iPSC-derived exosomes promote angiogenesis in naturally aged mice

iPSCs-derived exosomes promoted angiogenesis of aging aortic rings

Mouse iPSCs were generated by transducing mouse skin fibroblasts and confirmed by detecting the expression of the pluripotency markers Oct4, SOX2, and SSEA-1 (Figure 1A). Exosomes from iPSCs culture medium were isolated and characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA), western blotting, and immunofluorescence assay (Figure 1B–1E). Aged and young mice were injected with iPSC-derived exosomes for three months via the tail vein, and neovascularized area of aortic rings were examined. New blood vessels were observed around the aortic rings of aged mice treated with iPSC-derived exosomes after 7 d of treatment and peaked after 12 d, followed by neovascularization and apoptosis. Compared with untreated young mice, there was a significant decrease in linear angiogenesis around the aortic rings in untreated old mice at 9 d and 12 d (Figure 2A). At 9 and 12 d of treatment, there was a significant increase in capillary-like sprout area around aortic rings of aged mice in the iPSC-exosome group (old + exo in vivo), in the vitro culture group (old + exo in vitro), and in the iPSC-exosomes group (old + exo in vivo + exo in vitro) compared with the untreated group, with linear angiogenesis appearing around the rings and gradually increasing to form a network structure. The angiogenesis area in the iPSC-exosomes group (old + exo in vivo + exo in vitro) was significantly larger than those of the iPSC-exosome pretreatment (old + exo in vivo) and in vitro culture groups (old + exo in vitro) at 9 d after treatment (Figure 2B). Additionally, neovascularization increased significantly at 7–12 d in the young group; however, there was no significant difference in the cumulated capillary-like sprouts area between the treatment groups at the same time points (Figure 2C).

iPSCs-derived exosomes increased the clonality and proliferation of bone marrow cells

Adherent bone marrow cells were isolated from all mice and cultured, and the number of bone marrow cells in old and young mice was counted. Exosomes treatment increased the clonality of bone marrow cells from aged mice after 12 d regardless of whether the exosomes were cultured in vitro or pre-injected in vivo. Additionally, in vivo or in vitro administration of iPSC-exosomes significantly increased the proliferation of bone marrow cells from aged mice at 8 and 12 d of treatment but not at 4 d (Figure 3A). Compared with the old untreated group, in vitro, in vivo, and in vivo + in vitro treatment significantly increased cell concentrations at 8 and 12 d after treatment. In the case of bone marrow cells from young mice (Figure 3B), in vivo and in vivo + in vitro treatments with exosomes increased clonality at 8 d and 12 d after treatment (Figure 3B). However, in vitro exosome treatment did not significantly affect clonality at 8 or 12 d after treatment.

Intravenous administration of exosomes inhibited aging in old mice

Angiogenesis is necessary for the repair of parenchymal organs and to slow tissue aging. Persistently high p53 and p16 expression has been shown to promote cell senescence. Compared with the young mice, p53 expression was significantly higher in the kidney, muscle, liver, and lung tissues of aged mice (Figure 4). Intravenous injection of iPSC-exosomes for three months significantly decreased p53 expression levels in the kidney, skin, muscle, liver, and lung tissues of aged mice compared with that of untreated aged mice. Similarly, p16 protein expression was significantly higher in the skin, brain, muscle, lung, and spleen of aged mice than in the organs of young mice. Furthermore, intravenous injection of iPSC-exosomes for three months significantly decreased p16 expression in the kidney, skin, brain, muscle, lung, and spleen tissues of aged mice compared with that of untreated aged mice. However, there were no significant differences in p53 and p16 expression levels between untreated young mice and young mice administered iPSC-exosomes (Figure 4).

Intravenous administration of exosomes increased total antioxidant activity in tissues of aged mice

Healthy tissues and organs contain several antioxidative molecules and enzymes that can eliminate reactive oxygen species (ROS) to prevent oxidative stress. Therefore, the effect of exosome treatment on the TAOC of the kidney, skin, heart, brain, muscle, liver, spleen, and lungs of mice was examined using ABTS kit. The TAOC of the kidney, skin, heart, muscle, liver, and lung tissues of aged mice was significantly lower than that of young mice. Compared with untreated aged mice, treatment with iPSC-exosome increased the TAOC of the kidney, skin, brain, muscle, and lung tissues of aged mice by varying degrees (Figure 5).

