Mice

The BALB/c mice aged 4 to 8 weeks were purchased from Pasteur Institute (Tehran, Iran). All animal experiments and procedures have been approved by the ethics committee of Shenzhen Key Laboratory of Ophthalmology, Affiliated Hospital of Jinan University, Shenzhen, China. All the experiments were conducted according to the national specific ethical standards for biomedical research. The mice were kept in cages in ventilated rooms, providing plenty of food and water.

iPSC culture and exosome preparation

The iPSCs used in this study were obtained from the Cell Resource Center, Peking Union Medical College. The iPSCs were cultured in exosome-free media for 48 h before being processed for exosome isolation. According to the previous studies (Théry et al. 2006; Gao et al. 2023), the engineering exosomes were separated by ultracentrifugation. Specifically, 0.4 mL of the iPSC cell culture supernatant was centrifuged at 300 x g for 10 min, transferred to a clean tube, and centrifuged at 2000 g for 20 min to remove cell debris. Subsequently, the supernatant was transferred to a clean test tube, centrifuged at 10,000 g for 30 min, and then vacuum filtered with a 0.22 μm filter (EMD, billica, MA). Furthermore, the supernatant was transferred to an ultracentrifuge tube (Beckman Coulter, Braille, CA) and centrifuged in an ultracentrifuge (Beckman Coulter optima TM Xe) 100,000 × g for 70–120 min. The resulting precipitate was washed once with cold, sterile phosphate buffered saline (PBS, pH 7.4) and resuspended in 0.3–0.6 mL PBS containing 1% DMSO. The protein concentration of the isolated exosomes was then measured using the Pierce™ BCA Protein Assay.

Isolation and characterization of exosome

The Exosomes Isolation Reagent (Invitrogen, CA) was used to isolate exosomes from iPSCs. The iPSCs were first detached using Accutase (Innovative Cell Technologies, San Diego, CA) by incubating the cells at 37 °C for 5 min until they detached from the culture dish. The detached cells were collected by centrifugation at 300 g for 5 min and resuspended in PBS. After washing with PBS, the cells were resuspended in exosome-free media and cultured for 48 h. After that, the cells were centrifuged 3000 rpm for 15 min and the supernatants were collected. The supernatants were transferred to a fresh tube and exosome isolated reagent was added. The mixture was incubated overnight and then centrifuged at 15,000 rpm for 1 h to pellet exosomes. The pellet exosomes were resuspended with incomplete ESC medium or PBS. The morphology and the concentration particles of exosomes were detected by the transmission electron microscope (TEM) (FEI, USA) and nanoparticle tracking analysis (NTA) (ZetaView PMX110, Particle Matrix, Meerbusch, Germany), respectively.

Exosome labeling and loading

Exosomes were observed using the fluorescent probe PKH67 (Invitrogen, Carlsbad, California, USA). A total of 100 µL of purified exosome suspension (with a protein concentration of 10 µg/mL) was incubated with 5 µL PKH67 at 37 °C for 15 min. The mixture was then centrifuged at 120,000× g for 90 min to remove unbound probes. After two rounds of washing and centrifugation, the labeled exosomes were suspended in PBS and subsequently used for cell research.

For miRNA loading, exosomes with a total protein concentration of 10 µg/mL were mixed with 400 nm Cy5-labeled miRNA in 1 mL PBS. The mixture was subjected to electrophoresis under the following conditions: 400 V, 50 µF, 30 ms pulse/2 s, for three cycles. After loading miRNA, the exosome samples were diluted 10 times with PBS and centrifuged at 110,000× g for 70 min to remove the unbound miRNA. The incorporation of miRNA into exosomes was determined by RT-PCR.

Exosome uptake

iPSC-derived neuronal cells (3 × 105) were seeded in a 3.5 cm glass bottom dish and incubated until 70% confluency was achieved. Then the cells were washed with PBS and incubated with cell culture medium containing 108 particles/mL PKH67-labeled foreign bodies. The fluorescence signals of PKH67 were recorded by confocal laser scanning fluorescence microscope (CLSM), and the images were processed by Zen software (CLSM; Zeiss lsm710, Oberkochen, Germany).

