Time-lapse observation of the dedifferentiation process in mouse chondrocytes using chondrocyte-specific reporters Y. Minegishi, K. Hosokawa, N. Tsumaki

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Time-lapse observation of the dedifferentiation process in mouse chondrocytes using chondrocyte-specific reporters Y. Minegishi, K. Hosokawa, N. Tsumaki Osteoarthritis and Cartilage Volume 21, Issue 12, Pages 1968-1975 (December 2013) DOI: 10.1016/j.joca.2013.09.004 Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 The GFP and YFP fluorescence of Col11a2-EGFP transgenic mice and Col11a2-Cre; R26-stopflox-EYFP transgenic mice. (A) The hindlimb skeletons of 3-week-old mice were observed under a fluorescent microscope. Bars, 5 mm. (B) The knee joints of 3-week-old mice were harvested and processed for cryostat sectioning. Semiserial sections were observed under fluorescence microscope and stained with hematoxylin and eosin. Bars, 500 μm in the top panels and 50 μm in the bottom panels. (C) Dermal fibroblasts prepared from 3-week-old mice were observed under phase and fluorescent microscopes. Bars, 100 μm. Osteoarthritis and Cartilage 2013 21, 1968-1975DOI: (10.1016/j.joca.2013.09.004) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 The dedifferentiation of chondrocytes during monolayer expansion culture as indicated by the loss of GFP fluorescence. (A) The percentages and numbers of fluorescent-positive cells out of the total cell population. *P < 0.01 compared with Col11a2-EGFP cells on each day (n = 5). (B–E) Chondrocytes prepared from Col11a2-EGFP transgenic mice were cultured. Images taken on day 1 (B and C) and day 7 (D and E) are shown. Phase (B and D) and GFP fluorescent (C and E) images are shown. Bars, 100 μm. (F–I) Chondrocytes prepared from Col11a2-Cre; R26-stopflox-EYFP transgenic mice were cultured. Images taken on day 1 (F and G) and day 7 (H and I) are shown. Phase (F and H) and YFP fluorescent (G and I) images are shown. Bars, 100 μm. (J) The results of the real-time RT-PCR analysis of the expression of marker genes in chondrocytes during monolayer expansion culture. The levels of a fibroblast marker, Col1a1, and chondrocyte markers, Col2a1, Aggrecan, Col11a2, Sox5, Sox6 and Sox9 were examined. *P < 0.01 (n = 5). Osteoarthritis and Cartilage 2013 21, 1968-1975DOI: (10.1016/j.joca.2013.09.004) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 The redifferentiation of chondrocytes by the addition of Cytochalasin B or suspension culture. Dedifferentiated chondrocytes were reseeded in 6 cm culture dishes for monolayer culture or in 6 cm petri dishes for suspension culture. Cytochalasin B was added to the monolayer culture at a final concentration of 20 μM. Phase and GFP images were taken 4 days after reseeding. Osteoarthritis and Cartilage 2013 21, 1968-1975DOI: (10.1016/j.joca.2013.09.004) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 The dedifferentiation of chondrocytes treated with mitomycin C during monolayer culture, as indicated by the loss of GFP fluorescence. (A) The relative numbers of chondrocytes with or without treatment with mitomycin C during monolayer culture. *P < 0.01 compared with cells cultured in the absence of mitomycin C on each day (n = 5). (B) The percentages and numbers of fluorescence-positive cells compared to the total cell population. *P < 0.01 compared with Col11a2-Cre; R26-stopflox-EYFP cells on each day (n = 5). (C–F) Chondrocytes prepared from Col11a2-EGFP transgenic mice were treated with mitomycin C and cultured. Images taken on day 1 (C and D) and day 7 (E and F) are shown. Phase (C and E) and GFP fluorescent (D and F) images are shown. Bars, 100 μm. (G–J) Chondrocytes prepared from Col11a2-Cre; R26-stopflox-EYFP transgenic mice were treated with mitomycin C and cultured. Images taken on day 1 (G and H) and day 7 (I and J) are shown. Phase (G and I) and YFP fluorescent (H and J) images are shown. Bars, 100 μm. (K) The results of the real-time RT-PCR analysis of the expression of marker genes in chondrocytes treated with mitomycin C and cultured in monolayers. A fibroblast marker, Col1a1, and chondrocyte markers, Col2a1, Aggrecan and Col11a2, were examined. *P < 0.01 (n = 5). Osteoarthritis and Cartilage 2013 21, 1968-1975DOI: (10.1016/j.joca.2013.09.004) Copyright © 2013 Osteoarthritis Research Society International Terms and Conditions