Comparison of exosomes derived from induced pluripotent stem cells and mesenchymal stem cells as therapeutic nanoparticles for treatment of corneal epithelial defects

Characterization of iPSCs/MSCs-Exos

We collected exosomes from the supernatant of iPSCs and MSCs by differential ultracentrifugation and evaluated their size distribution by nanoparticle tracking analysis (NTA). We found that size distribution of iPSCs-Exos ranged from 30 to 120 nm while it was 60-400 nm in MSCs-Exos (Figure 1A). Transmission electron microscopy (TEM) revealed that the exosomes were round-shaped with a size distribution consistent with what was observed by NTA (Figure 1B). There is no significant difference between the size and morphology of exosomes derived from iPSCs and MSCs. Western-blot analyses revealed that the iPSCs-Exos and MSCs-Exos expressed exosomal markers including CD9 and CD63, while the intracellular protein Calnexin (negative markers for exosomes) was absent in the isolated exosomes (Figure 1C).

Uptake of iPSCs/MSCs-Exos by the corneal epithelium

To determine whether exosomes could be absorbed by HCECs, HCECs were incubated with 25 μg/ml DiI-labeled iPSCs/MSCs-derived exosomes for 24 h to optimize the internalization of exosomes by cultured cells. Photomicrographs showed that red fluorescent particles were present throughout the cell cytoplasm, meaning that the exosomes were taken up by HCECs (Figure 2A).

The uptake of exosomes was also verified in vivo following their topical application to the cornea of rats. After 24 h, whole mount imaging of the cornea showed a wide distribution of labeled exosomes throughout the mouse cornea indicating successful fusion and uptake of iPSCs/MSCs-Exos by the corneal epithelium in vivo (Figure 2B). There was no significant difference between iPSCs-Exos group and MSCs-Exos group in uptake. Exosomes and cell fusion were also verified by TEM. After adding drops of iPSCs/MSCs-Exos to the eyes of SD rats for 24 h, exosome-like vesicles were detected throughout the corneal epithelium. The diameters of vesicles were between 100-200 nm. We identified these vesicles as exosomes, rather than apoptotic bodies, based on their size (Figure 2C).

Effects of iPSCs/MSCs-Exos on HCECs in vitro

The cell morphology of HCECs was analyzed after HCECs were stimulated with iPSCs/MSCs-Exos or DMEM/F12 medium for 48 h. HCECs with DMEM/F12 exhibited signs of atrophy while those with iPSCs/MSCs-Exos showed a fuller shape (Supplementary Figure 1).

Annexin V-FITC/propidium iodide (PI) staining demonstrated the apoptotic rates of HCECs stimulated with iPSCs/MSCs-Exos (Figure 3A, 3B). The proportion of apoptotic cells was lowest in iPSCs-Exos group (7.027 ± 0.472%), HCECs apoptosis was less in MSCs-Exos group (12.65 ± 0.533%) than in control group (18.343 ± 1.732%).

Correspondingly, the proportion of living cell in iPSCs-Exos treated group is highest in three groups, which indicated that iPSC-Exos more strongly inhibited the apoptosis of HCECs.

In addition, to assess the effect of iPSCs/MSCs-Exos on corneal epithelial defect healing, a monolayer of confluent HCECs was scratched and then treated with 500 μg/ml iPSCs/MSCs-Exos for 12 h. Cell migration in monolayers treated with iPSCs-Exos was significantly accelerated, with 61.93 ± 5.487% migration area after 6 h, compared to that of the control which was 25.604 ± 4.212%. After 12 h, the migration area was 83.029 ± 2.454% in iPSCs-Exos group while it was 56.971 ± 3.531% in the control group. MSCs-Exos also increased the migration of HCECs (6 h: 49.467 ± 7.263%; 16 h: 71.374 ± 3.864%) compared with control (Figure 3C, 3D).

Moreover, we tested the cell viability of HCECs which showed a time-dependent growth trend in all groups. Treatment with iPSCs/MSCs-Exos increased cell viability at 24 h, 48 h and 72 h compared with the control. In each timepoint, cell proliferation was stronger in iPSCs-Exos group than that in MSCs-Exos group and was significantly different at 24 h (Figure 3E).

As the cell cycle is one of the major reasons for cell growth, then we further explored the effect of iPSCs/MSCs-Exos on cell cycle distribution of HCECs. Cells were treated with PI to stain DNA then sorted for PI levels by flow cytometry, generating a profile of the cell population indicative of their cell cycle stage. Treatment with 500 μg/ml iPSCs/MSCs-Exos for 24 h significantly increased the number of HCECs in the S phase, which was accompanied by a significant decrease in the number of cells in the G0/G1 phase (Figure 4A, 4B). Moreover, the proportion of cells in the S phase was higher in iPSCs-Exos group than it was in MSCs-Exos group. The results indicated that iPSCs-Exos promoted the cell cycle progression of HCECs, driving HCECs to exit the G0/G1 phase and enter the S phase.

