Journal: The Journal of Clinical Investigation

Article Title: Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva

doi: 10.1172/JCI93521

Figure Lengend Snippet: (A and B) Knockdown experiments of mTORC complex components assessed by GAG/DNA (A) and Alcian blue staining (B) in a 2DCI assay of FOP-iMSCs. Scale bar: 200 μm. (C and D) Phosphorylation of SMAD1/5/8 (C) and SMAD2/3 (D) in FOP-iMSCs. Serum-starved FOP-iMSCs were pretreated with 10 nM rapamycin (Rapa), 1 μM DMH1, or 1 μM SB-431542 for 1 hour, and after a 1-hour stimulation with Activin-A, the cells were harvested. (E) p-S6 was enhanced in FOP-iMSCs compared with resFOP-iMSCs In 2DCI assays, p-S6 was enhanced in Activin-A–stimulated FOP-iMSCs compared with levels detected in resFOP-iMSCs. The strength of resFOP at each time point was set at 1. (F) Enhanced p-S6 was inhibited by mTOR (Rapa), PI3K (LY), or AKT (Ipa) inhibitors 24 hours after treatment with Activin-A and inhibitors in FOP-iMSCs. Treatment with 10 μM DMH1, 10 μM BIRB 796 (BIRB), 10 μM LY294002 (LY), 1 μM ipatasertib (Ipa), or 10 nM rapamycin was used. Results represent the mean ± SEM. n = 3 (A and C–F). *P < 0.05, **P < 0.01, and ***P < 0.001, by Dunnett’s multiple comparisons t test compared with the DMSO-treated control stimulated with Activin-A (C, D, and F); with the siRNA-transfected negative control, with or without Activin-A stimulation (A); and by Student’s t test compared with resFOP-iMSCs stimulated with Activin-A (E). Data are representative of 2 independent experiments (A, B, E, and F).

Article Snippet: FOP- (right leg) and resFOP-iMSCs (left leg) (4 × 10 6 cells, respectively) were transplanted into the gastrocnemius muscle of 6- to 8-week-old NOD/ShiJic-scid Jcl (NOD/SCID) mice (CLEA Japan) with C3H-DoxOn-hINHBA (5 × 10 5 cells), which can continuously expose Activin-A to the transplanted iMSCs in vivo ( 38 ).

Techniques: Staining, Transfection, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva

doi: 10.1172/JCI93521

Figure Lengend Snippet: (A) Venn diagram of genes highly upregulated in FOP-iMSCs compared with resFOP-iMSCs 24 hours after chondrogenesis induction stimulated with Activin-A, BMP-7, or TGF-β3. (B) Genes highly upregulated in FOP-iMSCs 24 hours after chondrogenesis induction stimulated with Activin-A, but not with BMP-7 or TGF-β3. (C) Expression of ENPP2 during the chondrogenic induction of FOP-iMSCs and resFOP-iMSCs. The expression level of resFOP (0 h) was set at 1. (D and E) ENPP2 inhibitors (HA, HA130; PF, PF-8380) (D) and ENPP2 knockdown (E) reduced p-S6 levels. (D) The cells were harvested 24 hours after treatment with Activin-A and ENPP2 inhibitors, and (E) siRNA–transfected cells were harvested 48 hours after treatment with Activin-A. Rapa, 100 nM rapamycin; HA, 10 μM HA130; PF, 10 μM PF-8380. (F) Serum-starved FOP-iMSCs were treated with 100 μM LPA, and 30 minutes after stimulation, the cells were harvested. Results represent the mean ± SEM. n = 1 (A and B) and n = 3 (C–F). *P < 0.05, **P < 0.01, and ***P < 0.001, by Student’s t test compared with LPA (–) control (F), by Dunnett’s multiple comparisons t test compared with FOP-iMSCs stimulated with Activin-A (D), or with negative control siRNA-transfected FOP-iMSCs stimulated with Activin-A (E). Data are representative of 2 independent experiments (D and F).

Article Snippet: FOP- (right leg) and resFOP-iMSCs (left leg) (4 × 10 6 cells, respectively) were transplanted into the gastrocnemius muscle of 6- to 8-week-old NOD/ShiJic-scid Jcl (NOD/SCID) mice (CLEA Japan) with C3H-DoxOn-hINHBA (5 × 10 5 cells), which can continuously expose Activin-A to the transplanted iMSCs in vivo ( 38 ).

Techniques: Expressing, Transfection, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Definition of Synovial Mesenchymal Stem Cells for Meniscus Regeneration by the Mechanism of Action and General Amp1200 Gene Expression

doi: 10.3390/ijms251910510

Figure Lengend Snippet: Cell clustering by Amp1200. Lots of SyMSCs (4 donors: S1, S2, S3 and S4), ADSCs (3 donors: A1, A2 and A3), BMSCs (3 donors: B1, B2 and B3), RECs (3 donors: R1, R2 and R3), FBs, iMSCs, WPs, PAECs, T cells, and CD14+ monocytes were clustered by the expression level of genes obtained by Amp1200. The black lines at the top and left showed the results of the clustering. A similar clustering analysis using all 25,193 human genes is shown in .

Article Snippet: Human SyMSCs (Articular Engineering, Northbrook, IL, USA, CDD-H-2910-N), human ADSCs (Lonza, Basel, Switzerland, PT-5006), human BMSCs (Lonza, Basel, Switzerland, PT-2501), human RECs (PuREC, Izumo, Japan, 387-16591), and iMSCs (FUJIFILM Cellular Dynamics, Madison, WI, USA, iCell MSC R1098) were all cultured in MEMα (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.

Techniques: Expressing