Human umbilical cord mesenchymal stromal cell-derived exosomes protect against MCD-induced NASH in a mouse model

Reagents

Serum-free additive was purchased from Elitecell Biomedical Corp. (EPA-050, Texas, USA), complete medium was purchased from Darkway Biotechnology Co., Ltd. (6,114,021, 6,114,541, 6,114,551, 6,114,531, Beijing, China), PBS was purchased from HyClone Co. Ltd. (SH30406.05IR30-40, South Logan, UT, USA), and trypsin was purchased from Gibco Invitrogen Co. (New York, NY, USA). PKH26 was purchased from Sigma–Aldrich (PKH26PCL, San Francisco, CA, USA). Oxidized low-density lipoprotein (Ox-LDL) was purchased from Yiyuan Biotechnology (YB-001, Guangzhou, China). The primary antibody targeting F4/80 was purchased from Abcam (ab100790, Cambridge, UK). Antibody for FACS includes PE anti-human CD73 (clone AD2, Biolegend), FITC anti-human CD90 (clone 5E10, Biolegend), PE anti-human CD105 (clone 266, BD Pharmingen), FITC anti-human CD19 (clone HIB19, Biolegend), FITC anti-human CD34 (clone 581, Biolegend), FITC anti-human CD14 (clone M5E2, Biolegend) and FITC Mouse IgG1, κ (clone MOPC-21, Biolegend) PE Mouse IgG1, κ (clone MOPC-21, Biolegend) as isotype control. For Western blot, the primary antibody targeting both human and mouse PPARα was purchased from Boster Biological Technology Co. Ltd. (BA1691, Wuhan, China). The primary antibodies targeting histone H3, CD63, TSG101, CD9, GAPDH and alkaline phosphatase-conjugated secondary antibodies (SA00002-1 and SA00002-2) were purchased from Proteintech (Chicago, IL, USA). Cell lysis buffer and DAPI were purchased from Beyotime (Beijing, China). All enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). The methionine–choline-deficient (MCD) diet (MD12052) and control diet were purchased from Trophy Feed Technology Co., Ltd. (Jiangsu, China). The methionine and choline contents in the MCD feed are close to 0, and the fat content is 7%. The control feed had methionine and choline contents within the reference standard level and was otherwise matched with the model feed.

Human umbilical cord mesenchymal stromal cell (hUC-MSC) isolation and culture

The experimental hUC-MSCs were preserved in the International Joint Research Center of the National Human Stem Cell Bank. The cells were cultured to passages 4–7.

Identification of MSC adipogenic and osteogenic differentiation

Cells were seeded into 24-well culture plates at a density of 4 × 104 cells/well. After 2–3 days of culture, the growth of cells was observed. The lipid induction medium or osteogenic induction culture medium was replaced in the experimental cells, and the complete medium was replaced in the control cells. The 24-well plate was incubated in a CO2 incubator at a stable temperature, and the liquid medium was changed every 2–3 days. The growth, differentiation and morphological characteristics of the cells were observed. On the 16th day of induction culture, the cells were stained with either oil red O or Alizarin red, observed under an inverted microscope and photographed.

Identification of MSC differentiation into cartilage

The digested and collected cells were inoculated into a 15-mL centrifuge tube at a density of 2.5 × 105 cells/tube and centrifuged at 300 g at room temperature for 5 min. After forming microspheres, they were transferred to a 24-well cell culture plate. The chondrocyte induction medium was changed in the experimental wells. The liquid was changed every 2–3 days, and the growth, differentiation and morphological characteristics of the cells were observed. On the 21st day of induction culture, the tissue was stained with toluidine blue and washed once with PBS. Frozen sections of the tissue spheres were stained with toluidine blue for 5 min, washed with pure water twice, observed under an inverted microscope and photographed.

Preparation of exosomes

hUC-MSCs at passages 4–7 were cultured to 90% confluence in 10-cm-diameter dishes. The adherent cells were incubated in 5% vesicle-depleted fetal bovine serum (FBS) for at least 24 h. Then, the conditioned medium was collected and centrifuged at 4 °C at 2000 g for 10 min to remove the cells and at 10,000 g for 5 min to remove debris. hUC-MSC-derived exosomes were extracted using an exoEasy Maxi Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The morphology of MSC exosomes was examined using transmission electron microscopy (TEM). The expression of CD9, Tsg101 and CD63 was confirmed by western blot.

