Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human ipscs

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1 Stem Cell Reports Supplemental Information Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human ipscs Akihiro Yamashita, Miho Morioka, Yasuhito Yahara, Minoru Okada, Tomohito Kobayashi, Shinichi Kuriyama, Shuichi Matsuda, and Noriyuki Tsumaki 1

2 Supplemental Figures 2

3 Figure S1. Establishment of COL11A2-EGFP hipsc lines and determination of culture conditions for the chondrogenic differentiation of hipsc-derived mesodermal cells, related to Figure 1. Mouse ipscs have previously been generated from Col2a1-reporter transgenic mice and used to monitor the chondrogenic differentiation of mouse ipscs (Diekman et al., 2012; Saito et al., 2013). The mouse α2(xi) collagen chain gene (Col11a2) is another chondrocyte-specific gene whose promoter/enhancer sequences direct the chondrocyte-specific expression in transgenic mice (Tsumaki et al., 1996). However, it remains unknown whether the corresponding human COL2A1 or COL11A2 sequences direct the chondrocyte-specific expression in human cells, as the specificity of the reporter constructs cannot be examined in the human body. Therefore, we examined the specificity of the COL11A2-reporter construct by creating COL11A2-reporter transgenic hipscs, after which we induced teratoma formation and examined the reporter expression pattern in the teratomas. (A) Structure of the COL11A2-EGFP reporter transgene piggybac vector. A single-cell suspension of hipscs was transduced with the COL11A2-EGFP piggybac vector and transposase vector via Amaxa nucleofection. Colonies arising from single cells were picked up and subjected to genotype analysis of the COL11A2-EGFP transgene. (B) Stable hipsc lines bearing the COL11A2-EGFP transgene were transplanted into SCID mice. The teratomas that formed were harvested and subjected to histological analysis. Semiserial sections were stained with hematoxylin-eosin and safranin O-fast green-iron hematoxylin and immunostained with anti-gfp antibodies. Scale bars: 50 mm. (C) Expression analysis of hipsc-derived particles generated in the presence of the indicated supplements on day 28. RNAs were extracted from the particles on day 28 and subjected to real-time RT-PCR analysis of the expression of chondrocyte markers. The RNAs extracted from hipscs and redifferentiated chondrocytes were used as negative and positive controls, respectively. The RNA expression levels were normalized to the level of the β-actin expression (n = 3 technical replicates). (D) Histological sections of the particles generated in the presence of the indicated supplements on day 42. HE, Hematoxylin-eosin staining; Safranin O, safranin O-fast green-iron hematoxylin staining. Magnified images of the boxed regions in the top panels are shown in the bottom panels. Scale bars: 50 μm. 3

4 (E) FACS analysis of COL11A2-EGFP-positive cells in the ipsc-derived cell culture in the presence of the indicated supplements on day 14. **P<0.01 (n = 3 independent experiments). (F) Histological sections of the particles generated in the presence of the indicated supplements on day 42. HE, Hematoxylin-eosin staining; Safranin O, safranin O-fast green-iron hematoxylin staining. Magnified images of the boxed regions in the top panels are shown in the bottom panels. Scale bars: 50 μm. The error bars denote the means ± s.d.. 4

