Adiponectin stimulates exosome release to enhance mesenchymal stem cell driven therapy of heart failure in mice
Data files
Sep 21, 2020 version files 1.70 GB
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1_CH2-2.tif
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1_CH2-3.tif
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1_CH2.tif
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1_CH3-2.tif
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1_CH3-3.tif
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1_CH3.tif
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1_Overlay_1.tif
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1_Overlay-2.tif
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1_Overlay-3.tif
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1_Overlay.tif
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180524_TAC_hMSC_Ht.eds
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181211_hMSC_siExo.eds
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181211_siRNA_Exosome_machinary.xlsx
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190308_hAdMSC_cytokine_array_(siC_vs_siAlix).xlsx
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1st&2nd.xlsx
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2_sham3_×2.tif
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2_sham3_×40_01.tif
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2_TAC+hMSC2_×2_01.tif
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2_TAC+hMSC2_×40_02.tif
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2_TAC1_×2.tif
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2_TAC1_×40.tif
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200211_hMSC_Exo(Calnexin).xlsx
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2018-05-14_12h56m38s_37.555s_Chemiluminescence.scn
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2018-05-14_12h56m38s_37.555s_Chemiluminescence(Chemiluminescence).jpg
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2018-05-14_12h59m18s_0.886s_Chemiluminescence.scn
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2018-05-14_12h59m18s_0.886s_Chemiluminescence(Chemiluminescence).jpg
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2018-05-15_15h53m59s_1.173s_Chemiluminescence.scn
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2018-05-15_15h53m59s_1.173s_Chemiluminescence(Chemiluminescence).jpg
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2018-05-15_15h56m04s_4.186s_Chemiluminescence.scn
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2018-05-15_15h56m04s_4.186s_Chemiluminescence(Chemiluminescence).jpg
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2018-05-15_15h57m12s_0.375s_Chemiluminescence.scn
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2018-05-15_15h57m12s_0.375s_Chemiluminescence(Chemiluminescence).jpg
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2018-05-15_15h58m02s_0.690s_Chemiluminescence.scn
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2018-05-15_15h58m02s_0.690s_Chemiluminescence(Chemiluminescence).jpg
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2018-05-24_185803.xls
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2018-11-28_12h46m38s_47.683s_Chemiluminescence.scn
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2018-11-28_12h46m38s_47.683s_Chemiluminescence(Chemiluminescence).jpg
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2018-11-28_12h47m56s_17.599s_Chemiluminescence.scn
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2018-11-28_12h47m56s_17.599s_Chemiluminescence(Chemiluminescence).jpg
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2018-11-28_12h48m41s_1.145s_Chemiluminescence.scn
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2018-11-28_12h48m41s_1.145s_Chemiluminescence(Chemiluminescence).jpg
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2018-12-11_180233.xls
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2018-12-19_17h04m18s_0.324s_Chemiluminescence.scn
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2018-12-19_17h04m18s_0.324s_Chemiluminescence(Chemiluminescence).jpg
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2018-12-19_17h05m43s_3.759s_Chemiluminescence.scn
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2018-12-19_17h05m43s_3.759s_Chemiluminescence(Chemiluminescence).jpg
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2019-01-14_18h39m40s_1.127s_Chemiluminescence.scn
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2019-01-14_18h39m40s_1.127s_Chemiluminescence(Chemiluminescence).jpg
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2019-01-14_18h42m29s_8.278s_Chemiluminescence.scn
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2019-01-14_18h42m29s_8.278s_Chemiluminescence(Chemiluminescence).jpg
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2019-01-14_18h45m09s_25.717s_Chemiluminescence.scn
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2019-01-14_18h45m09s_25.717s_Chemiluminescence(Chemiluminescence).jpg
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2019-01-14_18h47m08s_31.052s_Chemiluminescence.scn
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2019-01-14_18h47m08s_31.052s_Chemiluminescence(Chemiluminescence).jpg
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2020-02-11_14h19m33s_1.865s_Chemiluminescence.scn
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2020-02-11_14h19m33s_1.865s_Chemiluminescence(Chemiluminescence).jpg
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2020-02-11_14h20m25s_0.155s_Colorimetric.scn
