Exosomes released from educated mesenchymal stem cells accelerate cutaneous wound healing via promoting angiogenesis

Abstract Objectives Skin serves as the major interface between the external environment and body which is liable to many kinds of injuries. Mesenchymal stem cell (MSC) therapy has been widely used and became a promising strategy. Pre‐treatment with chemical agents, hypoxia or gene modifications can partially protect MSCs against injury, and the pre‐treated MSCs show the improved differentiation, homing capacity, survival and paracrine effects regard to attenuating injury. The aim of this study was to investigate whether the exosomes from the educated MSCs contribute to accelerate wound healing process. Materials and methods We extracted the exosomes from the two educated MSCs and utilized them in the cutaneous wound healing model. The pro‐angiogenetic effect of exosomes on endothelial cells was also investigated. Results We firstly found that MSCs pre‐treated by exosomes from neonatal serum significantly improved their biological functions and the effect of therapy. Moreover, we extracted the exosomes from the educated MSCs and utilized them to treat the cutaneous wound model directly. We found that the released exosomes from MSCs which educated by neonatal serum before had the more outstanding performance in therapeutic effect. Mechanistically, we revealed that the recipient endothelial cells (ECs) were targeted and the exosomes promoted their functions to enhance angiogenesis via regulating AKT/eNOS pathway. Conclusions Our findings unravelled the positive effect of the upgraded exosomes from the educated MSCs as a promising cell‐free therapeutic strategy for cutaneous wound healing.


| INTRODUC TI ON
Skin is the largest organ of body which serves as the first defence line to the external environment. However, it makes the skin tissue liable to many kinds of injuries like incision, burn and infection. 1 Impaired cutaneous wound healing may become life-threatening and is a major public health issue worldwide. 2 To date, various strategies have been explored, and mesenchymal stem cell (MSC) therapy shows a great potential. 3 More and more studies have shown that MSC therapy could promote cutaneous wound healing and reorganized the structure of skin. 4,5 Meanwhile, pre-treatment of MSCs could enhance the function and efficacy of therapy, and many studies focus on how to optimize MSCs before their applications. [6][7][8] However, many studies have shown that only part of transplanted MSCs can eventually survive and incorporate into the host tissues. [9][10][11][12][13] Therefore, improving the functions of MSC before transplantation is a good choice to ensure the effect of therapy.
As we all known, young MSCs have a more powerful therapeutic effect, but whether it is affected by the cell microenvironment in serum is still eye-catching.
Several studies have pointed out a key class of mediators that function between MSCs and target cells: exosomes, a population of extracellular vesicles, 14 were shown as the important regulators of cell functions. 15,16 Exosomes are abundant in circulation system and could influence target cells by transmitting factors including proteins, mRNAs, microRNAs and so on. 17,18 The components of exosomes are varied in different conditions, which are likely to be involved in various pathophysiology environments in the body.
For example, the exosomes derived from aged cells are different from that derived from normal cells. 19 The specific changes in the components of exosomes have been adopted in disease diagnosis and cell-free therapy. 16,20 Moreover, local exosomes secreted by stem cells in hypothalamus have been revealed to affect ageing and locally delivering the exosomes secreted by young hypothalamic stem cells slows down the ageing of mice. 21 Meanwhile, the exosomes from the pre-treated MSCs emerged a therapeutic effect on wound healing, which reminded us using the upgraded exosomes for treatment and regulating tissue regeneration might be a good approach.
Thus, in this study, we selected the cutaneous wound healing model to observe the effect of the exosomes which from serum of neonatal and adult mice on MSCs, and the potential therapeutic effect of exosomes derived from the educated MSCs. In our present study, we firstly compared the differences between the exosomes from serum of neonatal and adult mice. Then, we investigated the biological effect of two exosomes on MSCs and compared the therapeutic ability in cutaneous wound repair between these two educated MSCs. We found that the MSCs educated by exosomes from neonatal serum had a better therapeutic effect on wound healing process. Moreover, we extracted the exosomes from two educated MSCs and utilized them to treat the cutaneous wound directly to compare the therapeutic efficacy. We found that two released exosomes both had the therapeutic effect, and the exosomes released from MSCs which educated by neonatal serum before had the more outstanding performance. Mechanistically, we revealed that the recipient endothelial cells were targeted, and the exosomes promoted their functions to enhance angiogenesis via regulating AKT/eNOS pathway. In conclusion, we verified the positive effect of exosomes from neonatal serum on MSCs and provided the upgraded exosomes from educated MSCs as a promising cell-free therapeutic strategy for cutaneous wound healing. cutaneous wound model directly. We found that the released exosomes from MSCs which educated by neonatal serum before had the more outstanding performance in therapeutic effect. Mechanistically, we revealed that the recipient endothelial cells (ECs) were targeted and the exosomes promoted their functions to enhance angiogenesis via regulating AKT/eNOS pathway.