However, the effect of iPSC-exosome on young mice varied among the organs; specifically, the TAOC of the brain and spleen tissues of exosome-treated young mice was significantly higher than those of untreated young mice. In contrast, the TAOC of the kidney, muscle, and lung of exosome-treated young mice was significantly lower than those of untreated young mice; however, there were no significant differences in the TAOC in liver, skin and heart between the groups. Regardless of iPSC-exosome treatment, TAOC was higher in young mice than in untreated old mice (Figure 5).

iPSC-derived exosomes or serum exosomes from mice treated with iPSC-derived exosomes decreased HUVEC injury

The effect of iPSC-derived exosomes and serum exosomes from aged mice treated with iPSC-derived exosomes (old-serum-exo) on HUVEC with H₂O₂ to induce endothelial cell injury was examined at 12, 24, and 48 h after co-culture (Figure 6A). Compared with the untreated H₂O₂ injured group, treatment with serum exosomes from untreated or iPSC-derived exosomes treated young mice, as well as iPSC-exosomes, promoted the proliferation of injured HUVEC after 24 and 48 h of co-culture. Similarly, serum exosomes from old mice treated with the iPSC-derived exosomes promoted the proliferative ability of injured cells at 48 h. Additionally, treatment with iPSC-derived exosomes or serum exosomes of young mice also increased the ability of endothelial cells to form capillary-like structure and to uptake acetylated low density lipoprotein (Figure 6B). Overall, these results indicated that iPSC-derived exosomes or serum exosomes from mice treated with iPSC-derived exosomes can restore endothelial function.

Protein profiles of exosomes

Exosomes have been confirmed to play a variety of roles mainly due to their source and the selective encapsulation of a variety of functional proteins. Therefore, we examined the protein profiles of serum exosomes of untreated old mice (old-serum-exo), old mice treated with iPSC-exosomes (old+iPSC-exo-serum-exo), untreated young mice (young-serum-exo), and young mice injected with iPSC-exosomes (young+iPSC-exo-serum-exo), using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, bioinformatics was performed to identify differentially expressed proteins, followed by functional annotation and pathway enrichment analysis of the proteins using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes databases (KEGG).

A total 30 proteins were differentially expressed in serum exosomes of untreated aged and young mice, among which 13 proteins were upregulated and 17 downregulated in serum exosomes of untreated aged mice. The top 10 significantly enriched GO terms by the 30 proteins are presented in Figure 7A, 7B. Specifically, the upregulated proteins were enriched in immune response and metabolic process related to higher immune/inflammatory response, while the downregulated proteins were involved in positive regulation of biological process and metabolic process related to increased cell vitality and decreased blood lipid levels in the “Biological Processes” (BP) category. Additionally, the upregulated proteins were enriched in calcium ion binding, while the downregulated proteins were enriched in lipid binding, including high-density lipoprotein particle receptor binding, in the “Molecular Function” (MF) category. Moreover, KEGG analysis revealed that the upregulated proteins were involved in complement and coagulation cascades, while the downregulated proteins were involved in PPAR signaling pathway (Figure 7C, 7D).

Furthermore, a total of 56 differentially expressed proteins were identified in the serum exosomes of young mice treated with iPSC-exosomes compared with the levels in the exosomes of old mice treated with iPSC-exosomes group, among which 41 were upregulated and 15 were downregulated in the exosomes of old mice treated with iPSC-exosomes. The top 10 significantly enriched GO terms are presented in Figure 7E, 7F. Specifically, the upregulated proteins were enriched in immune system response, while the downregulated proteins were enriched in immune system response, including complement activation and humoral immune response, in the BP category. Additionally, the upregulated proteins were enriched in antigen binding, while the downregulated proteins were enriched in enzyme regulator activity in the BP category. KEGG enrichment analysis showed that the upregulated and downregulated proteins were mainly enriched in nitrogen metabolism and complement and coagulation cascades, respectively (Figure 7G, 7H).

