3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFβRII-SMADS pathway

Background Exosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo. In our study, exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo. Results In vitro, PKH26 labeled-hESC-Exosomes were shown to be internalized and integrated into TGFβ-activated-LX2 cells, and reduced the expression of profibrogenic markers, thereby regulating cellular phenotypes. TPEF imaging indicated that PKH26-labeled-3D-hESC-Exsomes possessed an enhanced capacity to accumulate in the livers and exhibited more dramatic therapeutic potential in the injured livers of fibrosis mouse model. 3D-hESC-Exosomes decreased profibrogenic markers and liver injury markers, and improved the level of liver functioning proteins, eventually restoring liver function of fibrosis mice. miRNA array revealed a significant enrichment of miR-6766-3p in 3D-hESC-Exosomes, moreover, bioinformatics and dual luciferase reporter assay identified and confirmed the TGFβRII gene as the target of miR-6766-3p. Furthermore, the delivery of miR-6766-3p into activated-LX2 cells decreased cell proliferation, chemotaxis and profibrotic effects, and further investigation demonstrated that the expression of target gene TGFβRII and its downstream SMADs proteins, especially phosphorylated protein p-SMAD2/3 was also notably down-regulated by miR-6766-3p. These findings unveiled that miR-6766-3p in 3D-hESC-Exosomes inactivated SMADs signaling by inhibiting TGFβRII expression, consequently attenuating stellate cell activation and suppressing liver fibrosis. Conclusions Our results showed that miR-6766-3p in the 3D-hESC-Exosomes inactivates smads signaling by restraining TGFβRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESC-Exosome enriched-miR-6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease. These results also proposed a significant strategy that 3D-Exo could be used as natural nanoparticles to rescue liver injury via delivering antifibrotic miR-6766-3p. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-021-01138-2.


Methods hESC Culturing
The hESC line was cultured in 2D and 3D conditions respectively, brifly, hESCs dissected from culture condition with feeder cells [47] were moved to the Matrigel-coated plate for 2D culture and the ultra-low attachment plate (Corning) for 3D culture, and cultured with serum-free mTesR1 medium (STEMCELL).

Exosome Isolation
2D-Exo and 3D-Exo were isolated as previously described [20]. Supernatants were collected from hESC culture in mTesR1 medium without FBS and subsequently subjected to sequential centrifugation steps at 500 g for 10 minutes to remove cells, at 2000 g for 20 minutes to remove apoptotic bodies, and at 5000 g for 30 minutes to remove cell debris. The resulting supernatant was then filtered using 0.22 μm filters (Millipore, Merck, Germany), and hESC-Exosomes were harvested by centrifugation at 130000 g for 2 hours in a SW32 Ti rotor (Beckman Coulter, L-100XP Ultracentrifuge, CA). The pellet was resuspended in PBS and subsequently ultracentrifuged at 130000 g for another 2 hours to remove the contaminating proteins.

Transmission Electron Microscope (TEM)
TEM (Talos F200C, Thermo Fisher, MA) was performed at 200 kV to visualize and examine the morphology of hESC-Exosomes. Samples were deposited on copper grids covered with a carbon support film (Zhongjingkeyi Technology, Beijing, China) 7 and dried for 2 minutes at room temperature. The excess fluid was removed with a piece of filter, and the samples were negatively stained with 2% uranyl acetate for 30 seconds. Finally, these samples were air-dried for 60 minutes, and images were captured.

Nanoparticle Tracking Analysis
The size, concentration, and zeta potential of hESC-Exosomes were determined using NTA (Particle Metrix, Germany). The parameters of the measurement were set using 100 nm polystyrene-latex beads as standards. hESC-Exosomes were prepared by the dilutions at 4000-and 2000-folds using distilled deionized water respectively, to achieve the objects at the number between 20 and 100 per frame. Each sample was measured in triplicate at the camera setting with an acquisition time of 60 seconds.

