Nature Biotechnology: doi: /nbt Supplementary Figure 1. Generation of paraxial mesoderm from the H7 hesc line.

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1 Supplementary Figure 1 Generation of paraxial mesoderm from the H7 hesc line. H7 hescs were differentiated as shown in Figure 1a. (a) Flow cytometric analyses of the proportion of CD56+, PDGFRα+, and KDR+ cells in day 4 EBs. (b) Flow cytometric analyses showing the proportion of CD73+, CD105+, and PDGFRβ+ cells in day 15 populations specified as indicated (MOCK indicates no DM and no bfgf). qrt-pcr-based expression analyses of indicated genes on day 8 (c-d) and 15 (e-h) of culture. Each point represents the copy number mrna relative to TBP (n = 3 biological replicates), the line indicates the mean. (i-j) Percentage of cardiac troponin T (ctnt) positive cells on day 15 of culture was measured by intracellular flow cytometry. (i) Representative contour plots of day 15 cultures treated with indicated factors. (j) Quantification of ctnt-positive cells from three independent experiments (points), the line indicates the mean. Significance was calculated by student s t-test. *p<0.05, **p<0.01, ***p<0.001.

2 Supplementary Figure 2 Generation of paraxial mesoderm from human induced pluripotent stem cells (ibj hipscs). ibj hipscs were differentiated with the following modifications of the protocol shown in Figure 1a; the Wnt pathway agonist CHIR99061 (1 μm) was added to the stage 1 cultures and stage 1 was shortened from 3 to 2 days. The paraxial mesoderm fate was specified in the monolayer cultures by treatment with Dorsomorphin (DM) from day 3 to day 5, and FGF from day 3 to day 14 (Stage 2). Experimental cell treatment(s) during stage 2 include: no additional factors (MOCK), and Dorsomorphin and bfgf (DM+FGF). Inhibition of the Wnt signaling pathway from day 3 to 5 improved the efficiency of paraxial mesoderm specification in some cases. (a) Flow cytometric analyses of the proportion of CD56+, PDGFRα+, and KDR+ cells in day 3 EBs. qrt-pcr-based expression analyses of indicated genes on day 6 (b, n = 4) and 15 (c,d; MOCK n = 3, DM+FGF n = 5) of culture. Boxplots depict the copy number mrna relative to TBP. 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. (e) Flow cytometric analyses showing the proportion of CD73+, CD105+, PDGFRβ+, and CD31+ cells in day 14 populations specified as indicated. (f) Intracellular flow cytometric analysis of the percentage of cardiac troponin T+ cardiomyocytes in the day 14 monolayer populations. Points on graph indicate the average percentage of troponin T+ cells in the day 14 populations for 3 independent experiments, the line indicates the mean. Significance between Mock and DM+FGF groups was calculated by student s t- test. *p<0.05, **p<0.01, ***p<0.001.

3 Supplementary Figure 3 Generation of paraxial mesoderm and chondrocytes from human induced pluripotent stem cells (Y2-1 line). Y2-1 hipscs were differentiated with the following modifications of the protocol shown in Figure 1a; stage 1 was shortened from 3 to 2 days. The paraxial mesoderm fate was specified in the monolayer cultures by treatment with Dorsomorphin (DM) from day 3 to day 5,

4 and FGF from day 3 to day 14 (Stage 2). Experimental cell treatment(s) during stage 2 include: no additional factors (MOCK), and Dorsomorphin and bfgf (DM+FGF). Inhibition of the Wnt signaling pathway from day 3 to 5 improved the efficiency of paraxial mesoderm specification in some cases. (a) Flow cytometric analyses of the proportion of CD56+, PDGFRα+, and KDR+ cells in day 3 EBs. (b) qrt-pcr-based expression analyses of indicated genes on day 8 of culture. Boxplots depict the copy number mrna relative to TBP (MOCK, n = 8; DM+FGF, n = 10). (c) Flow cytometric analyses showing the proportion of CD73+, CD105+, and PDGFRβ+ cells in day 14 populations specified as indicated. (d) qrt-pcr-based expression analyses of indicated genes on day 14 of culture. Boxplots depict relative gene expression (MOCK, n = 4; DM+FGF, n = 5). 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. (e) Intracellular flow cytometric analysis of the percentage of cardiac troponin T+ cardiomyocytes in the day 14 monolayer populations. Histological images of micromass tissues derived from Y2-1 hipscs in the presence of TGFβ3 (f) or BMP4 (g) for 12 weeks. Sections stained metachromatically with Toluidine blue dye. Scale bar, 200 μm. (h-r) qrt-pcr-based expression analyses of indicated genes in micromass tissue cultured in the presence of TGFβ3 or BMP4 for 6 weeks (h-o) or 12 weeks (p-r). Points represent copy number mrna relative to TBP (n = 3), lines indicate the mean. Significance was calculated by student s t-test. *p<0.05, **p<0.01, ***p<0.001.