Cell culture

HepRG cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). HepRG cells were cultured in RPMI 1640 (72,400,120, Gibco, USA) supplemented with 10% FBS (04-001-1A, BI, Israel). The mouse liver cell line Huh1-6 was preserved in our laboratory, and the Raw264.7 mouse macrophage line was a gift from the Institute of Immunization, Jilin University. Huh1-6 and RAW264.7 cells were cultured in DMEM (01-052-1ACS, BI, Israel) supplemented with 10% FBS (04-001-1A, BI, Israel). All cells were incubated at 37 °C with 5% CO2.

Animal treatment

C57BL/6 J mice (male) with body weights of 22–25 g were purchased from Vital River Laboratories (Beijing, China). All mice were maintained under standard light conditions (12-h light/dark cycle) at room temperature. After 7 days of adaptive feeding, the mice were randomly divided into four groups. Group 1 was fed regular chow (wild-type). Group 2 was the disease model group which were fed a methionine–choline-deficient (MCD) diet without any intervention. In the other two groups, the mice were fed an MCD diet and received tail vein injections once a week of either conditioned medium extract (using an exosome preparation protocol) or exosomes. Each mouse was injected with 20 mg/kg body weight hUC-MSC-derived exosomes twice a week. MCD feeding lasted for 6 weeks. At the end of the experiment, animals were injected intraperitoneally with pentobarbital sodium (1% v/v) for anesthesia. Peripheral blood was collected and centrifuged to obtain serum for biochemical analyses and cytokine measurements. The fresh liver was excised and stored at − 80 °C for further use. Fixed tissues in each group were used to prepare pathological sections. The protocol was performed as indicated. All procedures were in accordance with the Institutional Guidelines for Animal Experiments.

Histological analysis

Liver samples from each mouse were fixed in 10% (v/v) formalin and then dehydrated in a series of gradient alcohol washes. The tissues were cleared in xylene and embedded in paraffin. Sections were approximately 3 μm and stained with hematoxylin and eosin (H&E). The levels of F4/80 and NF-κB(p65) in the tissues were analyzed by immunohistochemistry as previously described [24]. For the histological diagnosis of NASH, the NAFLD activity score was evaluated by a pathologist using standards as previously described [25].

Biochemical analyses

The levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in the serum were measured by a Roche Cobas testing assay.

Western blot analysis

Both tissue and cell protein concentrations and extracted exosomes were detected using a BCA assay kit. The lysates were separated by 12% (w/v) SDS–PAGE. After electrophoresis, the proteins were transferred to nitrocellulose (NC) membranes and blocked with 5% (w/v) bovine serum albumin (BSA) in phosphate-buffered solution for 1 h at room temperature. The membranes were probed with the indicated primary antibodies for 4 h at room temperature, washed three times and incubated with AP-conjugated secondary antibodies for 1 h at room temperature. The NC membranes were washed with PBST (PBS with 0.5% Tween-20) three times after each hybridization step. Protein levels were detected by a BCIP/NBT alkaline phosphatase color development kit.

Enzyme-linked immunosorbent assay (ELISA)

Inflammatory cytokine levels in plasma were measured using ELISA kits (R&D) specific for mouse TNF-α, IL-1 and IL-6, according to the manufacturer’s instructions. The absorbance of the plates was read at 450 nm. The cytokine levels were measured and calculated by standard curves prepared with the respective recombinant cytokines.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from mouse liver tissue, and single-stranded cDNA was synthesized with a reverse transcription kit (Takara Bio, Kusatsu, Japan). Real-time quantitative polymerase chain reaction (PCR) analysis was conducted with SYBR Green PCR master mix (Takara Bio, Kusatsu, Japan). GAPDH was used as a reference gene in each sample. All primers were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The primers are presented in Table 1.

Table 1 The primer name and the sequencesStatistical analysis

Statistical analyses were performed by Prism software (GraphPad 5.0). Student’s t test was performed to compare experimental and relative control groups. Statistical p values < 0.05 were considered significant, and p values < 0.01 were considered extremely distinct. Statistical p values > 0.05 were considered nonsignificant (NS).

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