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6 Figure S2. Histological analysis of hipsc-derived particles, related to Figure 2. (A) Histological analysis of the particles derived from three independent ipsc lines on day 42. Semiserial sections were stained with hematoxylineosin and safranin O-fast green-iron hematoxylin. (B, C) Histological analysis of the hipsc-derived particles continuously cultured in chondrogenic medium, rather than changing the medium to conventional medium on day 42. Semiserial sections were stained with hematoxylineosin and safranin O-fast green-iron hematoxylin and immunostained with anti-type II collagen antibodies and anti-type I collagen antibodies. (B) A particle on day 70. (C) A particle on day 140. (D, E) Histological analysis of the hipsc-derived particles. The medium was switched from chondrogenic medium to conventional medium on day 14 (D) or on day 28 (E). Particles were further cultured for 28 days. Semiserial sections were stained with hematoxylineosin and safranin O-fast green-iron hematoxylin and immunostained with anti-type II collagen antibodies and anti-type I collagen antibodies. (F) A particle derived from the 604B1 ipsc line was continuously cultured in chondrogenic medium until day 70. (G) A particle derived from the 604B1 ipsc line on day 70. The medium was switched from chondrogenic medium to conventional medium on day 42. (H) A particle on day 70. The medium was switched from chondrogenic medium to a medium supplemented with A, ABT, ABG or ATG on day 42 (I) Immunohistochemical analysis of type X collagen expression in hipsc-derived particles in the suspension culture. The growth plate cartilage of the proximal tibia obtained from an 8-week-old rat was used as a positive control. The blue color reflects DAPI. Scale Bars: 50 μm. (A-G) Magnified images of the boxed regions in the top panels are shown in the bottom panels. 6

7 Figure S3. Suspension culture facilitates the chondrogenic differentiation of ipscs and elimination of non-chondrocytic cells, related to Figure 2. (A) Histological analysis of the hipsc-derived cartilaginous nodules maintained in the adhesion culture until day 42 instead of being transferred to the suspension culture on day 14. Semiserial sections were stained with hematoxylin-eosin and safranin O-fast green-iron hematoxylin. Bars: 50 μm. 7

8 (B) Characteristics of the cells attached to the bottom of the dishes during the suspension culture of the COL11A2-EGFP hipsc-derived particles. After transferring the particles to a suspension culture in new dishes, the dish bottoms became gradually covered with cells, suggesting that some of the cells detached from the particles and attached to the dish bottoms. Phase and GFP images of the cells attached to the bottom of the dishes on day 42. Bars: 50 μm. (C) RNAs were extracted from the particles and cells attached to the dish bottoms on day 28 and subjected to real-time RT-PCR expression analysis of chondrocyte marker genes. RNAs extracted from redifferentiated chondrocytes and dermal fibroblasts were used as controls. The RNA expression levels were normalized to the level of β-actin expression (n = 3 technical replicates). The error bars denote the means ± s.d.. 8

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10 Figure S4. Real-time RT-PCR expression analysis of marker genes for the development of mesenchymal genes, related to Figure 3. The RNA expression levels were normalized to the level of β-actin (ACTB) expression. n = 3 technical replicates. The data are representative of two independent experiments. The error bars denote the means ± s.d.. 10

11 Figure S5. Cellular analysis of chondrogenic differentiation of hipscs, related to Figure 3. (A) FACS analysis of the COL11A2-EGFP fluorescence of hipsc-derived cells on days 0 and

12 (B) We divided and isolated cells on day 10 into two groups: a GFP (-) cell group, which showed lower GFP fluorescence intensity than the median, and a GFP (+) cell group, which showed higher GFP fluorescence intensity than the median. GFP (-) and GFP (+) cells were subjected to micromass culture for a further 10 days and subjected to FACS analysis for COL11A2-EGFP fluorescence. (C) Real-time RT-PCR expression analysis of chondrocyte-marker genes. Blue bars, GFP (-) and GFP (+) cells were collected on day 10 and immediately subjected to expression analysis. Red bars, GFP (-) and GFP (+) cells collected on day 10 were subjected to micromass culture for a further 10 days and subjected to expression analysis (n = 3 dishes). The error bars denote the means ± s.d.. (D) GFP (-) cells and GFP (+) cells collected on day 10 and subjected to micromass culture in ABTG medium for a further 28 days and then to histological analysis and safranin O-fast green-iron hematoxylin staining. Scale bar, 50 μm. 12

13 Figure S6. Real-time RT-PCR amplification of human β-actin sequences in samples in which various numbers of human cells (HDFs) were mixed with mouse cells (MEFs) (A) or rat cells (rat chondrosarcoma cells) (B), related to Figures 4 and 6. Ct values of 40 correspond to samples in which % (A) and % (B) of cells were human cells. n = 3 technical replicates. 13