Western Blot
The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL) according to the manufacturer's instructions.

hESC-Exosome Labeling
Purified 2D-Exo and 3D-Exo were labeled with PKH26 Red Fluorescent Cell Linker Kit according to the manufacturer's protocol. The extracted hESC-exosomes and lipophilic red fluorescent dye PKH26 (4 μM) (Sigma, MIDI26) were fully mixed and incubated at 37 ℃ for 30 minutes. After incubation, the mixture was sucked into a 100 KDa MWCO ultra-filtration centrifuge tube, 10 times the volume of PBS, was gently blown and mixed, centrifuged at 4 ℃ for 30 minutes, the unbound PKH26 dye was removed, the concentrated liquid in the ultra-filtration centrifuge tube was collected, and the bacteria were removed by a 0.22 μm filter. The filtered concentrated liquid was moved into the new EP tube and preserved at 4 ℃.

Activation of LX2 Cells and Treatment with hESC-Exosomes
In the logarithmic proliferation phase of LX2 cells, the cells were digested and subcultured by trypsin. 1.25×10 5 cells per well were seeded in the six-well plate, and 9 the microslide treated with polylysine were placed in the dish pool. When LX2 cells were adherent to the wall (> 6 hours), TGF-β1 (TGFβ, Peprotech, 100-21-50) was added to induce activation of LX2 cells at the final concentration of 10ng/mL for 48 hours. 20 μg of 2D-Exo and 3D-Exo labeled with PKH26 (4 μM; λex = 565 nm, λem = 594 nm) were respectively added. The same volume of PBS was added to the control group. The cells were examined 24 hours after treatment.

Scanning Microscopy (LSM)
LX2 cells were cultured in 6-well plates with the cell number at 1×10 5 cells per well and adhered overnight. PKH26-labeled 2D-Exo and 3D-Exo were respectively added into LX2 cells and incubated at 37 ℃ in dark for 24 hours, discarded culture medium, and washed with PBS for 3 times. Afterward, the cells were fixed using 4% paraformaldehyde (PFA) (Sangon Biotech, Shanghai, China) for 10 minutes, and then washed with PBS three times. The fixed cells were incubated with primary antibody against α-Tubulin (CST, 3873s) overnight at 4°C, and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (CST, 4412s). The cell nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) for 10 minutes and imaged using LSM (Ni-E-A1, Japan).

Determination of Intracellular Calcium Concentration
Differently treated-LX2 cells were washed three times with PBS, and incubated with Fluo-3 AM (Beyotime, S1056) at 5 mM at 20-37 ℃ for 10-60 minutes, then washed 10 for 3 times with PBS. Fluo-3 fluorescence was detected by flow cytometry to determine the changes in intracellular calcium concentration.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Eick-End Labeling (TUNEL) assay
A TUNEL assay was performed to evaluate the apoptotic cells of liver tissues. The tissues were fixed with 4% PFA and post-fixed in a pre-chilled solution containing ethanol and acetic acid mixed at 2:1 for 5 minutes at −20°C. After fixation, samples were processed according to the manufacturer's instructions (Beyotime, C1086) and imaged using LSM (Ni-E-A1, Japan). Quantitative analysis was accomplished using ImageJ software (National Institutes of Health, USA).

Wound Healing Assay
5×10 5 LX2 cells were seeded in the six-well plate, when the cells were adherent (> 6 hours), TGFβ was added to induce activation of LX2 cells at the final concentration of 10 ng per mL for 48 hours, 20 μg of 2D-Exo and 3D-Exo were respectively added and incubated with LX2 cells. After culturing for 24 hours, the LX2 cells were scratched with a pipette tip, and the cells were then washed three times with PBS to remove the scraped cells, finally, serum-free medium was added for culturing with 5% CO2 at 37 ℃. The scratched areas were imaged and measured using NIH Image J at 0 hour and 24 hours (n = 3) CFSE assay 11 LX2 cell proliferation rate in four different groups were also determined using flow cytometric analysis with a CellTrace CFSE Cell Proliferation Kit ( 1x10 6 cells/ml in 5μM of CFSE, 0.1% BSA in PBS for 10 minutes, Biolengend). The reaction was terminated by the addition of 10 volumes of cold RPMI 1640 with 10% FBS. Finally, the CFSE fluorescent staining was analyzed by flow cytometry. CFSE assays were performed in triplicate. When cells undertook divisions, the CFSE was distributed to daughter cells equally and CFSE fluorescence decreased in daughter cells. Briefly, the progressive loss of CFSE fluorescence is a hallmark of the cell proliferation.