It is evident that iPSCs/MSCs-Exos may induce proliferation of HCECs in vitro from the above results. To identify whether iPSCs/MSCs-Exos is strictly associated with cell proliferation on the molecular level, we used Ki-67 as a marker to determine the growth fraction of HCECs population. As shown in Figure 4C, nuclear protein Ki-67 was positively stained in HCECs and the fluorescence intensity was strongest in iPSCs-Exos group.

To determine which molecules participate in the regulation of iPSCs/MSCs-Exos on HCECs proliferation and cell cycle, HCECs treated with iPSCs/MSCs-Exos were subjected to qRT-PCR and western-blot analysis. As a result, we found that genes associated with HCECs proliferation marker PCNA were significantly increased at mRNA level within 24 h of exposure to 500 μg/ml iPSCs/MSCs-Exos. In addition, cyclin A and CDK2 which play important roles in the cell cycle, were robustly upregulated in response to iPSCs-Exos stimulation (Figure 4D), providing a molecular basis for the flow cytology results, while cyclin E showed no significant difference among the three groups. The protein levels of PCNA, cyclin A and CDK2 were consistent in terms of gene expression (Figure 4E).

iPSCs/MSCs-Exos accelerate corneal epithelial defect healing in vivo

Finally, we attempted to investigate the therapeutic potential of the iPSCs/MSCs-Exos for corneal epithelial defects in vivo using a mechanical injury model. iPSCs/MSCs-Exos or PBS were applied topically on the cornea following 2 mm epithelial debridement defect. Then, we monitored the defect of cornea for 48 h, with corresponding eye examinations at different time points (Figure 5A). We found that the defect area in iPSCs-Exos group decreased obviously compared with that in PBS group and had statistical significance at 12 h, 30 h, 36 h, 42 h and 48 h while the MSCs-Exos group had statistical significance only at 30 h. At 48 h, the healing area was 88.88 ± 2.925%, 84.38 ± 2.26% and 75.85 ± 2.875% in iPSCs-Exos group, MSCs-Exos group and control group respectively (Figure 5B, 5C).

Anterior segment optical coherence tomography (AS-OCT) scans were used to reveal the thickness of corneas during defect healing. The corneal thickness was restored after the removal of corneal epithelium. By measuring the central corneal thickness, we found that the corneas treated with iPSCs-Exos or MSCs-Exos were generally thicker than those in the vehicle group. The difference in central corneal thickness between iPSCs-Exos group and the vehicle group showed statistical significance at both 24 h and 48 h. At 48 h, the central corneal thickness was 241 ± 7.81 μm, 232.7 ± 8.11 μm and 217 ± 4.223 μm in iPSCs-Exos group, MSCs-Exos group and control group respectively (Figure 5D, 5E). With a 3D-pattern of OCT, the corneal epithelial defects were visualized stereoscopically after corneal epithelium deprivation for 36 h, the size and depth of corneal defects was smallest in iPSCs-Exos group (Figure 5F). The results demonstrated that iPSCs-Exos had a better effect on the restoration of corneal thickness after epithelium damage.

Hematoxylin and eosin (H&E) staining revealed the epithelial cell layers of the cornea. In PBS group, there were still some corneal epithelial defects in the central cornea, and 3-4 layers of corneal epithelial cells appeared in the peripheral cornea. In iPSCs/MSCs-Exos group, the corneal epithelium were integrated with 4-5 layers cells in MSCs-Exos group and 4-6 layers cells in iPSCs-Exos group (Figure 5G). This matched with the results of corneal thickness measured by AS-OCT.

Preparation of exosomes

Undifferentiated iPSC lines were maintained on Matrigel (Stemcell, US) treated six-well plates in PGM1 medium (Cellapy, China) at 37°C in a humidified incubator with 5% CO₂. Cells between passages 30 and 50 were used for exosomes collection. Human umbilical cord-derived MSCs were harvested and cultured as reported previously [42]. In brief, MSCs were cultured in mesenchymal stem cell serum-free medium (Yocon, China). MSCs between passages 3 and 7 were used for the subsequent experiments. HCECs were maintained in complete DMEM/F12 with 10% fetal bovine serum (HyClone, Australia).

