1. Replenishing exosomes from older bone marrow stromal cells with miR-340 inhibits myeloma-related angiogenesis
by Tomohiro Umezu, Satoshi Imanishi, Kenko Azuma, Chiaki Kobayashi, Seiichiro Yoshizawa, Kazuma Ohyashiki, and Junko H. Ohyashiki
BloodAdv
Volume 1(13):
May 23, 2017
© 2017 by The American Society of Hematology

2. Tomohiro Umezu et al. Blood Adv 2017;1:812-823
© 2017 by The American Society of Hematology

3. Characterization of BMSCs
Characterization of BMSCs. (A) Morphology of BMSCs derived from 2 young healthy donors (young BMSCs; passage 2; donor ages: 19 and 20 years old) and 2 older healthy donors (older BMSCs; passage 2; donor age: 68 and 72 years) by phase-contrast inverted microscopy.
Characterization of BMSCs. (A) Morphology of BMSCs derived from 2 young healthy donors (young BMSCs; passage 2; donor ages: 19 and 20 years old) and 2 older healthy donors (older BMSCs; passage 2; donor age: 68 and 72 years) by phase-contrast inverted microscopy. Scale bar, 50 µm. (B) BMSC growth measured after 24, 48, 72, 96, and 120 h in culture. Cell proliferation of young BMSCs (pink line) and older BMSCs (blue) was evaluated. Each value represents the mean ± SD (n = 6). (C-D) Representative images of SA-β-gal staining (arrows indicate positive cells) and quantification of cell senescence. Data represent the mean ± SD. *P < .01 in comparison with young BMSCs using a Student t test. Scale bar, 50 µm. (E-F) Telomere lengths in young and older BMSCs determined by Southern blotting of TRFs. Data represent the mean ± SD. *P < .05 in comparison with young BMSCs using a Student t test. (G) Transmission electron micrographs of exosomes derived from young and older BMSCs. Scale bar, 50 nm. (H) Western blots of exosomal lysates (CD63 and CD81 are common exosomal markers) derived from young and older BMSCs. Exosomes were isolated from 5 mL of culture medium collected from BMSCs (4 × 104 cells/cm2) grown for 48 h. Equal volumes of exosomal lysate (30 µL) were loaded in the lanes of gels. (I) Nanoparticle concentration and size distribution of exosomes derived from young BMSCs (pink line) and older BMSCs (blue line).
Tomohiro Umezu et al. Blood Adv 2017;1:
© 2017 by The American Society of Hematology

4. Healthy BMSC exosomes inhibit HR-MM cell-induced angiogenesis in Matrigel plugs.
Healthy BMSC exosomes inhibit HR-MM cell-induced angiogenesis in Matrigel plugs. (A) Schematic of the Matrigel plug assay. A mixture of Matrigel and 4 × 106 RPMI8226-HR cells was admixed with exosomes derived from young BMSCs (young BMSC exosomes; 4 × 107 particles per 200 µL Matrigel) or older BMSCs (older BMSC exosomes; 4 × 107 particles per 200 µL Matrigel). (B) After 3 weeks, the Matrigel plugs were harvested and photographed. Paraffin-embedded sections of Matrigel plugs were stained with hematoxylin and eosin and then subjected to immunostaining for CD138 (brown) and CD31 (red). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 500 µm. (C) Quantification of vessel density in Matrigel plugs by pixel density. Young and older BMSC exosomes inhibited tumor angiogenesis in Matrigel plugs compared with the control (RPMI8226-HR only) (*P < .01; **P < .001 vs control, Student t test, n = 6). Values represent the mean ± SD. (D) Effect of BMSC exosomes on the viability of HR-MM cells. RPMI8226-HR cells were cultured with young or older BMSC exosomes (2.5 × 107 particles/mL). Cell viability was assayed after 48 h using Cell Counting Kit-8. Values represent the mean ± SD. (E) The formation of tube-like structures was observed using the cell-permeable dye Calcein AM (green). Endothelial tube formation of HUVECs treated with young BMSC exosomes, older BMSC exosomes, or the control (without exosomes). Scale bar, 500 µm. (F) Quantification of tube-like structures by pixel density. Young and older BMSC exosomes significantly inhibited HUVEC tube formation in comparison with the control (control vs young BMSC exosomes; P < .001, control vs older BMSC exosomes; P = .038, Student t test). Values represent the mean ± SD.
Tomohiro Umezu et al. Blood Adv 2017;1:
© 2017 by The American Society of Hematology

5. Differential miRNA expression profiles of exosomes derived from young and older BMSCs. (A) Differential miRNA expression of young and older BMSC exosomes was determined by TaqMan miRNA array analysis.
Differential miRNA expression profiles of exosomes derived from young and older BMSCs. (A) Differential miRNA expression of young and older BMSC exosomes was determined by TaqMan miRNA array analysis. (B) Schematic of the modification of older BMSC exosomes. Young BMSC exosome-specific miRNA mimics were transfected directly into older BMSC exosomes by Exo-fect. (C) Quantification of tube-like structures to determine the antiangiogenic effect of modified exosomes. Older BMSC exosomes transfected with miR-340 mimics (miR-340 exosomes) and older BMSC exosomes transfected with miR-365 mimics (miR-365 exosomes) significantly inhibited HUVEC tube formation in comparison with the control (older BMSC exosomes transfected with negative control miR; control miR) (control vs miR-340 exosomes; *P < .01, Student t test; **P < .001, control vs miR-365 exosomes). Values represent the mean ± SD. Ct, comparative threshold cycle.
Tomohiro Umezu et al. Blood Adv 2017;1:
© 2017 by The American Society of Hematology