Among 48 differentially expressed proteins, 30 and 18 were significantly upregulated and downregulated, respectively, in the serum exosomes of old mice treated with iPSC-exosomes compared with the expression levels in the serum exosomes of untreated old mice. GO functional annotation showed that the 30 upregulated proteins were enriched in biological processes that are more active in vigorous cells, while the 18 downregulated proteins were enriched in metabolic processes and immune response in the BP category. Additionally, the upregulated proteins were enriched in binding (such as high-density lipoprotein particle receptor binding) and enzyme regulator activity, while the downregulated proteins were enriched in protein binding in the MF category. KEGG pathway analysis showed that the upregulated proteins were involved in nitrogen metabolism, while the downregulated proteins were enriched in complement and coagulation cascades (Figure 8A–8D). Additionally, nine proteins were upregulated and 21 were downregulated in the serum exosomes of aged mice treated with iPSC-exosomes compared with the expression levels in the serum exosomes of untreated young mice. GO functional annotation showed that the upregulated proteins were enriched in immune system response, while the downregulated 21 proteins were enriched in negative regulation of lipase activity and lipid catabolic process in the BP category. Additionally, the upregulated proteins were enriched in binding of many molecules, while the downregulated proteins were enriched lipid binding in the MF category. KEGG pathway analysis showed that the upregulated proteins were enriched in complement and coagulation cascades, while the downregulated proteins were enriched in PPAR signaling pathway (Figure 8E–8H). Moreover, all proteins were enriched in exosome-related components in the “Cellular Components” (CC) category, which further proved that the extracted proteins were exosome components.

To further demonstrate that serum exosomes of aged mice injected with iPSC-exosomes played a similar role to the serum exosomes of untreated young mice, Venn diagram showed that 208 differentially expressed proteins were common to both groups (Figure 9A). GO functional annotation showed that the proteins were enriched biological processes related to stronger responses to stress and immune/inflammatory injury in the BP category, and in antigen binding and inhibitor activity of key metabolic enzymes in the MF category (Figure 9B, 9C). KEGG signaling pathway showed that proteins were enriched in several pathways related to cellular metabolisms and complement and coagulation cascade (Figure 9D).

No teratoma in experiment mice

After sample harvesting, the whole body of the animals were examined morphologically to determine if there was any teratoma in the animals. Teratoma was not observed in the body of the experimental animals.

iPSCs culture and characterization

iPSCs were induced from the skin fibroblast of C57 mouse as described previously [29]. Briefly, iPSCs were recovered a medium containing Dulbecco’s modified eagle medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (FBS; Thermo Fisher SCientific, Waltham, MA, USA), 1% non-essential amino acid (NEAA; Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin and streptomycin (PS; Thermo Fisher Scientific, Waltham, MA, USA), 0.01% CHIR98014 (Selleck Chemicals, Houston, TX, USA), 0.01% mirdametinib (PD0325901; Selleck Chemicals, Houston, TX, USA), and 1 × 10⁷ U/mL of recombinant murine leukemia inhibitory factor (mLIF; Peprotech, Rocky Hill, NJ). The culture medium for iPSCs was changed to a medium containing 90% DMEM, 10% knockout serum replacements (KSR; Thermo Fisher Scientific, Waltham, MA, USA), 1% NEAA, 1% PS, 0.01% CHIR98014, 0.01% PD0325901, and 1 × 10⁷ U/mL of mLIF 1 d after cell recovery.

The medium containing KSR was collected daily for exosome purification. iPSCs were fixed in 4% paraformaldehyde at 37° C for 15 min and then incubated with 5% donkey serum and 0.3% Triton X-100 for 60 min. The cells were incubated with the pluripotency markers octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (SOX2), or Stage-Specific Embryonic Antigen-1 (SSEA-1) (rabbit anti-mouse, 1:1000; Abcam, Cambridge, UK) at 4° C overnight. Thereafter, the cells were washed and incubated with secondary antibodies at 22° C for 60 min, followed by staining with DPAI.