14 Table S1. Antibodies, Related to Figures 2, 4, 5 and 6 Antibody Cat. No. Anti-type I collagen (goat polyclonal) Southern Biotech ( ) Anti-type II collagen (goat polyclonal) Southern Biotech ( ) Anti-human Vimentin (rabbit monoclonal) Abcam (ab16700) Anti-SOX9 (rabbit polyclonal) Santa Cruz (sc-20095) Anti-type X collagen (mouse monoclonal) Quartett ( ) Anti-GFP (rabbit polyclonal) Cell signaling (#2555) 14

15 Table S2. Primer Sequences, Related to Figures 3, 4 and 6 Primer Sequence ACTB F TGGCACCACACCTTCTACAATGAGC ACTB R GCACAGCTTCTCCTTAATGTCACGC OCT3/4 F GACAACAATGAGAACCTTCA OCT3/4 R TTCTGGCGCCGGTTACAGAA NANOG F CAAAGGCAAACAACCCACTT NANOG R CTGGATGTTCTGGGTCTGGT BRACHYURY F TATGAGCCTCGAATCCACATAGT BRACHYURY R CCTCGTTCTGATAAGCAGTCAC KDR F GGCCCAATAATCAGAGTGGCA KDR R CCAGTGTCATTTCCGATCACTTT FOXF1 F GCGGCTTCCGAAGGAAATG FOXF1 R CAAGTGGCCGTTCATCATGC MESP1 F GCTCTGTTGGAGACCTGGAT MESP1 R GTCTGCCAAGGAACCACTTC NKX2-5 F CAAGTGTGCGTCTGCCTTT NKX2-5 R GCGCACAGCTCTTTCTTTTC HAND2 F ATGAGTCTGGTAGGTGGTTTTCC HAND2 R CATACTCGGGGCTGTAGGACA MESOGENIN F AACCTGCGCGAGACTTTCC MESOGENIN R GTCTGTGAGTTCCCCGATGTA TBX6 F AGGCCCGCTACTTGTTTCTTC TBX6 R GGGGTGAATGTAGACACGGT MEOX1 F GTTCCAGAACCGAAGGATGA MEOX1 R CTTGGAGAGGCTGTGGAGTC TCF15 F TCCTGGAGAGCTGTGAGGAT TCF15 R CACACCCTGTCACCAACAGT NKX3-1 F CCCACACTCAGGTGATCGAG NKX3-1 R GAGCTGCTTTCGCTTAGTCTT SCX F CTGGCCTCCAGCTACATCTC SCX R CGGTCCTTGCTCAACTTTCT MYF5 F TCACCTCCTCAGAGCAACCT MYF5 R ATTAGGCCCTCCTGGAAGAA 15

16 Primer Table S3. Primer Sequences, Related to Figures 3, 4 and 6 Sequence SOX5 F CAGCCAGAGTTAGCACAATAGG SOX5 R CTGTTGTTCCCGTCGGAGTT SOX6 F GGATGCAATGACCCAGGATTT SOX6 R TGAATGGTACTGACAAGTGTTGG AGGRECAN F TGAGGAGGGCTGGAACAAGTACC AGGRECAN R GGAGGTGGTAATTGCAGGGAACA COL2A1 F TTTCCCAGGTCAAGATGGTC COL2A1 R CTTCAGCACCTGTCTCACCA LUBRICIN F AAAGTCAGCACATCTCCCAAG LUBRICIN R GTGTCTCTTTAGCGGAAGTAGTC IHH F AACTCGCTGGCTATCTCGGT IHH R GCCCTCATAATGCAGGGACT COL10A1 F ATGCTGCCACAAATACCCTTT COL10A1 R GGTAGTGGGCCTTTTATGCCT COL1A1 F GTCGAGGGCCAAGACGAAG COL1A1 R CAGATCACGTCATCGCACAAC COL1A2 F AATTGGAGCTGTTGGTAACGC COL1A2 R CACCAGTAAGGCCGTTTGC OSTEOCALCIN F CACTCCTCGCCCTATTGGC OSTEOCALCIN R CCCTCCTGCTTGGACACAAAG Taqman human SOX9 Hs _gl Taqman human COL11A2 Hs _ml Taqman human ACTB Hs gl Taqman mouse Actb Mn sl Taqman rat Actb Rn ml egfp COL11A2 F CATACAATGGGGTACCTTCTGG egfp COL11A2 R GCATGGACGAGCTGTACAAGTAA 16