RNA extraction and quantitative RT-PCR assay

Trizol reagent (Invitrogen, Carlsbad, California, USA) was used to extract total RNA from the samples. The M-MLV Reverse Transcriptase (RNase H-) kit (Invitrogen, CA) was employed to synthesize cDNA from the extracted RNA. This kit was chosen for its efficiency in producing high-quality cDNA by preserving the integrity of the RNA template, owing to its lack of RNase H activity. RT-qPCR was performed using SYBR Green PCR Master Mix (Takara, Kusatsu, Japan). GAPDH was used as the housekeeping gene for mRNA detection, and U6 snRNA was used as the housekeeping gene for miRNA detection. The relative expression levels were calculated using the 2^-ΔΔCT method. The primers used in this study were synthesized with sequences as shown in Table 1.

Table 1 Primer sequences used in the studyWestern blot

Total proteins from tissues or cells were lysed in RIPA buffer and quantified by BCA analysis (Beyotime, Shanghai, China). The protein was analyzed by 10% twelve alkyl sulfate polyacrylamide gel (SDS-PAGE), and the gel was transferred to TBST sealed polyvinylidene fluoride (PVDF) membrane in 5% skim milk powder for 1 h. The PVDF membrane was incubated with the first antibody at 4 ℃ overnight: anti-TSG101, anti-CD63, anti-cytochrome C. β-actin was used as an internal control. All antibodies were purchased from (San Francisco, Santa Cruz, USA, 1:1000). The optical density of protein band was determined by Image J software, Inc.

Rodent model of spinal cord injury

Mice were anesthetized with pentobarbital and underwent laminectomy at the T11-12 level. A weight-drop injury was induced using a 5 g rod dropped from a height of 5 cm. After the injury, the incision was closed, and the mice were placed in a controlled environment for recovery. The SCI mice were divided into two groups, receiving either PBS or iPSCs-Exo treatment. Recovery was assessed through behavioral and morphological evaluations. For the behavioral assessment motor function recovery was evaluated using the Basso Mouse Scale (BMS) at multiple time points (days 1, 7, 14, 21, and 28) by blinded observers. For morphological assessment, histological analysis was performed on the spinal cords using hematoxylin and eosin (H&E) staining to assess tissue architecture and Luxol fast blue (LFB) staining to evaluate myelin integrity and apoptosis. Significant improvements in tissue morphology and reduced apoptosis were observed in the iPSCs-Exo treated groups, especially those treated with miRNA-loaded exosomes.

Flow cytometry assay

Cell apoptosis was measured by Annexin V-Fluos Apoptosis Detection Kit (Invitrogen, Catalog No. V13242). In detail, iPSC-derived neuronal cells in each treatment group were harvested and washed twice with PBS. Each precipitate was resuspended in 400 µL PBS at a concentration of 1 × 106 cells/mL. Then, 100 µL of incubation buffer containing 2 µL of Annexin V-Fluos and 2 µL of propidium iodide from the kit was added to the cells in each experimental group. The samples were gently mixed and incubated for 15 min at room temperature in the dark. Following incubation, each group was analyzed by BD flow cytometry within 1 h.

In vivo visualization of intravenously injected exosomes

To evaluate the biodistribution of miRNA (miR-23b, miR-21-5p and miR-199b-5p) engineered exosomes, 1 × 106 cells were injected into the ventral side of 6-8-week-old female BALB/ c mice. 30 µg PKH67-labeled exosomes were injected into SCI model mice and healthy control mice as controls. 200 µL PBS + 5% glucose was injected into SCI model mice as background control. 5% glucose supports cell viability and reduces potential osmotic stress during intravenous injection. SCI model mice were anesthetized with 2.5% isoflurane at 3, 12, 24 and 48 h after administration, and small animal imaging system (Kodak, Rochester, New York, USA) was used. The fluorescence signal of each tissue sample was quantified in the freehand drawn target area.

Tissue dissection and fluorescent microscopy

At the end of the experiment, the mice were euthanized by neck detachment. In addition to spinal cord tissues, tissues of interest (including lung, liver, kidney and spleen) were cut and cryopreserved in 5%, 10% and 14% sucrose, then frozen in OCT medium at -80 ℃ and sectioned with Leica CM1800 cryostat. The frozen tissue Sect. (8 μm thick) were fixed in acetone and their nuclei were stained with DAPI. To visualize the fluorescently labeled exosomes in the dissected tissues, we used the Cycling-3 cell imaging multimodal reader (Bio-Tek Instruments, Winooski, VT, USA). Fluorescent images were captured for further analysis.

Statistical analysis

The mean ± SD represented data from three independent experiments. GraphPad Prism version 5.0 software (GraphPad Software, Inc.) was used for statistical analysis of all data. Unpaired Student t-test was used for comparison between two groups, and One-way ANOVA followed by tukey test was used for comparison within multiple groups. P value < 0.05 indicated that the difference was statistically significant.