Cell Viability Assay
Briefly, LX2 cells in the above groups were seeded at 5,000 cells per well in a 96-well plate and allowed to settle overnight. Cell proliferation of LX2 cells was measured using a Cell Counting Kit-8 (Beyotime, C0037). CCK-8 reagent was added to each well and incubated for 1, 2, 4 and 6 hours respectively. Absorbance was read at 450 nm and recorded using a microplate spectrophotometer.

Isolation and Charactwerization of Mouse Primary Hepatic Stellate Cells
Primary mouse hepatic stellate cells (mHSCs) were isolated from ICR mice as described previously [27], and freshly-isolated primary mHSCs were cultured in DMEM supplemented with 10% FBS. mHSCs were characterized by Oil red staining and immunofluorescent staining for the deposit of the lipid droplets and cell type specific marker Desmin.
Teratomas generally developed within 6-8 weeks, and the animals were sacrificed before the tumor sizes exceeded 1.5 cm in diameter. The teratomas were then embedded in paraffin and processed for hematoxylin and eosin (HE) staining.

Immunofluorescence Staining
The cells were fixed in 4% PFA at room temperature for 15 minutes and blocked with blocking buffer that contained 0.2% Triton X-100 (Sigma-Aldrich, T8787) and 3% normal goat serum (Jackson Immuno Research, 017-000-121) in PBS at room temperature for 45 minutes, then the cells were incubated with primary antibodies at 4°C overnight. Next day, primary antibodies were replaced with secondary antibodies conjugated with fluorescences (CST, 7074s and 7076s) after washing with PBS, and the cells with antibodies were incubated at room temperature for 1 hour. The nuclei were stained with DAPI. The primary and secondary antibodies were listed in Supplemental Table S4.

Flow cytometric Analysis
Single cell suspensions from 2D-hESCs and 3D-hESCs culturing were prepared using GCDR (STEMCELL, 07174), washed in PBS, then resuspended in 0.1% BSA in PBS. Anti-TRA-1-81-PE and anti-SSEA4-PE (STEMCELL Technologies) and PE 13 Mouse IgG1 κ Isotype Control (BD) were added to cell suspensions to incubate at 4 ℃ for 30 minutes in the dark. Intracellular staining for OCT4 were performed according to the manufacturer's instructions (STEMCELL Technologies). Cells were examined by flow cytometry. Data were analyzed using Flowjo software (Flowjo, Ashland, USA).

Detection of Serological Markers and Liver Injury in Fibrotic Mice
4 weeks after intravenously receiving CCL4 and 56% alcohol gavage, the mice fasted 12 hours before they were sacrificed. Serum was collected by centrifugation of eyeball blood for testing Aspartate Transaminase (AST) (Solarbio, BC1560), Alanine Ek-M20152) and Total bilirubin (TBIL) (GTX ， YS01266B) using an automatic biochemical analyzer. Liver tissue samples were retained separately, and fixed in 4% PFA for histopathological analysis. In addition, the remaining liver tissues were first placed in liquid nitrogen for quick cryopreservation and then transferred into -80 ℃ for storage.

Two-photon excited fluorescence (TPEF) imaging of PKH26-lebeled hESC-Exosomes Distributed in Various Organs of Mice in vivo and ex vivo
The hepatic fibrosis model mice were injected intravenously with 100 μg of PKH26-labeled 2D-Exo and 3D-Exo as well as equal volume PBS (control group).
The mice anesthetized at differetn time points, and tissue samples from mice were imaged to collect fluorescence in stack scan mode by TPEF (Ni-E-A1RMP) with a 14 laser at wavelengths of 1080 nm for PKH26 imaging. During the imaging process, each sample were scanned continuously (a frame of a 2D image) for PKH26 simultaneously at a speed of 10 second per frame. The exposure time of the samples was limited within 30 minutes. The intensities of the signals were analyzed using Image J software.

