Research ArticleHEALTH AND MEDICINE

Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes

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Science Advances  25 Mar 2020:
Vol. 6, no. 13, eaay6721
DOI: 10.1126/sciadv.aay6721
  • Fig. 1 Characterization of HASC-proliferating EVs (P-EV), HASC-white adipogenic differentiating EVs (D-EV), and HASC-beige adipogenic differentiating EVs (BD-EV).

    (A) TEM and (B) cryo-TEM images of P-EV, D-EV, and BD-EV. (C) Immunoblotting for CD9, CD63, ALIX, and TSG101 of P-EV, D-EV, and BD-EV. (D) The size distributions of P-EV, D-EV, and BD-EV were analyzed by dynamic light scattering. (E) Confocal laser scanning microscopic images of HASCs after the 3-hour incubation with 1 × 108 particles of DiD-labeled D-EV and PKH26-labeled P-EV and BD-EV. The nontreated group was used as control. Scale bar, 20 μm. (F) WST-1–based colorimetric assay to quantify proliferation and viability of HASCs after 1 and 3 days of culture with D-EV and BD-EV (*P < 0.001). Data are shown as the means ± SD from three separate experiments. GM, growth medium; BDM, beige adipogenic differentitation medium.

  • Fig. 2 Adipogenic capacity of D-EV and BD-EV.

    (A) Oil red O–stained and (B) phase-contrast microscopic images of HASCs treated with 1 × 109 particles/ml of P-EV, D-EV, and BD-EV for 2 weeks. Differentiation medium (DM) and growth medium were used as positive control and negative controls, respectively. Scale bars, 100 μm. (C) Oil red O intensity of lipid droplets in each group. All values are expressed as the means ± SD of four independent trials (**P < 0.0001). (D) Adipokines within P-EV versus D-EV and BD-EV measured using adipokine array (*P < 0.05). Both down-regulated and up-regulated adipokines were identified in each EV. (E) Fluorescent plots for adipokine array. N.S., not significant; a.u., arbitrary units; ANGPTL4, angiopoietin-like 4.

  • Fig. 3 In vivo adipose tissue regeneration using D-EV–containing hydrogels.

    (A) Schematic illustration of in vivo hydrogel injection into the back of each BALB/c mouse (6 weeks old, n = 9). (B) At 4 weeks after subcutaneous injection, the mice were euthanized, and the grafts were explanted. Scale bar, 1 cm. (Photo credit: Youn Jae Jung and Kyoung Soo Lee, ExoStemTech Inc. and Hanyang University ERICA.) (C) Histological evaluation of the grafts and normal mouse skin tissues stained by Oil red O at 4 weeks after injections. Scale bars, 100 μm. (D) Histological evaluation of the grafts and normal mouse skin tissues stained by H&E staining. Scale bars, 100 μm. (E) qRT-PCR analysis for relative mRNA expression of adipogenic Leptin, Fabp4, and Pparg. Data are shown as means ± SD from three separate experiments (*P < 0.05; **P < 0.01; ***P < 0.001).

  • Fig. 4 Adipose tissue browning induced by treatment with BD-EV.

    (A) Body weights of C57BL/6 mice at indicated times under high-fat diet (HFD) (n = 8 each group). (B) Body weights of C57BL/6 mice at 8 weeks after injection. (C) Photographs of C57BL/6 mice at 8 weeks after injection. (Photo credit: Youn Jae Jung and Chang Hee Woo, ExoStemTech Inc. and Hanyang University ERICA.) (D) Representative images of H&E staining of interscapular brown adipose tissue (iBAT) from 16-week-old HFD-fed mice treated with PBS, P-EV, and BD-EV for 8 weeks. Normal chow diet (NCD)–fed mice used as controls. (E) Representative images of sections stained with anti-UCP1 antibody of iBAT from 16-week-old HFD-fed mice treated with PBS, P-EV, and BD-EV for 8 weeks. (F) qRT-PCR analysis for relative mRNA expression of adipose tissue browning (Ucp1, Dio2, Ppargc1a, and Prdm16). These experiments were performed in triplicate. P values are shown in the figures.

  • Fig. 5 Administration of BD-EV ameliorates hepatic steatosis in HFD-fed mice.

    Photographs (A) and H&E-stained images (B) of liver tissue from 16-week-old mice treated with PBS, P-EV, and BD-EV for 8 weeks. NCD-fed mice used as control. (Photo credit: Youn Jae Jung and Chang Hee Woo, ExoStemTech Inc. and Hanyang University ERICA.) (C) Glucose tolerance test was performed after 12 hours of fasting of 16-week-old mice. (D) Calculated area under curve during glucose tolerance test (n=5 per group). (E) Quantification of liver tissue weight from 16-week-old mice treated with PBS, P-EV, and BD-EV for 8 weeks. (F) Liver triglycerides were quantified from liver tissues from 16-week-old mice treated with PBS, P-EV, and BD-EV for 8 weeks. NCD-treated mice were used as controls (n = 3 per group). P values are shown in the figures.

  • Fig. 6 EV miRNA profiling of P-EV and BD-EV.

    (A) Hierarchical cluster analysis of the significantly expressed miRNAs from P-EV and BD-EV. (B) Volcano plot of small RNA sequencing–identified miRNAs in BD-EV versus P-EV. (C) Numbers of differentially up-regulated and down-regulated count by fold change and P value. (D) GO and KEGG pathway analyses of the target genes of the up-regulated and down-regulated miRNAs. (E) Expression levels of miRNA was confirmed in P-EV and BD-EV using qPCR (n = 4). P values are shown in the figures. BP, biological processes; CC, cellular components; MF, molecular functions.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/13/eaay6721/DC1

    Supplementary Materials and Methods

    Fig. S1. White/beige adipogenic differentiation of HASCs.

    Fig. S2. Human adipogenesis PCR array.

    Fig. S3. UCP1 assay.

    Fig. S4. Immunohistochemistry of the grafts treated with P-EV and D-EV.

    Fig. S5. High-performance liquid chromatography.

    Fig. S6. Adipose tissue browning in inguinal white adipose tissue.

    Fig. S7. Overall schematic diagram for exosomes derived from human adipose stem cells during white/beige adipogenic differentiation for cell-free therapeutic systems.

    Table S1. The information of HASCs purchased from Cefo Bio Co. Ltd.

    Table S2. Sequences of RT-PCR primers.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. White/beige adipogenic differentiation of HASCs.
    • Fig. S2. Human adipogenesis PCR array.
    • Fig. S3. UCP1 assay.
    • Fig. S4. Immunohistochemistry of the grafts treated with P-EV and D-EV.
    • Fig. S5. High-performance liquid chromatography.
    • Fig. S6. Adipose tissue browning in inguinal white adipose tissue.
    • Fig. S7. Overall schematic diagram for exosomes derived from human adipose stem cells during white/beige adipogenic differentiation for cell-free therapeutic systems.
    • Table S1. The information of HASCs purchased from Cefo Bio Co. Ltd.
    • Table S2. Sequences of RT-PCR primers.

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