Exosomes were isolated as previously described with minor modification [27]. Briefly, conditioned medium samples were centrifuged at 300 g for 10 min followed by 1500 g for 10 min at 4°C to remove cells and cellular debris. Next, the supernatant was filtered through a 0.22 μm bacterial filter (Corning Incorporated, US) for further purification of exosomes, and then the filtrate was ultracentrifuged at 110,000 g for 70 min at 4°C to pellet exosomes. The exosomes were then washed in a large volume of phosphate-buffered saline (PBS) and ultracentrifuged again at the same high speed for 70 min. The pelleted exosomes were resuspended in PBS.

Validation of exosomes

For subsequent TEM examination, exosome samples were prepared by a routine “negative staining” method. Exosomes were directly adsorbed onto Formvar-carbon-coated 400 mesh copper EM grids (Pelco, US) and were negatively stained using 2% aqueous phosphotungstic acid (PTA), then, they were air dried, prior to examination by JEM-1220 TEM (JEOL, Japan), operating at an accelerating voltage of 80 kv. To measure the size of exosomes, NTA was performed using the Nanosight system (Malvern Zetasizer, England) as described previously [43]. The capture settings and analysis settings were established manually according to the manufacturer’s instructions. Exosomal surface marker proteins CD63 and CD9 and the negative marker Calnexin were analyzed by western-blot [44]. HCECs were used as a negative control.

Exosome uptake

Cellular uptake of exosomes was assessed by confocal microcopy [45]. iPSCs-Exos and MSCs-Exos were labeled with DiI fluorescent dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Beyotime, China), which labels the plasma membrane. Briefly, exosomes were incubated with DiI dye for 1 h in the dark. Then labeled-exosomes were resuspended in 4 ml PBS followed by ultracentrifugation at 110,000 g for 1 h. The pellets were resuspended in 100 μl of PBS. HCECs were incubated with labeled exosomes for 24 h and then washed with PBS to remove unbound exosomes. Mounting medium with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, US) was used to stain nuclei, and images were captured under a confocal microscope (Carl Zeiss, Germany).

To examine the uptake of iPSCs/MSCs-Exos by the corneal epithelium in vivo, DiI-stained exosomes were applied topically to the cornea of rats, and assessment occured 24 h later. The cornea was then obtained from rats and stained with DAPI, mounted on a slide, and imaged with confocal microscopy. The corneal epithelium of rats was also analyzed by TEM with the same method described above.

Corneal epithelial defect model

Corneal defect experiments in rats were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Committee on the Ethics of Animal Experiments of the First Affiliated Hospital of Harbin Medical University. Six-week-old male SD rats were anesthetized by an intraperitoneal injection of with a mixture of ketamine (75 mg/kg) and xylazine (13.5 mg/kg) with only one eye treated under topical anesthesia (0.5% proparacaine). A circle defect (2 mm in diameter) was produced in the corneal epithelium of SD rats by an AlgerBrush II (The Alger Company, US) and reaver blade as previously described [46]. The wounded corneas were then treated topically every 6 h with 5 μl of exosome suspension, which contained 500 μg/ml iPSCs/MSCs-Exos or PBS. Defect closure was monitored with fluorescein staining and photographed with a camera-equipped slit lamp every 6 h until recovery. The percentages of defect closure were calculated based on the baseline for each mouse using ImageJ software (Rewak Software, Germany).

AS-OCT (Optovue, USA) was applied to rats at baseline, 24 h and 48 h after treatment to measure the variation in corneal thickness. Once anesthetized, rats were placed on the animal imaging mount. Horizontal lines scans through the corneal vertex were carried out on central fixation to show sections across the central and peripheral cornea. Corneal thickness was measured for the central region [47]. Thickness (μm) was measured as the distance from epithelium to endothelium, at the point where a vertical line was orthogonal to the anterior corneal curvature. For a better perspective of cornea epithelial wound morphology, each wound pattern was measured by rotating the 3D-reconstructed images for the central regions at 36 h.

Hematoxylin and eosin staining

The eyeballs harvested from rats after they were sacrificed were immediately fixed in 4% paraformaldehyde (PFA, Beyotime, China) overnight. The fixed samples were embedded in paraffin for performing the histological analysis. 6 μm thick sections were stained with H&E and examined under a light microscope. The whole cornea and the local corneal epithelium were observed under 4 × magnification and 40 × magnification, respectively.

Cell proliferation assay

To evaluate the proliferative capacity of HCECs treated with exosomes, HCECs were seeded at 1x10⁴ per well in 96-well culture plates. iPSCs/MSCs-Exos were added at a concentration of 500 μg/ml to the DMEM/F12 culture medium after the cells were attached. Untreated HCECs exposed to the same volume of culture medium served as a normal control. A total of 10 μl of Cell Counting Kit-8 (CCK-8; Beyotime, China) reagent was added after iPSCs-Exos treatment. Following a 4 h incubation, the plate was measured at 450 nm with a spectrophotometer (Bio-Rad, US).

