptor-like kinase (ALK) receptors SB431542 (5.4 µM), bFGF (20 ng/ml) or the type I BMPR inhibitor DM (2–4 µM), as indicated. On day 6 (day 5 for iPSC), monolayer cultures were maintained in StemPro-34 containing no additional factors or bFGF (20 ng/ml) until day 15 (day 14 for iPSC). Cultures were maintained in a 5% CO2, 5% O2, 90% N2 environment for 12 days and then transferred into a 5% CO2/air environment for the remainder of the culture period. Cartilage tissues were generated from hPSC-derived mesoderm cultures on day 14/15 using micromass assays. Briefly, 200–500,000 cells were seeded into wells of 24-well tissue culture plates (Falcon) in 20 µl of base chondrogenic media consisting of high glucose DMEM with 1× ITS supplement, ascorbic acid (50 µg/ml), proline (40 µg/ml) and dexamethasone (0.1 µM) supplemented with 2% serum or bFGF (20 ng/ml) to allow the cells to adhere to the tissue culture-treated plastic. Base media containing TGFβ3 (10 ng/ml) was added to cover the adherent cells after 1 h. Micromasses were either (i) maintained in the base chondrogenic media supplemented with TGFβ3 (10 ng/ml) for the duration of the experiment or (ii) for 10 day at which time the TGFβ3 was replaced with BMP4 (50 ng/ml) for the duration of the experiment. In a series of experiments, TGFβ3 was replaced with TGFβ1 (10 ng/ml), TGFβ2 (10 ng/ml), Gdf5 (50 ng/ml), soluble (s) Bmpr1α (500 ng/ml) or cyclopamine (0.25 µM), as indicated. Recombinant human activin A, BMP4, TGFβ1-3, IL1β, bFGF, and mouse Gdf5 and sBmpr1α were purchased from R&D Systems; Y-27632 and KAAD-cyclopamine were obtained from Toronto Research Chemicals. DM was purchased from Sigma, and CHIR99021 was purchased from Stemgent. Dissociation procedure for human adult cartilage and fetal tissues. Use of primary human tissues was approved by the Mount Sinai Hospital Research Ethics Board. Human fetal cartilage was harvested under sterile conditions from fetal knee joints within 24 h of termination of pregnancy. The cartilaginous zone was dissected from the fetus (~16.5–19 weeks of gestation). Cells from one fetus were used for each experiment. Healthy adult articular cartilage and chondrocytes were ethically obtained as unsuitable transplant material from surgical procedures. Chondrocytes were isolated from tissues using enzymatic digestion with 0.1% collagenase A (Roche Diagnostics) for 18 h at 37 °C. Cells were resuspended in serum-free chondrogenic media. Flow cytometry and cell sorting. Embryoid bodies and monolayer cultures generated from hPSC differentiation cultures were dissociated with 0.25% trypsin/EDTA. Micromass tissues were dissociated by 0.2% w/v collagenase treatment (Sigma) for several hours at 37 °C. Cells were stained with anti-CD56phycoerythrin (BD, cat. no. 555518; 1:100), anti-PDGFRα-allophycocyanin (R&D Systems, cat. no. FAB1264A; 1:20), anti-KDR–phycoerythrin (R&D Systems, cat. no. FAB357P; 1:20), anti-CD73-allophycocyanin (BD, clone AD2; 1:400), anti-CD105-phycoerythrin (eBioscience clone SN6; 1:500), antiPDGFRβ- phycoerythrin (BD, cat. no. 558821; 1:100), anti-CD31-biotin (Pierce clone MEM-05, 1:1,600), anti-cardiac isoform of troponin T (cTnT; Thermo Scientific, clone 13-11; 1:1,000), goat anti-mouse IgG–allophycocyanin (BD, cat. no. 550826; 1:200), streptavidin-phycoerythrin-Cy7 (BD, cat. no. 557598;

