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Proteomic characterization of hUC-MSC extracellular vesicles and evaluation of its therapeutic potential to treat Alzheimer’s disease – Scientific Reports

Stable passage and identification of hUC-MSCs

Primary hUC-MSCs were obtained from Jianyuan Precision Medicine (Zhangjiakou) Co., Ltd. All the donors provided informed consent. All procedures were approved by the ethics committee. Three samples of cells from different donor sources were digested for 2 min at room temperature with 0.25% trypsin until 80% of the cells were detached and floated, after which the cell suspension was terminated with culture media. The terminated cell suspension was collected in a centrifuge tube and centrifuged at 1000 rpm for 10 min. Next, 2 × 106 cells were transferred to a new T175 culture vial, maintained in complete MEM (HyClone, China) supplemented with 5% EliteGro-Adv (EliteCell, China) and incubated at 37 °C and 5% CO2. The culture flask was observed under a microscope every day and passaged when the cell confluence reached 80–90%. The cells from 3 different donors were stably passaged to the third (P3) or fourth generation (P4). Cell morphology was observed under an inverted microscope. The cell surface markers CD73+, CD90+, CD105+ ≥ 95%, CD45+, and CD34+ ≤ 5% were analysed by flow cytometry. Cell subculture was stopped at this time.

Extraction and identification of hUC-MSC-EVs

In this study, EVs were enriched from typical hUC-MSCs culture with EV-free and serum-free media. Specifically, the third and fourth generations of hUC-MSCs were cultured in serum-free medium (5% EliteGro-Adv + 95% MEM) until the fusion rate reached to 90%. After washing with PBS twice, the fresh serum-free medium was added and the samples were incubated for 48 h. The culture supernatant was collected under aseptic condition, followed by centrifugation at 300×g for 10 min to remove dead cells. The resulting culture supernatant was subject to further centrifugation at 2000×g for 10 min using an F15-8 × 50cy rotor (Thermo, USA) and the resulting supernatant was filtered with 0.22-μm membrane filter (Navigator, China). After filtration, the supernatant was subject to ultracentrifugation at 4 °C and 100,000×g for 70 min using a P100AT2 rotor (Himac, Japan). The resulting pellets were further re-suspended with pre-cooled PBS, followed by ultracentrifugation at the same parameters. The final pellets were collected and re-suspended with pre-cooled PBS for the next analysis. Transmission electron microscopy (Hitachi, Japan) was used to detect the morphology of EVs. The EVs (1 μg/10 μl PBS) were dropped onto the sample copper net and let stand at room temperature for 2 min. The liquid was removed from the side of the filter with filter paper, and then negatively stained with 10 μL of 2% uranyl acetate solution at room temperature for 1 min. The negative control dye was absorbed by filter paper, after which the mixture was dried at room temperature. After natural drying, the samples were examined using transmission electron microscopy (Hitachi, Japan) at 60 kV. The particle size and concentration of the EVs were determined using a Zetasizer Lab nanometre particle size and Zeta potential analyser (Malvern Panalytical, UK). The surface markers of EVs, CD81, and CD63, were analysed by flow cytometry. Western blot was used to assess the presence of negative EV markers.

LC‒MS/MS analysis of the proteome

Extraction and preparation of EV proteins and hippocampal tissue proteins

The exudates of 1-P3, 1-P4, 2-P3, 2-P4, 3-P3, and 3-P4 from the hUC-MSC-EVs were extracted. Then, 50 μL was added to 2% SDS, 4,300 μL was added for ultrasonic comminution, and the samples were centrifuged at 8000×g. The supernatant was collected.

Mice were anaesthetized with sodium pentobarbital. The chest was opened from the xiphoid process with scissors to expose the heart’s position. The needle of the infusion device punctured the apex of the heart at a depth of approximately 4 mm. The needle was fixed with tweezers, and the right atrial appendage was cut. The blood vessels were washed with normal saline until there was no blood outflow from the right atrial appendage. The hippocampal tissue was separated, stored in a frozen tube and frozen in liquid nitrogen. The hippocampal tissue of the mice was fully ground in a homogenizer with PBS and protease inhibitor. The grinding solution was stored at 4 °C and the samples were centrifuged at 12,000×g for 5 min, after which the supernatant was collected. All the sacrifice procedures were performed under sodium pentobarbital anaesthesia, and efforts were made to minimize animal suffering.