6. Antiangiogenic effects of modified exosomes in vivo.
Antiangiogenic effects of modified exosomes in vivo. (A) Exosomal miR-340 expression levels in young BMSC exosomes, older BMSC exosomes, and miR-340 exosomes were quantified by reverse transcription polymerase chain reaction (young BMSC exosomes vs older BMSC exosomes; *P < .01, young BMSC exosomes vs miR-340 exosomes; **P < .001, Student t test). Values represent the mean ± SD. (B-C) Recipient cells were treated with exosomes directly transfected with Cy3-miR-340 mimics. Cy3-miR-340 signals were detected in the cytoplasm of RPMI8226-HR cells (B) and HUVECs (C). Nuclear and cytoplasmic staining were performed with DAPI (blue) and Calcein AM (green), respectively. Scale bar, 25 µm. (D) A mixture of Matrigel and 4 × 106 RPMI8226-HR cells was admixed with older BMSC exosomes directly transfected with miR-340 mimics (miR-340 exosomes; 4 × 107 particles per 200 µL Matrigel), miR-365 mimics (miR-365 exosomes; 4 × 107 particles per 200 µL Matrigel), or negative control miR (control miR exosomes). The Matrigel mixture was injected subcutaneously into nude mice. Three weeks after implantation, the Matrigel plugs were harvested. Paraffin-embedded sections of Matrigel plugs were stained with hematoxylin and eosin and then subjected to immunostaining for CD31 (red). Scale bar, 500 µm. (E) Quantification of vessel density in Matrigel plugs by pixel density. Both mir-340 and miR-365 exosomes inhibited tumor angiogenesis in Matrigel plugs in comparison with the control (*P < .001 vs control, Student t test, n = 6). Values represent the mean ± SD. Exo, exosomes.
Tomohiro Umezu et al. Blood Adv 2017;1:
© 2017 by The American Society of Hematology

7. p-cMET and HGF protein levels vary in HUVECs cocultured with HR-MM cells.
p-cMET and HGF protein levels vary in HUVECs cocultured with HR-MM cells. (A) Western blots showing the expression of cMET and HGF in MM cells (RPMI8226 and RPMI8226-HR) and HUVECs. (B) The intensities of cMET and HGF bands in panel A were quantified and normalized to the levels of β-actin (*P < .01 vs RPMI8226, Student t test, n = 3). (C) Schematic of the nonadherent coculture system of MM cells (blue) and HUVECs (green). MM cells and HUVECs were cocultured separately using a Transwell filter (polycarbonate membrane insert, 0.45-µm pores). (D) cMET, p-cMET, and HGF levels in HUVECs were measured by Western blotting after coculture with RPMI8226 or RPMI8226-HR cells under hypoxic conditions (1% O2). (E) The intensities of cMET, p-cMET, and HGF bands in panel D were quantified and normalized to the levels of β-actin (*P < .01; **P < .001 vs control (HUVECs only), Student t test, n = 3).
Tomohiro Umezu et al. Blood Adv 2017;1:
© 2017 by The American Society of Hematology

8. Regulation of HGF/cMET signaling in endothelial cells by exosomal miR-340 derived from young BMSCs. (A) Representative Western blots of the expression of cMET and HGF in HUVECs after treatment with older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) or negative control miR (control miR exo.).
Regulation of HGF/cMET signaling in endothelial cells by exosomal miR-340 derived from young BMSCs. (A) Representative Western blots of the expression of cMET and HGF in HUVECs after treatment with older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) or negative control miR (control miR exo.). (B) The hsa-miR-340-binding site spanning nucleotides 1487–1508 (accession no. NM_000245) of the human cMET 3′-UTR was predicted to be evolutionarily conserved. The seed sequence of hsa-miR-340 (AAAUAU) is shown in red. (C) Luciferase reporter assay using a reporter plasmid containing the cMET 3′-UTR with the miR-340-binding site. The luciferase reporter vector to assess miR-340-specific activity contained complementary miR-340 sequences in its 3′-UTR. Normalized firefly luciferase activity was obtained according to firefly luciferase activity/β-gal activity. Sensor vector: the luciferase activity of HUVECs/Luc/β-gal (HUVECs co-transfected with luciferase reporter and β-gal control vectors) treated with young BMSC exosomes was significantly reduced in comparison with control cells (HUVECs only, *P < .01, n = 3). miR-340 exosomes (older BMSC exosomes transfected with miR-340 mimics) also reduced the luciferase activity in HUVECs in comparison with control miR exosomes (older BMSC exosomes transfected with negative control miR) (#P < .01, Student t test, n = 3). Using the mutated sensor vector, there was no difference in luciferase activity with or without exosomes. (D) Schematic of the endothelial cell tube formation assay to confirm the antiangiogenic effect of miR-340 exosomes. HUVECs were cultured on Matrigel in 3 types of medium (basal medium, basal medium supplemented with conditioned medium from RPMI8226-HR cells, or basal medium supplemented with HGF). (E) Endothelial tube formation in response to older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) observed under a bright field by phase-contrast microscopy. Scale bar, 500 µm. (F) Quantification of tube formation as shown in panel E. The conditioned medium from HR-MM (RPMI8226-HR) cells and HGF induced endothelial tube formation in vitro. Induction of tube formation by conditioned medium of HR-MM cells and HGF was inhibited by older BMSC exosomes transfected with miR-340 mimics (miR-340 exosomes) in comparison with the control (older BMSC exosomes transfected with negative control miR; control miR exosomes) (*P < .01, Student t test). Values represent the mean ± SD. (G) Immunohistochemical staining of CD31 (red) and p-cMET (green) in Matrigel plugs seeded with RPMI8226-HR cells at 3 weeks after transplantation into nude mice. Scale bar, 200 µm. med., medium.
Tomohiro Umezu et al. Blood Adv 2017;1:
© 2017 by The American Society of Hematology