Stepwise preconditioning enhances mesenchymal stem cell-based cartilage regeneration through epigenetic modification S. Lin, W.Y.W. Lee, L. Xu, Y. Wang,

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Stepwise preconditioning enhances mesenchymal stem cell-based cartilage regeneration through epigenetic modification S. Lin, W.Y.W. Lee, L. Xu, Y. Wang, Y. Chen, K.K.W. Ho, L. Qin, X. Jiang, L. Cui, G. Li Osteoarthritis and Cartilage Volume 25, Issue 9, Pages 1541-1550 (September 2017) DOI: 10.1016/j.joca.2017.05.008 Copyright © 2017 Terms and Conditions

Fig. 1 Changes of cell morphology and surface markers of MSCs and M-MSCs. (A) Cell morphology of untreated MSCs, MSCs incubated in CIM for 10 days (CIM-10), or withdrawal of induction medium for 10 days (M-MSCs). Scale bar: 10 μm. (B) Surface antigen profile in MSCs or M-MSCs as determined by flow cytometry. The data are expressed as mean and 95% CIs (n = 2 donors with three replicates each donor). Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions

Fig. 2 Potential of lineage differentiation of MSCs and M-MSCs. (A) Staining of osteogenic differentiation by Alizarin Red S, adipogenic differentiation by Oil Red O, and chondrogenic differentiation by Safranin O and Fast Green on 21 days or 31 days. Scale bar: 10 μm. Expression levels of osteogenic (B), adipogenic (C), and chondrogenic (D) differentiation markers in MSCs and M-MSCs determined by Real time PCR assays. The data are expressed as mean and 95% CIs (n = 2 donors with three replicates each donor). ∗P < 0.05, ∗∗P < 0.01 vs MSCs. Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions

Fig. 3 Colony formation, cell proliferation, and cell survivability of MSCs and M-MSCs. (A) Crystal violet staining of colony-forming assay after 10 days of incubation at low-density plating. (B) Quantitative data of colony-forming assay. Colonies that equivalent or larger than 2-mm in diameter were counted. (C) Cell proliferation was measured on day 0, 3, or 5 by BrdU assay. (D) Cell morphology after challenging by hydrogen peroxide at the concentration of 250 μM for 24 h. Scale bar: 10 μm. (E) Cell viability of MSCs and M-MSCs challenged by hydrogen peroxide at 0, 150, 250, or 350 μM for 24 h. The data are expressed as mean and 95% CIs (n = 2 donors with three replicates each donor). ∗∗P < 0.01 vs MSCs. Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions

Fig. 4 Expression level and the methylation status of Nanog and Oct4 in MSCs and M-MSCs. (A & B) The relative expression levels of Nanog and Oct4 as measured by qRT-PCR. Total RNA was extracted from MSCs proceeding 3 days of chondrogenic differentiation followed by 3 days of expansion (M-MSCs3-3). RNA was also extracted from M-MSCs7-7, M-MSCs10-10, M-MSCs10-7, and M-MSCs10-14 in a similar fashion. MSCs at p3–p5 were taken as controls. (C) DNA methylation status of Nanog and Oct4 promoters. The top panel indicates the CpG dinucleotide position and the numbers show positions of CpGs relative to the translation start site. Ten or seven representative sequenced clones were depicted by filled (methylated) and open (unmethylated) circles for each CpG site in Nanog or Oct4, respectively. The data are expressed as mean and 95% CIs (n = 2 donors with three replicates each donor). ∗P < 0.05, ∗∗P < 0.01 vs MSCs; #P < 0.05, ##P < 0.01 vs (M-MSCs10-10). Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions

Fig. 5 The effect of Nanog silencing on potency of M-MSCs. (A) Relative expression level of Nanog and Oct4 after Nanog silencing. (B) Cell proliferation, (C) survivability, (D) colony formation, and (E) chondrogenic potential of M-MSCs infected with shNanog or scrambled control. The data are expressed as mean and 95% CIs (n = 2 donors with three replicates each donor). ∗P < 0.05, ∗∗P < 0.01 vs Scrambled. Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions

Fig. 6 The fate of injected GFP positive MSCs or M-MSCs in knee joint of OA animal model. (A) Representative images of GFP-positive cells in knee joints at day 15 and day 30 after injection. (B) Quantitative data of GFP positive cells in knee joints. 10 fields above patellar tendon from each animal were randomly selected for measurement. The data are expressed as mean and 95% CIs (n = 5). ∗P < 0.05 vs (OA + MSCs). Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions

Fig. 7 The therapeutic effects of MSCs or M-MSCs on cartilage destruction in the surgically induced-OA rat model. (A) Representative images of Safranin O and Fast Green staining sections of the medial compartment of the knee joint, GFP signals (orange arrows) in medial meniscus, MMP13 signals (blue arrows) in articular cartilage of tibiae. Black arrows point to the damaged area of cartilage. Quantitative results of OA score (B), GFP positive cells in meniscus (C), and MMP13 signals in articular cartilage (D). The data are expressed as mean and 95% CIs (n = 10). ∗∗P < 0.01 vs Sham; #P < 0.05, ##P < 0.01 vs (OA + PBS); ▲P < 0.05, ▲▲P < 0.01 vs (OA + MSCs). Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions

Fig. 8 The effects of MSCs or M-MSCs on microstructure of the tibial subchondral bone. (A) Representative images of tibial subchondral bone structure in the rats after treatment. (B) Quantitative results of tibial subchondral bone structural parameters, including BV/TV (trabecular BV per TV) and BMD. The data are expressed as mean and 95% CIs (n = 10). ∗P < 0.05 vs Sham; #P < 0.05 vs (OA + PBS). Osteoarthritis and Cartilage 2017 25, 1541-1550DOI: (10.1016/j.joca.2017.05.008) Copyright © 2017 Terms and Conditions