Cell migration assay

A wound healing assay was performed for the migration of HCECs. HCECs were seeded into 12-well plates and grown until they reached confluence. A sterile 200-μl pipette tip was used to scratch the cell layers. After washing with PBS for three times, the culture medium was replaced with DMEM/F12 basal medium containing 500 μg/ml iPSCs/MSCs-Exos. Control dishes were similarly scratched and cultured in the same volume of culture medium. After 6 h and 12 h of adding exosomes, the cells were photographed. The distance of cell migration was measured using ImageJ software.

Evaluation of apoptosis

To compare the apoptosis of HCECs under different treatments, 0.5×10⁶ cells were stimulated with iPSCs/MSCs-Exos at a concentration of 500 μg/ml. After 48 h, cells were harvested by transferring them with complete medium into 1.5 ml reaction tubes. Cells were centrifuged at 500 g for 5 min at 4°C and the supernatant was discarded. Cells were washed with 1 ml PBS and resuspended in 100 μl of staining buffer (Annexin V-FITC/PI Apoptosis Detection Kit, Meilunbio, China); then, they were mixed with 5 μl of Annexin-V-FITC and 10 μl of PI in incubation buffer according to the manufacturer's instructions. The cells were incubated for 15 min at room temperature and were protected from light. After centrifugation at 500 g for 5 min at 4°C, cells were resuspended in 100 μl PBS and analyzed by a flow cytometer (Beckman Coulter, US).

Cell cycle assay

Flow cytometry was also used to evaluate the cell cycle of HCECs. HCECs (1×10⁶) that were untreated or were treated with 500 μg/ml iPSCs/MSCs-Exos were harvested, washed with PBS, and fixed in 75% ethanol overnight at 4°C. Prior to analysis, cells were washed again with PBS, resuspended and treated with 0.25 mg/ml ribonuclease A (Cell Cycle and Apoptosis Analysis Kit, Meilunbio, China) for 30 min at 37°C. Then, they were incubated with 20 mg/ml ribonuclease A in the dark for 30 min. Finally, the samples were immediately analyzed.

Quantitative real-time polymerase chain reaction (qRT- PCR) analysis

Total RNA was extracted using TRIzol reagent (Invitrogen, China) according to the manufacturer’s instructions. The isolated RNA was reverse-transcribed to generate synthesize cDNA in a 20 μl reaction mixture. The following RT-PCR conditions were used: 15 min at 95°C, followed by 40 cycles for 10 s at 95°C, 30 s at 60°C, and 1 s at 72°C,; then, there was 1 cycle of cooling for 30 s at 50°C. The primers were synthesized by Co Invitrogen Ltd. (China). The sequences of the primers are shown in Supplementary Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control.

Western-blot assay

Protein extraction and western-blot analysis were performed according to a previously described protocol [16]. In brief, treated cells or exosomes were homogenized in RIPA buffer with protease inhibitor. The protein level was determined using a BCA protein assay kit (Beyotime, China). Samples were run on 12% SDS-PAGE gels, blotted onto PVDF membranes, and then blocked with 5% nonfat dried milk for 2 h at room temperature Afterward, the PVDF membranes were incubated overnight at 4°C with the following antibodies: mouse anti-Cyclin A1+Cyclin A2, anti-CDK2, anti-PCNA (Abcam, US) and rabbit anti-GAPDH (Cell Signaling Technology, US). The membranes were then rinsed five times with Tris-buffered saline containing 0.5% Tween-20, and were then incubated with goat anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology, US) for 1 h at room temperature. An Odyssey fluorescent scanning system (LI-COR, US) and Quantity One software were used to detect the immunoreactive proteins.

Immunofluorescence staining

HCECs were untreated or were treated with 500 μg/ml iPSCs/MSCs-Exos for 48 h. Cultured HCECs grown on coverslides were fixed in 4% PFA, permeabilized via 0.1% Triton X-100 (Sigma-Aldrich, US) for 4 min, then washed three times in PBS. Cells were blocked with 5% goat serum (Sigma-Aldrich, US) for 1 h and incubated with mouse anti-Ki67 antibody (Abcam, US) overnight at 4°C. The Cy^TM3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, US) was used as a secondary antibody. DAPI was used for nuclear counterstaining and images were captured under a confocal microscope (Carl Zeiss, Germany).

Statistical analysis

All quantitative data are reported as the mean ± standard error of the mean (SEM). Multiple group comparisons were performed by one-way ANOVA using GraphPad Prism 6 software. P values<0.05 were considered statistically significant.