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2020-02-11_14h20m25s_0.155s_Colorimetric(Colorimetric).jpg
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2020-02-11_14h21m17s_6.347s_Chemiluminescence.scn
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2020-02-11_14h21m17s_6.347s_Chemiluminescence(Chemiluminescence).jpg
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2020-02-11_14h22m07s_0.159s_Colorimetric.scn
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2020-02-11_14h22m07s_0.159s_Colorimetric(Colorimetric).jpg
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2020-02-11_14h23m02s_5.632s_Chemiluminescence.scn
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2020-02-11_14h23m02s_5.632s_Chemiluminescence(Chemiluminescence).jpg
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2020-02-11_14h23m51s_0.151s_Colorimetric.scn
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2020-02-11_14h23m51s_0.151s_Colorimetric(Colorimetric).jpg
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2020-02-11_14h24m57s_19.492s_Chemiluminescence.scn
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2020-02-11_14h24m57s_19.492s_Chemiluminescence(Chemiluminescence).jpg
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2020-02-11_14h25m49s_0.149s_Colorimetric.scn
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2020-02-11_14h25m49s_0.149s_Colorimetric(Colorimetric).jpg
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2020-02-13_13h35m40s_106.802s_Chemiluminescence.scn
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2020-02-13_13h36m39s_0.085s_Colorimetric.scn
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2020-02-13_13h36m39s_0.085s_Colorimetric(Colorimetric).jpg
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3_TAC+hMSC2_×40_02.tif
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36B4-2.PDF
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36B4-2.txt
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36B4-3.PDF
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36B4-3.txt
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36B4.PDF
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36B4.txt
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Ad-APN_2nd.jmp
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Alix_2018-10-22_12h55m16s_13.371s_Chemiluminescence.scn
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Alix_2018-10-22_12h55m16s_13.371s_Chemiluminescence(Chemiluminescence).jpg
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Alix_2019-02-05_16h39m14s_40.825s_Chemiluminescence.scn
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Alix_2019-02-05_16h39m14s_40.825s_Chemiluminescence(Chemiluminescence).jpg
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Alix_2019-02-09_11h45m37s_54.941s_Chemiluminescence.scn
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Alix_2019-02-09_11h45m37s_54.941s_Chemiluminescence(Chemiluminescence).jpg
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Alix_Analysis.scn
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Alix_user_2019-03-21_15h15m25s.scn
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Alix_user_2019-03-21_15h15m25s(Chemiluminescence).jpg
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Analysis.scn
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Analysis.xlsx
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ANP.PDF
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ANP.txt
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APN-1_x200_2-002-report.pdf
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APN-1_x200_2-002-summary.csv
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apn-2_X200-002-report.pdf
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apn-2_X200-002-summary.csv
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apn-3_X200-002-report.pdf
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apn-3_X200-002-summary.csv
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apn+1_X200-2_SONIC_CAM12-001-report.pdf
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apn+1_X200-2_SONIC_CAM12-001-summary.csv
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apn+2_X200_SONIC_CAM15-001-report.pdf
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apn+2_X200_SONIC_CAM15-001-summary.csv
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APN+3_x200_sonic_cam15-001-report.pdf
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APN+3_x200_sonic_cam15-001-summary.csv
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BNP.PDF
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BNP.txt
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Calnexin.scn
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Canonical1.xls
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CD63_2019-02-05_16h37m45s_8.554s_Chemiluminescence.scn
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CD63_2019-02-05_16h37m45s_8.554s_Chemiluminescence(Chemiluminescence).jpg
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CD63_2019-02-09_11h42m49s_10.653s_Chemiluminescence.scn
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CD63_2019-02-09_11h42m49s_10.653s_Chemiluminescence(Chemiluminescence).jpg
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CD63_Analysis.scn
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CD63_user_2019-03-15_17h59m23s.scn
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CD63_user_2019-03-15_17h59m23s(Chemiluminescence).jpg
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CD63_user_2019-03-21_15h11m41s.scn
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CD63_user_2019-03-21_15h11m41s(Chemiluminescence).jpg