doi:10.10.38/nbt.3210

1:200). Most staining was carried out at 4 °C in PBS containing 5% (v/v) serum. For cell sorting, antibody staining was performed in IMDM containing 0.2% bovine serum albumin (Sigma). Cells were acquired using an LSR II flow cytometer (Becton Dickinson) or sorted using a FACSAria II (Becton Dickinson). Analysis was performed using FlowJo (Tree Star). Quantitative real-time PCR. Total RNA was prepared with the RNAqueousMicro Kit with DNase treatment. RNA (0.1–1 µg) was reverse transcribed using random hexamers and oligo(dT) with Superscript III reverse transcriptase (Invitrogen). Real-time quantitative (Q)-PCR was performed on a MasterCycler EP RealPlex (Eppendorf) using Quantifast SYBR Green PCR kit (Qiagen). Genomic DNA standards were used to evaluate the efficiency of the PCR and calculate the copy number of each gene relative to the expression of the gene encoding TATA-box binding protein (TBP). All data represent three biological replicates or more as indicated in the figure legend. Student’s t-test (one-sided) or one way ANOVA followed by Tukey’s post hoc test were used to evaluate statistical significance, as indicated. Heat map generated using Multiexperiment viewer (MeV)41. Oligonucleotide sequences are provided upon request. Cell transplantations. Animal studies were performed in compliance with ethical regulations and were approved by Ontario Cancer Institute Animal Care Committee (AUP2909). Micromass cultures (~8–10 weeks) were dissociated, and chondrocytes were resuspended in growth factor–reduced Matrigel (Becton Dickinson). 500,000 cells were injected in a volume of 30 µl subcutaneously near the mammary fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) male mice aged 6–8 weeks. Grafts were harvested as indicated (n = 12 grafts per sample from four independent experiments for 4-week analyses; n = 8 grafts from TGFβ3-treated chondrocytes and n = 5 grafts from BMP4-treated chondrocytes from three independent experiments for 12-week analyses; an additional experiment including n = 7 grafts from BMP4-treated chondrocytes after 8 weeks had similar histological characteristics to 12-week grafts derived from BMP4-treated chondrocytes, not shown). hPSC-derived chondrocyte populations lacked tumorigenicity when transplanted in vivo. Randomization and blinding were not used for animal studies. Histology and immunohistochemistry. In vitro–derived cartilage tissues, grafts from in vivo studies, and primary human samples were fixed in 10% formalin and embedded in paraffin. 5-µm sections were stained with toluidine blue, von Kossa, safranin O (counterstained with fast green) or for TRAP-positivity, as indicated. Immunohistochemical analysis was performed using antibodies recognizing type II collagen (MS306-P; Labvision, Fremont CA), type X collagen (X53; Quartett, Berlin, Germany), type I collagen (C2456; Sigma), CD31 (ab28364, Abcam) and lubricin (Matthew Warman, Boston Children’s Hospital). Sections were counterstained with Mayer’s hematoxylin. Statistical analyses. All data represent three biological replicates (separate experiments or individual primary samples) or more as indicated in the figure legend. Where box plots are shown, the box extends from the 25th to 75th percentiles, the line indicates the median, and the Tukey method was used to plot the whiskers and the outliers. Where dot plots are shown, each point represents an individual value and the line indicates the mean. Error bars, where present, indicate the s.d. unless otherwise indicated in the figure legend. Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test was performed for all analyses as indicated in the figure legend. P values are categorized as *P < 0.05, **P < 0.01, ***P < 0.001. No statistical method was used to predetermine sample size. 37. Kennedy, M., D’Souza, S.L., Lynch-Kattman, M., Schwantz, S. & Keller, G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 2679–2687 (2007). 38. Witty, A.D. et al. Generation of the epicardial lineage from human pluripotent stem cells. Nat. Biotechnol. 32, 1026–1035 (2014). 39. Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008). 40. Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008). 41. Saeed, A.I. et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374–378 (2003).

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