17 Supplemental Experimental Procedures Cell counts The adherent cells and nodules (until day 14) and suspended particles (from day 15) were collected and digested with 0.25% Trypsin/EDTA at 37 C for one hour followed by 4% collagenase at 37 C overnight to prepare single-cell suspensions. Following treatement with trypan blue, the numbers of cells were counted using a Countess Automated Cell Counter (Invitrogen). Cells that incorporated trypan blue were considered dead. Histological analysis Particles in the suspension culture were collected, fixed with 4% paraformaldehyde, processed and embedded in paraffin. Semiserial sections were stained with hematoxylin-eosin and safranin O-fast green-iron hematoxylin staining. For immunohistochemistry, semiserial histological sections were immunostained with the antibodies listed in Supplemental Table S1. For goat anti type II collagen antibody and goat anti-type I collagen antibody, immune complexes were detected by N-Histofine Simple Stain MAX PO (GO) (Nichirei Biosciences Inc, ) and DAB (DAKO, K3468) as a chromogen. For the anti-gfp antibodies and anti-sox9 antibodies, immune complexes were detected using a CSAII Biotin-free Tyamide Signal Amplification System kit (Dako, ) and DAB as a chromogen. For the anti-gfp antibodies, anti-human vimentin antibodies and anti-type X collagen antibodies, immune complexes were detected using secondary antibodies conjugated to Alexa Fluor 488 or 546. RNA isolation and quantitative real-time RT-PCR During the period of adherent cell culture until day 14, total RNA was isolated from whole cell lysates using the RNeasy Mini Kit (Qiagen) according to the manufacturer s instructions with on-column DNase I digestion. After day 14, when the cells were 17

18 transferred to the suspension culture in petri dishes, the suspended particles were collected and homogenized using mortar before RNA extraction. Total RNAs were extracted from human dermal fibroblasts (HDFs) and differentiated hipscs for two weeks in convential medium (DMEM with 10% FBS, 50 units and 50 mg/ml of penicillin and streptomycin, respectively). Total RNAs were extracted from normal-looking articular cartilage and bone cells of the posterior condyle of the distal femur that were discarded at the time of total knee replacement surgery. Total RNAs prepared from the redifferentiated human fetal chondrocytes were purchased from Cell Applications, Inc. (402RD-R10f). A total of 500 ng of total RNA was used as a template for cdna synthesis using the ReverTra Ace (TOYOBO). A total of 1/400 of the product was used for quantitative real-time PCR. Real-time PCR was performed in a Step One system (ABI) using a KAPA SYBR FAST qpcr kit Master Mix ABI prism (KAPA BIOSYSTEMS). The primer sequences are described in Supplementary Tables S2 and S3. The amplified products were used to derive standard curves for quantitative real-time PCR. Transplanation of hipsc-derived cells into the subcutaneous spaces of SCID mice hipsc-derived particles obtained on days 28, 42 and 70 were transplanted into the subcutaneous spaces of SCID mice (C.B-17/lcr-scid/scid Jcl). We transplanted a hipsc-derived cartilaginous particle obtained on day 42 composed of approximately 1 x 10 6 cells per site. The mice were sacrificed 4 weeks, 12 weeks and 12 months later. The transplanted sites were dissected from the mice, and the samples were fixed in 4% paraformaldehyde, processed and embedded in paraffin for histological analysis. Transplanation of hipsc-derived cells into defects created in articular cartilage in SCID rats and mini-pigs. The skin and joint capsules of the knee joint of 6-week-old SCID rats (F344-Il2rgtm2kyo)(Mashimo et al., 2012) were opened. A drill hole with a diameter of 1 mm with a depth of 0.5 mm was created at the femoral groove. We transplanted a hipsc-derived cartilaginous particle obtained on day 28 composed of approximately