Exosome extraction, identification, and storage

Cell culture media were centrifuged at 200 g for 10 min and then at 2000 g for 20 min at 4° C to remove debris and dead cells. Subsequently, the cell-free culture supernatant (4 mL) was incubated with 1 mL of ExoQuick-TC solution (System Biosciences, Palo Alto, CA, USA) overnight at 4° C, and exosomes were isolated by centrifugation at 1500 g for 30 min. The precipitates were resuspended in 1 mL of PBS, further purified by ultracentrifugation at 100,000 g for 70 min, and finally resuspended in 100 μL of PBS. Exosomes were detected using high-resolution transmission electron microscopy and nanosight tracking analysis (NTA; NanoSight NS300, UK). The expression of TSG101 and CD63 (specific protein markers of exosomes) were determined using western blotting analysis and the respective primary antibodies TSG101/HSP90/CD63 (rabbit anti mouse, 1:1000; Abcam, Cambridge, UK) and secondary antibody IgG (donkey anti rabbit, 1:10000; Jackson Laboratory, Bar Harbor, ME, USA). The isolated exosomes were stored at -80° C until use. For the uptake assay, exosomes labeled with PKH26 (Sigma-Aldrich, Merck KGaA, Germany) were incubated with HUVECs for 24 h and nuclei were counterstained with DAPI (blue), and the cells were then photographed.

Animal arrangement

The animal protocol was approved by the University of Fudan Animal Care Committee in conformity with the Guide for the Care and Use of Laboratory Animals (National Research Council of United States). The mice were maintained on a standard rodent chow and water ad libitum. A total of 12 mice comprising of six aged mice (42 months old) and six young mice (6–8 weeks) were used for this study. Three aged and three young mice were injected with PBS via the tail vein once every 3 d for three months as the control groups, while the remaining three aged and three young mice were injected with exosomes derived from iPSCs (doses 200 μg) for 3 months as the experimental group.

The in vitro experiments using aortic rings and nucleated bone marrow from aged and young mice were separately subdivided into four groups. Old mouse group: untreated group (old); in vivo pretreatment with iPSC-exosomes (old + exo in vivo); in vitro culture with iPSC-exosomes (old + exo in vitro); in vivo pretreatment and in vitro culture with iPSC-exosomes (old + exo in vivo and exo in vitro). Young mice: untreated group (young); in vivo pretreatment with iPSC-exosomes group (young + exo in vivo); in vitro culture with iPSC-exosomes group (young + exo in vitro); and in vivo pretreatment and in vitro culture with iPSC-exosomes group (young + exo in vivo and exo in vitro).

Aortic ring extraction

Aortic ring extraction was performed according to previously described method [30]. Briefly, the ventral skin was opened with single cut and blunt dissected using dissection scissors. The thoracic cavity was cut open using fresh sterile scissors, and the heart and lungs were removed to expose the aorta along the spine. The anterior end of the aorta was gently grasped, and the aorta was separated from the spinal column by blunt dissection, ending before the artery branching into the iliacs in the abdomen. The dissected aorta was transferred into sterile PBS. The tunica adventitia of the artery was gently removed as cleanly as possible in sterilized PBS using micro-forceps sterilized in 75% alcohol for 1 h. The aorta pectoralis was placed in another dish with sterilized PBS and then cut into approximately 10 aortic rings using micro-scissors.

Teratoma examination

After sample harvesting, the whole body of the animals were examined morphologically to determine if there was any teratoma in the animals.

Aortic ring culture

Thawed Matrigel (No.356237; BD Biosciences, Bedford, MA, USA) (4 mg/mL) was diluted in DMEM (6 mg/mL), and 50 μL of diluted Matrigel was transferred into one well of 96-well plate. Subsequently, the 96-well plates were incubated in a cell incubator for 15 min. The ring was carefully transferred into Matrigel using micro-forceps, and 50 μL of additional diluted Matrigel was added into the well, followed by incubation for 15 min. Finally, 100 μL of ECM was added into the well to culture the aortic ring. Rings from each mouse were divided into two categories: ECM with exosomes (derived from iPSCs; total amount: 10 μg per well) and ECM without exosomes. The medium was changed after 5 d and then every 2 d, and the rings were continuously observed and photographed. All images were processed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to calculate the area of sprout vessels and the number of clones.

Bone marrow cell extraction and culture

Bone marrow cells (BMCs) were isolated from the bone marrow of sacrificed mice and cultured in a medium containing 80% α-MEM and 20% FBS. The cells were treated with 10 μg of exosomes and the number of adherent cell clones was calculated at 4, 8, and 12 d after treatment. Cloned BMCs were digested with trypsin on 4, 8, and 12 d after treatment and the number of cells in each well was then counted using a cell counter.