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Cell_APN_2019-04-16_16h26m37s_24.948s_Chemiluminescence.scn
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Cell_APN_2019-04-16_16h26m37s_24.948s_Chemiluminescence(Chemiluminescence).jpg
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Cell_Tcad_2019-04-16_16h27m40s_12.534s_Chemiluminescence.scn
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Cell_Tcad_2019-04-16_16h27m40s_12.534s_Chemiluminescence(Chemiluminescence).jpg
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Cell_Tub_2019-04-16_16h28m51s_6.720s_Chemiluminescence.scn
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Cell_Tub_2019-04-16_16h28m51s_6.720s_Chemiluminescence(Chemiluminescence).jpg
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Control(-)x10_01.tif
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Control(-)x10_scale.tif
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Control(-)x2_02.tif
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Control(-)x2_03.tif
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Control(-)x2_scale.tif
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Control(+)x2.tif
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DDBJ_No.txt
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Dose_Alix_2019-04-13_12h46m59s_20.243s_Chemiluminescence.scn
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Dose_Alix_2019-04-13_12h46m59s_20.243s_Chemiluminescence(Chemiluminescence).jpg
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Dose_CD63_2019-04-13_12h44m52s_0.672s_Chemiluminescence.scn
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Dose_MFG-E8_2019-04-13_12h45m49s_2.236s_Chemiluminescence.scn
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Dose_MFG-E8_2019-04-13_12h45m49s_2.236s_Chemiluminescence(Chemiluminescence).jpg
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Dose_Tsg101_2019-04-15_13h09m09s_11.800s_Chemiluminescence.scn
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Dose_Tsg101_2019-04-15_13h09m09s_11.800s_Chemiluminescence(Chemiluminescence).jpg
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Echo_180328.jpg
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Echo_180521.jpg
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Echocardiography1-2.jpg
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Echocardiography1-3.jpg
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Echocardiography1.jpg
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Echocardiography2-3.jpg
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Echocardiography4.jpg
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Echocardiography5-2.jpg
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Echocardiography5.jpg
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Functional.genomics_(in_vitro).txt
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Functional.genomics_(in_vivo).txt
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gluc_Time_course.wgd
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gluc_Time_course.xlsx
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hAdMSC_APN_dose.jmp
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hAdMSC_siTcad+APN.jmp
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hCD63_2018-10-22_12h52m50s_2.824s_Chemiluminescence.scn
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hCD63_2018-10-22_12h52m50s_2.824s_Chemiluminescence(Chemiluminescence).jpg
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hCD63_Analysis.scn
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hMFG-E8_2018-10-22_12h47m55s_6.002s_Chemiluminescence.scn
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hMFG-E8_2018-10-22_12h47m55s_6.002s_Chemiluminescence(Chemiluminescence).jpg
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hMFG-E8_Analysis.scn
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hMSC_APNdose.xlsx
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hMSC_Exo_Alix_2018-06-06_11h04m32s_42.215s_Chemiluminescence.scn
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hMSC_Exo_Alix_2018-06-06_11h04m32s_42.215s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exo_APN_2018-06-06_11h05m38s_1.218s_Chemiluminescence.scn
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hMSC_Exo_APN_2018-06-06_11h05m38s_1.218s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exo_CD63_2018-06-05_17h40m18s_41.533s_Chemiluminescence.scn
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hMSC_Exo_CD63_2018-06-05_17h40m18s_41.533s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exo_MFG-E8_2018-06-05_17h41m20s_0.377s_Chemiluminescence.scn
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hMSC_Exo_MFG-E8_2018-06-05_17h41m20s_0.377s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exo_synthenin_2018-06-05_17h38m28s_0.682s_Chemiluminescence.scn
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hMSC_Exo_synthenin_2018-06-05_17h38m28s_0.682s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exo_Tcad_2018-06-05_17h36m38s_59.066s_Chemiluminescence.scn
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hMSC_Exo_Tcad_2018-06-05_17h36m38s_59.066s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exo_Tsg101_2018-06-06_11h08m03s_85.676s_Chemiluminescence.scn
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hMSC_Exo_Tsg101_2018-06-06_11h08m03s_85.676s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exo_Tsg101_2018-06-06_11h10m22s_93.437s_Chemiluminescence.scn
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hMSC_Exo_Tsg101_2018-06-06_11h10m22s_93.437s_Chemiluminescence(Chemiluminescence).jpg