19 x 10 6 cells per joint defect, press fitting the particle into the osteochondral defect of the articular cartilage. The joint capsule and skin were closed, and the rats were sacrificed one and four weeks later. Knee samples were collected, fixed in 4% paraformaldehyde, decalcified with EDTA, processed and embedded in paraffin for the histological analysis. Similarly, the skin and joint capsules of the knee joints of 10-month-old male mini-pigs weighing kg were opened. Defects with a diameter of 6 mm and depth of 1 mm were created at the femoral groove. We transplanted 10 hipsc-derived cartilaginous particles obtained on day 56 into the defect of the articular cartilage and fixed the particles with fibrin-glue (Bolheal, Teijin Pharma, Japan). The joint capsule and skin were closed, and the mini-pigs were sacrificed four weeks later. Knee samples were collected, fixed in 4% paraformaldehyde, decalcified, processed and embedded in paraffin for the histological analysis. Mini-pigs were treated with 15 mg/kg/day cyclosporine. PCR amplification of the human sequence in various organs of SCID animals that underwent transplantaion of hipsc-derived particles To detect ectopic tissue formation, various tissues of the mice and rats were dissected. The samples were subjected to RNA extraction, and 500 ng of total RNA was subsequently used as a template for cdna synthesis. A total of 1/10 of the product was used for quantitative real-time PCR amplification of the human and mouse or rat -actin cdna sequences. The primers are listed in Supplementary Table S3. In order to obtain information on the level of dilution of human cells detectable in this assay, we also prepared cell samples in which various numbers of HDFs were mixed with mouse cells (mouse embryonic fibroblasts, MEFs) or rat cells (rat chondrosarcoma cells) and subjected them to real-time RT-PCR amplification. A total of 40 cycles of amplification was performed because Taq polymerase may be deactivated at over 40 cycles. FACS analysis 19

20 COL11A2-EGFP-positive cells were analyzed on an AriaII flow cytometer (BD Bioscience). Data were collected from 10,000 events. Data were analyzed with DIVA software (BD Bioscience). We divided cells on day 10 into two groups based on FACS analysis: a GFP (-) cell group, which showed lower GFP fluorescence intensity than the median, and a GFP (+) cell group, which showed higher GFP fluorescence intensity than the median. We isolated both cell groups and subjected them to micromass culture in chondrogenic medium for a further 10 days. 20

21 Supplemental References Diekman, B.O., Christoforou, N., Willard, V.P., Sun, H., Sanchez-Adams, J., Leong, K.W., and Guilak, F. (2012). Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells. Proc Natl Acad Sci U S A 109, Mashimo, T., Takizawa, A., Kobayashi, J., Kunihiro, Y., Yoshimi, K., Ishida, S., Tanabe, K., Yanagi, A., Tachibana, A., Hirose, J., et al. (2012). Generation and characterization of severe combined immunodeficiency rats. Cell reports 2, Saito, T., Yano, F., Mori, D., Ohba, S., Hojo, H., Otsu, M., Eto, K., Nakauchi, H., Tanaka, S., Chung, U.I., et al. (2013). Generation of Col2a1-EGFP ips cells for monitoring chondrogenic differentiation. PLoS ONE 8, e Tsumaki, N., Kimura, T., Matsui, Y., Nakata, K., and Ochi, T. (1996). Separable cis-regulatory elements that contribute to tissue- and site-specific alpha 2(XI) collagen gene expression in the embryonic mouse cartilage. J Cell Biol 134,