The same volume of Tris-saturated phenol was added to the collected hUC-MSC-EVs and mouse hippocampal supernatant. After mixing, the solution was centrifuged at 12,000×g for 5 min to achieve stratification. The upper aqueous phase was removed while keeping the liquid organic phase intact. The same volume of 50 mM Tris–HCl was added, and the solution was mixed well. The mixture was stored at 4 °C and centrifuged at 12,000×g for 15 min. The above centrifugation steps were subsequently repeated. Five times the volume of precooled ammonium acetate methanol was added to the retained organic phase, the mixture mixed well, and the mixture was precipitated overnight at − 20 °C. The solution was centrifuged under the same conditions, after which the precipitate was retained. One millilitre of methanol was added to the precipitate, and the sample was mixed well and transferred to a 1.5 mL EP tube. This solution was centrifuged under the same conditions, and the supernatant was discarded. This step was repeated twice. Afterwards, 10 μL of DTT was added to the precipitated protein sample for 30 min at 37 °C. IAA (20 μL) was subsequently added to the precipitated protein sample, after which the mixture was protected from light at 26 °C for 30 min. Then, 500 μL of trypsin was added at a ratio of 1:50 (v:v) at the end of reductive alkylation, after which the mixture was hydrolysed at 37 °C for 10 h. The above enzymatically hydrolysed peptides were desalted via solid-phase extraction with C18 SPE (Anpu, China), after which the mixture was dried and set aside.

LC‒MS/MS analysis

The polypeptide samples were redissolved in pure water (Fisher, USA) containing 0.1% formic acid, and the iRT reagent (Biognosys, Switzerland) was added to prepare for subsequent MS identification. The polypeptide samples were separated by UPLC (Mtel Classwork Waters, USA). The separation was carried out on a C18 reversed-phase chromatographic column (1.8 μm particle size, 75 μm ID × 250 mm length; Waters, USA). Phase A was pure water (Fisher, USA) containing 0.1% formic acid, and phase B was acetonitrile (Fisher, USA) containing 0.1% formic acid. The elution flow rate was 300 nL/min, and the elution gradient was mobile phase B from 2% Mel to 35% for 2 h. The polypeptide samples were sprayed into Q Exactive HF mass spectrometer (Thermo, USA) via an ion source for DIA-based quantitative analysis. The MS parameters were set as follows: (A) The DIA mode was based. The full scanning range was set at 350 to 1200 m/z. The scanning resolution of the parent ion was set to 60,000. The AGC was set to 3e6, and the maximum ion implantation time was set to 50 ms. (B) HCD was used for fragmentation, and the collision energy was set to 27%. (C) The DIA method was set as follows: full MS (350 to 1250 m/z), followed by 20 DIA MSMS. The DIA isolation window (IW) was set at 59.0 m/z, 25.0 m/z, 19.0 m/z, 17.0 m/z, 13.0 m/z, 13.0 m/z, 11.0 m/z, 12.0 m/z, 12.0 m/z, 11.0 m/z, 12.0 m/z, 9.0 m/z, 10.0 m/z, 10.0 m/z, 11.0 m/z, 10.0 m/z, 10.0 m/z, 9.0 m/z, 10.0 m/z, and 10.0 m/z; then Full MS (350–1250 m/z), followed by 20 DIA MSMS (IW) at 10.0 m/z, 9.0 m/z, 10.0 m/z, 8.0 m/z, 9.0 m/z, 9.0 m/z, 10.0 m/z, 10.0 m/z, 10.0 m/z, 10.0 m/z, 9.0 m/z, 10.0 m/z, 10.0 m/z, 10.0 m/z, 10.0 m/z, 10.0 m/z, 11.0 m/z, 10 m/z, 10 m/z, and 11 m/z; then, full MS (350–1250 m/z), followed by 20 DIA MSMS (IW) at 10 m/z, 11 m/z, 12 m/z, 11 m/z, 13 m/z, 13 m/z, 13 m/z, 14 m/z, 13 m/z, 14 m/z, 14 m/z, 19 m/z, 18 m/z, 20 m/z, 27 m/z, 24 m/z, 33 m/z, 45 m/z, 56 m/z, and 91 m/z. (D) The MS2 scan resolution was set to 30,000 and the AGC target was set to 1e6 23.

Database searching and bioinformatics analysis

The original data were analysed with Spectronaut software version 15.0 (Biognosys AG, Schlieren, Switzerland) and standard DIA analysis (FDR < 1%). The database of mouse proteins (UniProt, download date, protein quantity) was used to search for DIA. To rule out the possibility of contamination in the results, the human keratin sequence was used as the contaminated database for proteomic searching. The selection of the search parameters was as follows: (A) digestion at two misfolded sites; (B) variable modifications adjusting the oxidation of methionine; and (C) fixed changes adjusting cysteine carbamidomylation. The proteomics data from mass spectrometry were stored in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) using the iProX partner repository with the dataset identifier PXD037877 23.