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hMSC_Exosome_miRNA(NGS_Hiseq).xlsx
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hMSC_Exosome.xlsx
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hSyntenin_Doseresponse_SerumExo_2018-07-19_13h05m29s_17.129s_Chemiluminescence.scn
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hSyntenin_Doseresponse_SerumExo_2018-07-19_13h05m29s_17.129s_Chemiluminescence(Chemiluminescence).jpg
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Htx20-2.oib
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Htx20-2.txt
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sham2-3_2020-02-21_18-37-31-ExperimentReport.pdf
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siT1→MSC3_2020-02-13_20-22-22_Summary.csv
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siT2_2020-02-13_20-25-47-ExperimentReport-2.pdf
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siT2_2020-02-13_20-25-47-ExperimentReport.pdf
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siT2_2020-02-13_20-25-47-ExperimentSummary.csv
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siT2_2020-02-13_20-25-57_Summary-2.csv
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siT2_2020-02-13_20-25-57_Summary.csv
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siT3_2020-02-13_20-29-55-ExperimentReport-2.pdf
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siT3_2020-02-13_20-29-55-ExperimentReport.pdf
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siT3_2020-02-13_20-29-55-ExperimentSummary.csv
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siT3_2020-02-13_20-30-06_Summary-2.csv
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Abstract
Mesenchymal stem/stromal cells (MSCs) are cultured adult stem cells originally reside in virtually all tissues and the gain of MSCs by transplantation has become the leading form of cell therapy in various diseases. However, there is limited knowledge on the alteration of its efficacy by factors in recipients. Here we report that the cardioprotective properties of intravenously injected MSCs in a mouse model of pressure-overload heart failure largely depend on circulating adiponectin, an adipocyte secreted factor. The injected MSCs exerted their function through exosomes, extracellular vesicles of endosome-origin. Adiponectin stimulated exosome biogenesis and secretion through binding to T-cadherin, a unique glycosylphosphatidylinositol-anchored cadherin, on MSCs. A pharmacological or adenovirus-mediated genetic increase in plasma adiponectin enhanced the therapeutic efficacy of MSCs. Our findings provide novel insights into the importance of adiponectin in mesenchymal progenitors-mediated organ protections.
Methods
Cell transplantation. hMSCs were freshly prepared before each experiment. The cells [prepared at three doses: low (0.5x105 cells), mid (1.67x105 cells), and high (5.0x105 cells)] were mixed with saline at one-third dilution, and injected using a 27-gauge needle inserted through the tail vein at 2-3 day intervals within a period of 2 weeks. In each session, the cells were injected slowly over a period of at least 30 seconds. For PKH-labeled hMSC transplantation experiments, hMSCs were labeled with a PKH26 red fluorescent dye (Sigma) according to the protocol supplied by the manufacturer. For RNAi experiments, hMSCs were transfected with silencer select siRNA (Ambion) using Lipofectamine RNAiMAX reagent (Life Technologies) according to the protocol supplied by the manufacturer, followed by injection of the cells the next day. For adenovirus-infected cell injection, hMSCs were infected with Gaussia luciferase-fusion MFG-E8 adenovirus, and the infected cells were injected via the tail vein on the next day. The injected cells were used within five passages in all experiments.
Adiponectin purification. HMW-APN purification was performed as reported previously11. Briefly, serum samples were obtained from WT mice at 4-days after infection with Ad-APN and applied onto T-cadherin-Fc conjugated with Protein G sepharoseTM (GE Healthcare). Adiponectin was eluted with 5 mM EDTA.
Exosome isolation. Exosomes were isolated from the cell culture supernatant as described previously32. Briefly, hMSCs were cultured with a xenofree hMSC culture medium with or without HMW-APN for 48 hours. Then, the conditioned medium was collected and centrifuged at 800 x g for 10 min to deplete floating cells, and at 10,000 x g for 30 minutes to remove cell debris. The plasma sample was mixed with thrombin (500 U/mL) for 10 min to remove fibrin, followed by centrifugation at 1,2000 x g for 20 minutes. For exosome isolation, the supernatant, plasma, and serum were ultracentrifuged at average 110,000 x g for 2 hours, followed by a washing step of the exosome pellet with Dulbecco’s phosphate-buffered saline with calcium and magnesium [PBS (+)] at average 110,000 x g for 2 hours (TLA100.1 rotor, Beckman Coulter). The exosome pellets were solubilized directly in Laemmli sample buffer. Essentially none of the mouse serum treatments, overexpression, or RNAi treatment significantly affected cell viability. Concentration and size-distribution of exosomes were analyzed by Nanoparticle Tracking Analysis (NanoSight LM10 system, Quantum Design). Serum exosomes were purified by a phosphatidylserine affinity magnetic resin, MagCapture™ Exosome Isolation Kit PS (Fujifilm Wako Pure Chemical). The recovery rate of exosomes was estimated as 16% by spiking the known amounts of gLuc-fusion MFG-E8 labeled exosomes into mouse serum and purifying by the affinity resin as above.