RNA Extraction and Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from cells and tissues using RNAiso Plus (Takara,9109) according to the manufacturer's manuals, MiRNAs of 2D-Exo and 3D-Exo were isolated using the SeraMir Exosome RNA Purification Column Kit (EZBioscience, EZB-exo-RN1). For mRNA detection, complementary DNA (cDNA) was synthesized from 500 ng of total RNA using a PrimeScript™ RT Master Mix (Takara); for miRNA expression analysis, 500 ng total RNA was reverse-transcribed into cDNA using Mir-X™ miRNA First Strand and Synthesis kit (Takara) as described by the manufacturer's protocol. All qPCR experiments were performed on a real-time PCR machine (ABI, ABI7500) with the QuantiTect SYBR Green PCR Kit (Takara) and specific primers of the genes of interest, and amplification efficiencies were checked by standard curves. Gene expression was quantified by the comparative cycle threshold (Ct) method. The relative amounts of target gene expression were determined by subtracting the Ct values of these genes from the Ct value of the housekeeping gene GAPDH and the abundance of U6 which was used as an internal control for comparison of relative changes in miRNA among different groups (△Ct). 15 The data were presented as 2 −(ΔΔCT) . The primer sequences used in this study were listed in Supplemental Table S1.

Histology, Immunohistochemistry (IHC) and Immunofluorescence (IF) Assays
On day 7, 14, 21 and 28 after administration of PBS, 2D-Exo and 3D-Exo, all mice were sacrificed, and the livers were isolated. Liver tissues were immediately fixed in 4% PFA overnight. Afterward, these tissues were embedded in paraffin and sectioned with a thickness of 5 μm, followed HE or Masson's trichrome staining, or ICH assay.
The slices obtained were examined with an optical microscope (Nikon, Japan).
Furthermore, another fraction of liver tissue was embedded into OCT compound (Sakura Finetek, Japan) and cut into micron-thick frozen sections with the thickness at 10 μm for IF staining. In brief, the sections for ICH and IF staining were incubated with primary antibodies, and then incubated with secondary antibodies. Cell nuclei in IF staining were counterstained with DAPI. Image J software was used to binarize the immunofluorescence images taken as previously described. 25 The numbers of positive cells were counted by blinded investigators in 6 randomly selected areas. The primary and secondary antibodies were listed in Supplemental Table S4.

miRNA Microarray and Data Analysis
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v14.9 software package (Agilent Technologies). After quantile normalization of the raw data, miRNAs that at least 3 out of 6 samples have 16 flags in Detected ("All Targets Value") were chosen for further data analysis.
Differentially expressed miRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed miRNAs between the two samples were identified through Fold Change filtering. Hierarchical Clustering was performed using the R scripts. Fold change ≥ 2.0 or ≤ 0.5 and P<0.05 were the threshold to filtrate differentially expressed miRNAs. The primer sequences used in this study were listed in Supplemental Table S2 and S3.

Target Prediction
Microarray analysis of miRNAs from 2D-Exo and 3D-Exo, including sample labeling,

Dual Luciferase Reporter Assay
For identifying the binding site between miR-6766-3p and its target TGFβRII, the 3'

RNA Interference and Transfection
The LX2  Quantification of cell cycle analysis by flow cytometry which was used to measure the growth of mHSCs and activated mHSCs exposed to 2D-Exo or 3D-Exo. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. were quantitated in the liver tissues of the fibrosis mice with different treatments.
Gapdh was used as a loading control. n=3, Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (a) Cell cycle analysis showed that the growth of activated LX2 cells exposed to (e-f) The decrease of wound recovery rates of active LX2 cells was inhibited by the knockdown of miR-6766-3p with the inhibitor, compared with the cells treated with miR-6766-3p inhibitor NC, as determined by cell scratch assays. n=3, Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. The expression level of SMADs genes was increased in activated LX2 cells after co-culture with miR-6766-3p inhibitor for 48 hours when compared to those co-culture with miR-6766-3p inhibitor NC. n=3, Data represent the mean ± SEM.