5 Supplementary Figure 4 TGFβ3 and BMP4 specify distinct populations of chondrocytes and cartilage tissues from hipsc-derived progenitors (ibj hipsc line). (a) Photomicrographs of chondrocytes generated following 6 weeks of culture in TGFβ3 or BMP4. Scale bar, 100 μm. (b) Flow cytometric analysis of forward (cell size) and side scatter (cell granularity) parameters of chondrocytes isolated from 12-week-old tissue. (c-n) qrt-pcr-based expression analyses of indicated genes in micromass tissue cultured in the presence of TGFβ3 or BMP4 for 6 weeks (c-k, n = 4) or 10 weeks (l-n, n = 5). Boxplots depict the copy number mrna relative to TBP. 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. Significance calculated by student s t-test. *p<0.05, **p<0.01, ***p<0.001.

6 Supplementary Figure 5 Primary human fetal chondrocytes generate cartilage tissues in vitro. Primary fetal (F) chondrocytes (passage 0) isolated from the knee joints at 16.5 and 18 weeks of gestation were cultured as micromass in the presence of TGFβ3 or BMP4 (a) Flow cytometric analysis of forward (FSC-A) and side (SSC-A) scatter parameters of primary

7 fetal chondrocytes isolated from micromass tissue cultured in TGFβ3 or BMP4 for 12 weeks. (b-t) qrt-pcr-based expression analyses of indicated genes in micromass tissue at 1, 4, 6, 8, and 12 weeks of culture. Values represent copy number mrna relative to TBP (n = 6 from two biologically independent experiments). Bars represent standard error of the mean. Significance calculated by student s t-test and compared TGFβ3 treated micromass cultures to BMP4-treated micromass cultures at indicated time points. *p<0.05, **p<0.01, ***p<0.001.

8 Supplementary Figure 6 hesc-derived paraxial/chondrogenic mesoderm expresses CD73, CD105 and PDGFRβ. (a) Flow cytometric analyses of DM+FGF-treated monolayer cultures during stage 2 of differentiation (T6 to T15). T (time) indicates day of differentiation. (b) Cells that expressed the combination of CD73 and either CD105 or PDGFRβ generated tissues in vitro. Double positive (CD73 + CD105 + and CD73 + PDGFRβ + ) populations from day 15 (T15) cultures were isolated from double negative populations by cell sorting and cultured as micromass. Micrographs of cells derived from sorted populations after 14 days (d, Scale bar, 200 μm) of micromass culture. (c) Tissues derived from sorted populations after 5 weeks of micromass culture in standard 24-well tissue culture plates.

9 Supplementary Figure 7 Gene expression analyses of hesc-derived chondrocytes (HES2 hesc line). qrt-pcr-based expression analyses of indicated genes in day 15 populations and derivative micromass tissue at different times. Values represent copy number mrna relative to TBP (n = 4-9 biological replicates), and are compared to primary fetal chondrocytes (aged weeks, n = 4), primary healthy adult articular chondrocytes (n = 2), and hypertrophic chondrocytes isolated from the iliac crest of a pediatric patient (n = 1). T15 Mesoderm indicates day 15 hesc-derived paraxial mesoderm (DM+FGF-treated). Bars represent standard error of the mean. n.d. indicates no data due to unavailability of primary tissue sample. Significance between TGFβ3 and BMP4 treatments at indicated time points was calculated by student s t-test. *p<0.05, **p<0.01, ***p<0.001.