Animal Procedure. C57B6/J male mice were purchased from CLEA Japan. Adiponectin knockout (AKO) mice intensively backcrossed to C57BL/6J background were used32. Pioglitazone (30 mg/kg BID, Takeda Pharmaceutical Company) was administered orally for 15 days. Mice were housed in cages in a room set at 22°C under a 12:12hr light-dark cycle (lights of from 8:00 AM to 8:00 PM). Animals randomly allocated after sham or TAC operation in all experiments. Data were analyzed in a blinded fashion.
Cell culture. Human adipose tissue-derived mesenchymal stem cells (hMSCs) were obtained from Lonza Ltd. and maintained in a xenofree hMSCs culture medium (ROHTO Pharmaceutical Co.). For RNAi experiments, hMSCs were transfected with silencer select siRNA (Ambion) by using Lipofectamine RNAiMAX reagent (Life Technologies) according to the protocol supplied by the manufacturer. Incubation with adiponectin-containing media started 36 hours after transfection. The multilineage differentiation of hMSCs to adipocytes, osteoblasts, and chondrocytes was tested by using Mesenchymal Stem Cell-Adipogenic Differentiation Medium 2, -Osteogenic Differentiation Medium, and -Chondrogenic Differentiation Medium (PromoCell) according to the protocol supplied by the manufacturer. After the incubation with differentiation medium, staining with Oil Red O, Alizarin Red S, and Alcian Blue was performed to detect the adipocytes, osteoblasts, and chondrocytes, respectively.
Antibodies. The following primary antibodies were used: goat polyclonal anti-adiponectin (AF1119, R&D Systems); goat polyclonal anti–T-cadherin (AF3264, R&D Systems); rabbit monoclonal anti–α-tubulin (11H10, Cell Signaling Technology); sheep polyclonal anti-human MFG-E8 (AF2767, R&D Systems); mouse monoclonal anti-human CD63 (H5C6, BD); rabbit monoclonal anti-Tsg101 (ab125011, R&D Systems); rabbit polyclonal anti-syntenin (ab19903, Abcam); and mouse monoclonal anti-ALIX (3A9, Santa Cruz Biotechnology). The following secondary antibodies were used: horseradish peroxidase-conjugated (HRP-conjugated) rabbit anti-sheep IgG (Invitrogen); HRP-conjugated donkey anti-goat IgG (R&D systems); HRP-conjugated sheep anti-mouse IgG antibodies and donkey anti-rabbit IgG antibody (GE Healthcare).
Western blotting. Whole-cell lysates were loaded onto 4-20% gradient SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with Block-OneTM blocking reagent (Nakarai Tesque) and then incubated with primary antibodies using Can Get SignalTM solution 1 (TOYOBO) overnight at 4°C, followed by incubation with secondary antibodies conjugated with HRP using Can Get SignalTM solution 2 (TOYOBO) for 60 minutes at room temperature. Chemiluminescence signals developed with Chemi-Lumi One SuperTM (Nakarai Tesque) were visualized by ChemiDoc Touch and quantitated using Image Lab software (Bio-Rad).
Quantitative RT-PCR. Total RNA was isolated from mouse tissues by using RNA STAT-60T (Tel-Test, Inc., Friendswood, TX) according to the protocol supplied by the manufacturer. First-strand cDNA was synthesized using ReverTra AceTM qPCR RT Master Mix (TOYOBO). Real-time quantitative PCR amplification was conducted with QuantStudio7 (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems) according to the protocol recommended by the manufacturer. The sequences of primers used for real-time PCR were as follows; Rplp0 (36B4), Fw5’- GGCCAATAAGGTGCCAGCT-3’, Rv5’- TGATCAGCCCGAAGGAGAAG-3’; ANP, Fw5’- GCTTCCAGGCCATATTGGAG-3’, Rv5’- GGGGGCATGACCTCATCTT-3’; BNP, Fw5’- GAGGTCACTCCTATCCTCTGG-3’, Rv5’- GCCATTTCCTCCGACTTTTCTC-3’.
Transverse aortic constriction (TAC) operation. TAC operation was performed using the Minimally Aortic Transverse Banding method described in detail by Martin et al 54. In brief, C57BL/6 male mice (8–9 week-old, 23-27 g) were anesthetized with a mixture of pentobarbital sodium (50 mg/kg ip) and ketamine (25 mg/kg ip), as described previously 16. The thymus was gently put away to expose the aortic arch. After isolation of the transverse aorta, it was constricted by a 7-0 silk suture ligature fastened stiffly to a 27-gauge needle to yield a constriction of 0.4 mm in diameter. Sham-operated mice underwent a similar surgical procedure, including the exposure of the transverse aorta but without the constriction. The chest was closed with a 5-0 silk suture, and mice were allowed to recover. The procedure was performed under a surgical microscope and was completed in 10 min.