Tissues extraction and Western blotting

Twelve mice were sacrificed after three months, and skin, muscle, kidney, liver, spleen, lung, heart, and brain tissues were sampled using 1.5 mL tubes and stored in a refrigerator at -80° C. Proteins were extracted from the tissues using radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the proteins were denatured, resolved on a 12% polyacrylamide gel, and transferred onto a polyvinylidene fluoride membrane (PVDF; Millipore, Bedford, MA, USA). The membranes were incubated overnight with primary antibodies against p53, p16, and β-actin (rabbit anti mouse, 1:1000; Abcam, Cambridge, UK) at 4° C. Thereafter, the PVDF membranes were washed with PBST (Thermo Fisher Scientific, Waltham, MA) three times for 15 min each, and incubated with donkey anti-rabbit (H+L)-peroxidase conjugated secondary antibody (Jackson Laboratory, Bar Harbor, ME, USA) for 1 h at 22° C. Protein bands were visualized using an electrogenerated chemiluminescence ECL (Thermo Fisher Scientific, Waltham, MA) bioluminescence method, and images were obtained using a Tanon image system (Tanon, Beijing, China).

Antioxidant assay

Briefly, approximately 0.25 mg of skin, muscle, kidney, liver, spleen, lung, heart, and brain tissues were transferred into 1.5 mL tubes containing 200 μL of PBS and two steel balls, and the tubes were vortexed in an ice box for 3 min at 65 HZ, followed by centrifugation at 12000 g for 15 min. The supernatant was carefully transferred into a 96-well plate without disturbing the precipitate by adding 2;2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Beyotime, China). The OD values at 450 nm were detected using a microplate reader (BioTek, Winooski, VT, USA).

Cell proliferation assay

HUVEC were seeded in 96-well plates at a density of 4,000 cells/well and cultured for 24 h. Cell culture media were removed, and the cells were treated with H₂O₂, iPSC-derived exosomes, and serum exosomes from iPSC-exosome-treated and untreated mice under different conditions for 24, 48, and 72 h. Cell viability was determined using the Cell Counting Kit-8 (CK04; Dojindo Molecular Technologies, Inc., Kyushu, Japan), and the absorbance was measured at 450 nm using a microplate reader. The assays were performed using six replicates and each experiment was independently repeated five times.

Measurement of capillary-like structure

HUVEC-GFP were seeded in 24-well plates (1 × 10⁴ cells/well) for 24 h and treated with H₂O₂, iPSC-derived exosomes, and serum exosomes from iPSC-exosome-treated and untreated mice for an additional 48 h. Thereafter, HUVEC were digested and seeded into 48-well plates (1 × 10⁵ cells/well) that had been pre-coated with 150 μL of Matrigel (10 mg/mL; BD Biosciences, Franklin Lakes, NJ, USA). After incubation for 12 h, capillary-like structures formed by the endothelial cells were observed using an epifluorescence microscope (BX51; Olympus, Tokyo, Japan). Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LD) were used to measured LDL uptake. The fluorescence intensities of Dil were calculated using Image J software.

LC-MS/MS and bioinformatics

Serum-derived exosome was collected from old mice, old mice pre-treated with iPSC-derived exosomes via tail vein injection, young mice and young mice pre-treated with iPSC-derived exosomes via tail vein injection. Exosomal protein profiling was performed using LC-MS/MS. MS/MS data were analyzed using Mascot (Matrix Science, London, UK; version 2.3) to search against database. The Search parameters were set as previously described. GO functional annotation of differentially expressed proteins between groups was performed using OmicsBean (http://www.omicsbean.com:88/). Gene expression clusters were correlated with biological process categories, identifying statistically over- and under-represented biological processes among the genes in each cluster by binomial test.

Data analysis

All the data were analyzed using STATA (StataCorp LLC, College Station, TX, USA) and Prism GraphPad 5.0 software. Significant difference among groups was determined using ANOVA, interaction effect was determined using factorial analysis, and repeated sampling of two samples was analyzed using mixed model analysis. Mean values were considered statistically significant at p < 0.05.