10 Supplementary Figure 8 Proliferation and apoptosis during micromass culture. (a) Growth curve of hpsc-derived cells during micromass culture (Stage 4). The initial number of cells plated in micromass for each experiment was 200,000 (week 0). Micromass tissues were digested with 0.2% collagenase for 1-4 hours at 37 degrees Celcius at indicated timepoints (n 3 biologically independent samples per timepoint). Bars indicate standard error of the mean. Significance calculated by student s t-test, **p<0.01, ***p< Analysis of BMP4-treated cultures began at 2 weeks of culture (approximately 4 days after BMP4 treatment began). (b) Apoptotic cells were quantified by annexin V staining using flow cytometry after 10 weeks of micromass culture. Representative contour plots are shown.

11 Supplementary Figure 9 Generation of chondrocytes from the H7 hesc line. H7 hescs were differentiated as shown in Figure 1a. (a-m) qrt-pcr-based expression analyses of indicated genes in micromass tissue cultured in the presence of TGFβ3 or BMP4 for 6 weeks. Boxplots depict the copy number mrna relative to TBP (n = 4 biological replicates). 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. Significance was calculated by student s t-test. *p<0.05, **p<0.01, ***p<0.001.

12 Supplementary Figure 10 TGFβ1, TGFβ2, and TGFβ3 have similar effects on generating articular chondrocytes from hpsc-derived paraxial mesoderm. COL2A1, PRG4, and CILP2 gene expression after 12 weeks of micromass culture in the presence of TGFβ agonists as indicated (10 ng/ml). Points on graphs indicate the copy number mrna relative to TBP (n = 3). Lines indicate the mean. Significance was calculated by one-way ANOVA followed by Tukey s post-hoc test (no significant differences were observed).

13 Supplementary Figure 11 hesc-derived chondrocytes derived in the presence of GDF5 do not acquire an articular chondrocyte phenotype. (a-d) qrt-pcr-based expression analyses of indicated genes after 6 weeks of micromass culture in the presence of indicated factors. Day 15 DM+FGF treated chondrogenic mesoderm was plated in micromass culture in the presence of TGFβ3 for 10 days. Micromasses were then either maintained in TGFβ3-containing media or switched to media containing BMP4 (50 ng/ml), GDF5 (50 ng/ml) or a combination (G1aC) of GDF5, soluble BMPR1a (500 ng/ml) and cyclopamine (2.5 μm; a Hedgehog pathway inhibitor) for 5 additional weeks. Points on graphs indicate the copy number mrna relative to TBP (n = 3 biological replicates), lines indicate the mean and error bars indicate the standard deviation. Significance calculated by one-way ANOVA followed by Tukey s post hoc test. *p<0.05, **p<0.01, ***p< Toluidine blue staining of cartilage tissues derived in the presence of GDF5 (e) or G1aC (f) for 6 weeks. Scale bar, 200 μm.

14 Supplementary Figure 12 BMP4-treated chondrocytes have the capacity to generate grafts containing developing bone ossicles and hematopoietic cells after 12 weeks in vivo. Hematoxylin and eosin (H & E; a-c) and TRAP (d, e) staining of graft 2 shown in Figure 5. (d, e) TRAP staining indicates the presence of osteoclasts (purple stain). Double arrowheads indicate hypertrophic chondrocytes, grey filled arrowheads indicate developing bone ossicles, black filled arrows indicate hematopoietic cells, black stylized arrowheads indicate erythrocytes (red blood cells). Scale bar (a) 200 μm, (b, d) 100 μm, (c, e) 50 μm.

15 Supplementary Figure 13 Presence of osteoclasts and vascularization within grafts generated by BMP4-treated chondrocytes after 12 weeks. Grafts generated by TGFβ3-treated chondrocytes (a, b, f, g) and BMP4-treated chondrocytes (c-e, h-j) were analyzed histologically after 12 weeks. Two representative high magnification fields are shown for BMP4 grafts. (a-e) Identification of osteoclasts by TRAP staining (purple, arrows). (f-j) The presence of CD31 (PECAM)-positive endothelial cells by immunohistochemistry (brown staining) indicates vascularization of the graft. No TRAP-positive or CD31-positive cells were observed in grafts generated by TGFβ3-treated chondrocytes. Scale bar (a, c, f, g), 100 μm; Scale bar (b, d, e, g, i, j), 50 μm.