Echocardiography. Transthoracic echocardiography was performed as described in detail previously 55. Briefly, it was performed in each mouse using LOGIQe ultrasound system with a 4.0–10.0 MHz linear probe (i12L-RS) (GE Healthcare). The mouse was first anesthetized with isoflurane and laid on a heating pad to maintain body temperature at 35–37°C. After obtaining the long-axis two-dimensional image of the left ventricle (LV), a two-dimensional guided M-mode trace crossing the septal and posterior walls was recorded. The following parameters were measured on the M-mode tracings: interventricular septal thickness, LV posterior wall thickness, LV end-diastolic diameter (LVDd), LV end-systolic diameter (LVDs), and LV fractional shortening [LVFS = (LVDd − LVDs)/LVDd × 100].
Measurement of Gaussia luciferase activity. The exosome fraction was prepared as described in the exosome isolation section. The Gaussia luciferase activity in the exosome was measured by Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific) according to the protocol supplied by the manufacturer.
RNA-seq. miRNA sequencing was performed at the NGS core facility of the Genome Information Research Center at the Research Institute for Microbial Diseases of Osaka University. Exosomal RNAs derived from hMSCs were isolated using a miRNeasy mini kit (Qiagen). Small RNA libraries were constructed according to the instructions provided by the manufacturer using the NEBNext Small RNA Library Prep Set for Illumina (NEB) and sequenced by the HiSeq 2500 platform (Illumina) in 75-base-pair (bp) single-end reads. The miRNA-seq analysis was conducted using a StrandNGS version 3.0 software (Strand life science) according to the small RNA alignment and small RNA analysis pipeline using the default parameters. Before analysis of the small RNA-Seq data, reads were trimmed of the adapter sequences and mapped to the human hg19 reference genome. The relative small RNA expression levels were calculated using the DESeq algorithm. The RNA sequencing library was prepared using a TruSeq Stranded mRNA sample prep kit (Illumina, San Diego, CA) according to the instructions supplied by the manufacturer. Sequencing was performed on an Illumina HiSeq 2500 platform in a 75-base single-end mode. Illumina Casava1.8.2 software was used for base calling. The sequenced reads were mapped to the human reference genome sequences (hg19) using TopHat version 2.0.13 in combination with Bowtie2 version 2.2.3 and SAMtools version 0.1.19. The fragments per kilobase of exon per million mapped fragments (FPKMs) were calculated using Cuffnorm version 2.2.1.
Cytokine array. The conditioned medium was collected and centrifuged at 800 x g for 10 minutes to deplete floating cells, and the pooled sample of four separate cells was analyzed by Cytokine Array -Human Cytokine Antibody Array (Membrane, 42 Targets, Abcam) based on the protocol supplied by the manufacturer.
Measurement of plasma adiponectin levels by ELISA. Blood samples were collected from the respective mice, and plasma adiponectin levels were measured by adiponectin ELISA (Otsuka Pharmaceutical Co.) according to the protocol supplied by the manufacturer.
Histochemistry. The heart tissue was collected from each mouse and the left ventricle was embedded in paraffin following 10% formalin-fixation. Mouse heart sections (4-μm thick) were prepared and stained with hematoxylin and eosin (H&E). Other sections were also stained with CFTM594-conjugated wheat germ agglutinin (WGA; Biotium) to evaluate the myocyte cross-section area (CSA). For the quantification of CSA, the area was detected and analyzed using a BZ-X700 microscope and the built-in software (Keyence). Mice lung tissue was collected from mice injected by PKH-labeled hMSCs and the tissue was embedded in Tissue-Tek® O.C.T. Compound (Sakura). Tissue sections were prepared 20-μm thick and stained with DAPI.
Ethical considerations. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University School of Medicine. This study also conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Statistical analysis. Data were expressed as mean±SEM. Differences between the experimental groups were assessed by Student’s t-test or one-way ANOVA, followed by post hoc Dunnet’s and Tukey’s test. P values <0.05 were considered statistically significant. All analyses were performed with JMP Software 13.0 (SAS Institute, Cary, NC).