Data analysis

To screen the retrieved data, unique peptides ≥ 2, a Q value < 0.05, and a CV < 20% were selected as parameters for further analysis. The UniProt database (https://www.uniprot.org/) searches proteins based on molecular weight, isoelectric point (PI), and the ExPASy (ProtParam section of https://www.ExPASy.org/) website (https://www.ExPASy.org/resources/protparam). These data were used to calculate the grand average of hydropathicity (GRAVY) for each protein. Six groups of EV proteins were subsequently analysed with a Venn diagram (http://bioinformatics.psb.ugent.be/webtooLs/Venn/). The TOP100 EV marker proteins were downloaded from the ExoCarta database (http://www.exocarta.org/).

Bioinformatics analysis

Functional enrichment analysis of the hUC-MSC-EV proteome was carried out on the official GO website (http://www.exocarta.org/), and the BP, CC, and MF terms were evaluated. The KEGG website (https://www.kegg.jp/kegg/) was used to analyse the enrichment of the protein signalling pathways.

Grouping and administration to mice

All the mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., and all the animal experiments were conducted in accordance with ethical standards. All mice were provided free access to food and water and were kept in a colony room under a 12-h dark–light cycle in the Hebei Normal University Animal Center. All the animal procedures were performed in strict conformity with the Guidelines for Care and Use of Laboratory Animals of Hebei Normal University, approved by the Science and Technology Ethics Committee of Hebei Normal University, and complied with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. Twelve 3-month-old male APP swe/PS1dE9 double transgenic mice were randomly divided into a model group and an EV group of 6 animals each, and 6 litters of the same male mice with a C57BL/6J background were used as a control group; the mice weighed 25–30 g each. The protein concentrations of the six batches of hUC-MSC-EVs that were determined by the BCA assay kit were 10, 9, 9.5, 8, 9, and 10 μg/μL. Six batches of EVs were mixed and diluted to 2.5 μg/μL for subsequent animal experiments. The mice in the EV group were injected with 40 μL of hUC-MSC-EVs, and the mice in the model and control groups were given the same volume of PBS. All the mice were injected every 5 days for a total of 6 injections.

Behavioural experiments in mice

The Morris Water Maze experiment

This experiment was designed to examine spatial learning and memory in mice by allowing them to swim to find a platform hidden in water. The Morris water maze is composed of a pool with a diameter of 120 cm and a depth of 50 cm, a platform, and a video analysis system. The water temperature was held at a constant (24 ± 2) °C, and the samples were surrounded by curtains to prevent external interference. The experiment was divided into a positioning navigation experiment and a space exploration experiment. The first 6 days were used for the positioning navigation experiment. Mice were put into the water with their back to the pool wall in each quadrant every day. The experiment ended automatically if the mouse found the platform and remained there for 10 s. If the platform was not found in the first 60 s, the mouse was guided to the platform in the next 30 s. On the 7th day, the space exploration experiment was conducted. The platform was removed, and the mice entered the water from any quadrant. The swimming paths within the first 60 s were recorded, and the number of times the mice crossed the platform was counted.

Rotarod test

In this study, a YSL-4C rotary-type fatigue metre was used for testing. The maximum rotation speed of the rotary-type fatigue metre was 40 revolutions per minute (RPM), and the maximum test time was 5 min. Behavioural experiments were conducted in a quiet environment, so the training was conducted and tested in a special behavioural laboratory. In the first two days, training occurred three times a day. The rotation speed was set to 40 RPM, and the rotation time was set to 5 min. At the beginning of training, the mice often fell off the rotary metre as the speed of the rotary metre increased. At this time, the mice were returned to the corresponding track and allowed to continue moving until the end of their 5 min of training. On the third day, the formal experiment was carried out, and each mouse was tested three additional times. Twenty minutes were left between each experiment for the mice to rest. The number of times the mice were on the rod spinner before they fell was recorded, and the number of mice that did not fall in the first 5 min was recorded, and the experiment was ended. The average of the three tests provided the daily test results.

Voluntary activity experiment

The mice were placed in the reaction tank of the ZZ-6 mouse autonomous activity tester, and the lid was closed for 2 min after adaptation. The activity times of the mice in the first 5 min were recorded. When the mice were active, the infrared probe under the reaction tank was used to detect the signal change, and the data were automatically recorded and displayed on the display screen. Each experiment required cleaning of the box to minimize the influence of the previous mouse on the next mouse.

Statistical analysis

Statistical analysis was performed using SPSS 25.0 and Prism 5. The statistical significance (p < 0.05) was determined using Student’s t test. The data are presented as the mean ± SEM.

Ethics approval

All the animal procedures were performed following the Guidelines for Care and Use of Laboratory Animals of Hebei Normal University and approved by the Science and Technology Ethics Committee of Hebei Normal University (2023LLSC037).