1. Proposal of patient-specific growth plate cartilage xenograft model for FGFR3 chondrodysplasia 
T. Kimura, T. Ozaki, K. Fujita, A. Yamashita, M. Morioka, K. Ozono, N. Tsumaki 
Osteoarthritis and Cartilage 
Volume 26, Issue 11, Pages (November 2018)
DOI: /j.joca
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2. Fig. 1
Characterization of 409B2 induced pluripotent stem cell (iPSC)-derived cartilage. hiPSC-derived cartilage was created in vitro by the chondrogenic differentiation of iPSC line 409B2 for 6 weeks and subjected to histological analysis. A. Left, Gross appearance of hiPSC-derived cartilage. Right, Histology of hiPS-derived cartilage. Safranin O-fast green-iron hematoxylin staining (safranin O). Scale bars: left, 5 mm; right, 100 μm. B. hiPSC-derived cartilage was stained with safranin O and immunostained using antibodies that recognize the indicated proteins. Right bottom, Magnification of the boxed region in the middle bottom panel. Arrowheads indicate nuclei positive for Ki67. Counterstaining with hematoxylin. Scale bars, 100 μm. The data are representative of three independent experiments.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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3. Fig. 2
Tissues formed after xenografting 409B2 hiPSC-derived cartilage into subcutaneous spaces of SCID mice. hiPSC-derived cartilage was created in vitro by the chondrogenic differentiation of iPSC line 409B2 for 6 weeks and subsequently grafted into subcutaneous spaces of 8-week-old SCID mice. Grafts were recovered 3 months after transplantation. A. Left, Image of the transplanted site at the time of recovery. Right, The graft was easily separated and recovered from the surrounding subcutaneous tissue. We detected a distinct portion (dotted line) in the middle of the graft. B. 3D reconstructed micro-CT images of the graft are shown in three directions. Bone was detected in the middle of the graft. Scale bars, 2 mm. C. Histological analysis of the graft. Top: hematoxylin and eosin (HE) staining. Bottom: safranin O staining highlights cartilage (red). Scale bars, 500 μm. D. Left, Magnification of the boxed region in c. Right, A semi-serial section was immunostained with anti-COL10 antibody. Scale bars, 100 μm. E. Immunohistochemical analysis of the graft. Semi-serial sections were stained with safranin O and immunostained with antibodies that recognize the proteins indicated at the left of each panel. Scale bar, 100 μm. F. Percentage of Ki67-positive cells per total number of cells in the specific zones of cartilage in the recovered grafts after transplantation and hiPSC-derived cartilage before transplantation. **P < 0.01 compared with RZ, HZ or hiPSC-Cart by one-way analysis of variance (ANOVA) with Tukey's HSD test (n = 3 mice) (RZ vs FZ, [95% CI: 5.3–14.8]; FZ vs HZ, [95%CI: 6.9–16.4]; FZ vs hiPSC-Cart, [95%CI: 6.1–15.6]). RZ, round-shaped chondrocyte zone; FZ, flat-shaped chondrocyte zone; HZ, hypertrophic chondrocyte zone; hiPSC-Cart, hiPSC-derived cartilage before transplantation. A–E. The data are representative of three mice.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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4. Fig. 3
Time course of bone formation in grafts. 4-week-old SCID mice received subcutaneous transplantations of iPSC line 409B2-derived (A, B, C) and 604B1-derived (D) cartilage. Into each mouse was transplanted four hiPSC-derived cartilage (four xenografts in each mouse). Four mice were sacrificed at 14, 28 and 42 days, respectively, after transplantation. Ossification of the xenografts were analyzed by X-ray and micro-CT images. A. X-ray images of mice 14, 28 and 42 days after transplantation. Arrowheads indicate ossification. B. Magnification of the boxed region in (A). C, D. Total bone volume of the four xenografts in each mouse was measured by micro-CT analysis (each point represents one mouse.). *P < 0.05 and **P < 0.01 vs day 42 by one-way ANOVA with Tukey's HSD test. (C) Grafts of 409B2 hiPSC-derived cartilage: day 14 vs day 42 [95%CI: 4.4–43.1]; day 28 vs day 42 [95%CI: 2.7–41.3]. (D) Grafts of 604B1 hiPSC-derived cartilage: day 14 vs day 42 [95%CI: 8.7–54.0]; day 28 vs day 42 [95%CI: 6.0–51.3].
