Fig 2: Generation of COL10A1-KO cell lines from human iPS cell lines. (A) Schematic diagram of the COL10A1 locus before (upper) and after (lower) genome editing using N- and C-sgRNA. Gray and hatched boxes indicate noncoding and coding regions, respectively. F1, F2, R1, and R2 indicate the location of PCR primers for the evaluation of the genome editing. (B) Demonstration of an 8.1 kb deletion in the COL10A1 locus by genomic DNA PCR. Deletion is confirmed by the loss of a longer fragment (9.3 kb). Shorter fragments from N-terminal and C-terminal regions are amplified in cases with a heterozygous deletion but not with homozygous deletions. (C) Sequences of the longer and shorter fragments demonstrated in (B). Solid and dotted lines indicate conserved and deleted regions, respectively. Inserted sequences are highlighted in red.

Fig 3: Induction of hypertrophic chondrocytes from parental (414C2), COL10A1 +/-, and COL10A1 -/- iPSC lines. (A) Phase contrast images and (B) Safranin-O and Fast-Green staining of cell pellets at different days during the induction. Scale bar = 500 µm. Similar results were obtained in three independent experiments. (C) mRNA expression of growth plate–related genes during the induction. RNAs were extracted from 3D chondrocyte pellets at each time point and assessed for the expression of each gene by qRT-PCR. The expression level was normalized to that of parental iPSC-derived pellets at day 0, except COL10A1, for which the level at day 28 was used for the normalization. Statistical analysis was performed using a two-way ANOVA. Data are presented as boxplots presenting all points from the minimum to maximum values (n = 3, independent experiments).

Fig 4: Expression of cartilage matrix at day 42 in parental (414C2), COL10A1 +/-, and COL10A1 -/- iPSC-derived 3D chondrocyte pellets. (A) Histological evaluation of parental iPSC-derived pellets. Cell pellets at each time point were stained with Safranin-O and Fast-Green (SOFG) and antibodies against COL I, COL II, or COL X. Scale bar = 500 µm. (B) Histological evaluation of parental, COL10A1 +/-, and COL10A1 -/- iPSC-derived pellets at day 42. Cell pellets were stained with SOFG and antibodies against COL I, COL II, or COL X. Scale bar = 500 µm. (C) Capillary-based immunoassay of proteins extracted from cell pellets. Cellular proteins were extracted from pellets at day 42 and analyzed by Western blot using antibodies against COL I, COL II, or COL X. The experiments were performed three times with similar results.

Fig 5: Cdc5l promotes early chondrocyte differentiation.A, ATDC5 cells were transfected with siCont or siCdc5l overnight, induced with BMP-2 + ITS for the indicated time points, and subjected to immunoblotting for Cdc5l. Tubulin served as a loading control. B, ATDC5 cells were stimulated with or without BMP-2 + ITS for 4 days and subjected to RT-qPCR for miR-542-3p (n = 3). C, ATDC5 cells, primary (pr.) chondrocytes, or C3H/10T1/2 cells transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS and subjected to alcian blue staining on day 8, 13, or 16, respectively. D and E, ATDC5 cells (D) or primary (pr.) chondrocytes (E) transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS and subjected to RT-qPCR analysis for Sox9 and Col2a1 on day 7 or 6, respectively (n = 3). F and G, C3H/10T1/2 cells transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS for 10 days and subjected to RT-qPCR for Sox9 and Agc1 (F) or Scx and Tnc (G) (n = 3). H, primary (pr.) mesenchymal stem cells (MSCs) transfected with siCont or siCdc5l were stimulated with or without TGF-ß3 and a cocktail of dexamethasone, ascorbic acid, and ITS (DAI), and subjected to RT-qPCR analysis for Sox9 and Col2a1 on day 6 (n = 3). I, primary (pr.) chondrocytes transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS and subjected to RT-qPCR analysis for Col10a1 on day 6 (n = 3). J, ATDC5 cells transfected with siCont or siCdc5l were infected with AAV-LacZ or AAV-CDC5L for 2 days, followed by BMP-2 + ITS application for 4 days. RT-qPCR for Sox9 and Col2a1 was analyzed (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.