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 The Authors Terms and Conditions

5. Fig. 4
Origin of cells in endochondral bone formation in grafts of 409B2 hiPSC-derived cartilage. hiPSC-derived cartilage were created in vitro by the chondrogenic differentiation of hiPSC line 409B2 for 6 weeks and subsequently grafted into the subcutaneous spaces of 4-week old SCID mice. Grafts were recovered 6 weeks after transplantation and subjected to histological analysis. A–D. Left, A section was immunostained with antibodies that recognize human nuclear antigen (HNA) (dark brown). Right, Semi-serial sections were stained with safranin O (A), HE (B, D) and tartrate-resistant acid phosphatase (TRAP) (C). The data are representative of two independent experiments. A. Cartilage in the graft. B. Bone in the graft. C. Junction between cartilage and bone in the graft. Cells indicated by arrows are shown in the insets in the respective panels. Nuclei of TRAP-positive cells did not react with anti-HNA antibodies, as indicated by the red arrowheads. Right panel, Dotted lines indicate junctions between hypertrophic cartilage and marrow (M) space where osteoclastic resorption of hypertrophic cartilage occurred. D. Adipose tissue surrounding the graft. E. Semi-serial sections were stained with HE (left top) and safranin O (right top) and immunostained with anti-mouse CD31 (left bottom) and anti-human CD31 (right bottom) antibodies (brown). Blood vessels in the bone marrows in the graft were recognized by anti-mouse CD31 antibody but not by anti-human CD31 antibody. Signals were visualized with DAB (dark brown). Counterstaining was done with hematoxylin. Scale bars: A–D, 50 μm; E, 100 μm. The data are representative of three mice.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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6. Fig. 5
FGFR3 chondrodysplasia patient-specific growth plate cartilage xenograft model. A. hiPSC-derived cartilage were created in vitro by the chondrogenic differentiation of HCH-hiPSCs in the absence or presence of statin for 6 weeks. Safranin O staining is shown. Data are representative of four independent experiments. B, C and D. WT- and patient-specific-hiPSC-derived cartilage were created in vitro by chondrogenic differentiation in the presence of statin for 6 weeks and subsequently xenografted into the subcutaneous spaces of 4-week-old SCID mice. 409B2 and thanatophoric dysplasia (TD)1-714 lines were used to prepare WT- and TD-hiPSCs. Grafts were recovered 4 weeks after transplantation and subjected to histological examination. B. Immunohistochemical analysis of the graft after transplantation of TD (TD1-714) hiPSC-derived cartilage. Semi-serial sections were stained with safranin O and immunostained with antibodies that recognize the proteins indicated at the left of each panel. Scale bar, 100 μm. C. Safranin O staining. Data are representative of four mice. D. Left, Images of immunostaining with anti-MMP13 antibody. Right, Diameters of hypertrophic chondrocytes indicated by MMP13 expression. **P < 0.01 vs WT by one-way ANOVA with Tukey's HSD test. n = 4 mice (each point represents a different mouse) (WT vs TD, P =  ; WT vs ACH, P =  ). Scale bars, 100 μm.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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7. Fig. 6
Rescue of abnormalities in TD-hiPSC-derived growth plate cartilage xenografts by treatment of mice with FGFR inhibitor NVP-BGJ398. TD-hiPSC-derived cartilage was created in vitro by chondrogenic differentiation in the presence of statin for 6 weeks. 4-week-old SCID mice received xenografts of the TD-hiPSC-derived cartilage and were then treated with 2 mg/kg NVP-BGJ398 or vehicle for 4 weeks. Xenografts were recovered and subjected to histological analysis. A. Histological sections of a xenograft of TD (TD1-714)-iPSC-derived cartilage were stained with safranin O. Mice were treated with vehicle (left top) or NVP-BGJ398 (right top). Left bottom, As for control, cartilage derived from TD (TD1-714)-iPSCs in which FGFR3 was knocked-down were xenografted into control mice, which were treated neither with vehicle nor NVP-BGJ398. B. Semi-serial sections in (A) were immunostained with anti-MMP13 antibody. Diameters of hypertrophic chondrocytes indicated by MMP13 expression were measured. Vehicle vs TD1-shFGFR3, 95%CI: 17.8–25.0. The raw data (histological images of all samples) are shown in Supplemental Fig. 7. C. Histological sections of a xenograft of TD (TD1-329N)-iPSC-derived cartilage were stained with safranin O. Mice were treated with vehicle (left top) or NVP-BGJ398 (right top). D. Semi-serial sections in (C) were immunostained with anti-MMP13 antibody. Diameters of hypertrophic chondrocytes indicated by MMP13 expression were measured. **P < 0.01 vs vehicle by one-way ANOVA with Tukey's HSD test (B) and Student's t test (D). n = 4 mice (each point represents a different mouse).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2018 The Authors Terms and Conditions