Conclusions:
Our findings unravelled the positive effect of the upgraded exosomes from the educated MSCs as a promising cell-free therapeutic strategy for cutaneous wound healing.

| Isolation and characterization of exosomes
The neonatal mice (5-7 g) that were 14 days old and the adult mice (20-22 g) that were 12 weeks old were used. Firstly, the mice underwent anaesthesia by an intra-peritoneal injection of pentobarbitone sodium (40 mg/kg). Then, the whole blood was collected from heart and approximately 150-200 μL whole blood could be collected from each neonatal mouse, and 1-1.3 mL whole blood could be collected from each adult mouse. The serum was separated from whole blood and combined for further exosomes isolation. Approximately 40 μL serum could be separated from 100 μL whole blood. Exosomes were isolated by using the optimized protocol. The serum was mixed with an equal volume of phosphate-buffered saline (PBS) and centrifuged at 2000 g for 30 minutes; then, the supernatant was centrifuged at 12 000 g for 45 minutes. The supernatant was transferred into a fresh tube, filtered with a 0.22 μm filter and pelleted by ultracentrifugation (Beckman Optima L-100 XP, Beckman Coulter) at 110 000 g for 120 minutes. Exosome pellets were washed in a large volume of PBS and recovered by centrifugation at 110 000 g for 70 minutes ( Figure S1).
Firstly, the primary MSCs were cultured in α-MEM with 20% exosome-free FBS and all conditions of culturing MSCs were uniform before pre-treatment. Then, the second passage of MSCs was pre-treated with exosomes (20 μg/mL) from neonatal or adult serum for 24 hours under basic medium without FBS. After pre-treatment, the fresh FBS-free medium was changed and MSCs were cultured for another 48 hours. Finally, the conditioned medium was collected to isolate exosomes by ultracentrifugation. Briefly, cell culture supernatant was centrifuged at 2000 g for 10 minutes. Next, the supernatant was collected and centrifuged at 10 000 g for 30 minutes. The final supernatant is then ultracentrifuged at 100 000 g for 70 minutes. The pellet was washed in a large volume of PBS to remove contamination of proteins and ultracentrifuged at 100 000 g for 70 minutes once more ( Figure S2).
The collected exosomes were resuspended in PBS and quantified by BCA assay (TIANGEN, PA115) before using and stored at −80°C for further study if necessary. Approximately 25 μg exosomes could be isolated from 1 mL serum. In all experiments, neonatal and adult serum exosomes were used with the same and uniform concentrations. Exosomes were fixed in 4% paraformaldehyde (Sigma-Aldrich, 16005), washed and loaded onto copper grids. After washing, exosomes were post-fixed in 2% glutaraldehyde for 2 minutes, washed and contrasted in 2% phosphotungstic acid for 5 minutes. Samples were washed and dried, and images were obtained with an electron microscope (HITACHI, H7500). The size distribution was measured

| Colony-forming unit assays
To assess the colony-forming efficiency of MSCs, single-cell suspensions with α-MEM containing 20% FBS were seeded in 5 cm diameter culture dishes (Corning, 430166) at a density of 1 × 10 2 cells per well and cultured at 37°C in a humidified atmosphere containing 5% CO 2 . The medium was refreshed every other day. After culturing for 5 days, the dishes were rinsed with PBS and the cells were fixed by 4% paraformaldehyde (Sigma-Aldrich, 16005). The cells were stained with 0.2% crystal violet (Sigma-Aldrich, C6158), washed with distilled water and dried for evaluation under the inverted optical microscope (Leica, M205FA).

| Osteogenic differentiation assay
Mesenchymal stem cells were seeded in six-well plates at a density of 5 × 10 5 cells per well. When cells reached 100% confluence, the basal medium was changed into osteogenic induction medium: α-MEM containing 20% FBS, 1% penicillin/streptomycin, 5 mM β-glycerophosphate, 50 μg/mL ascorbic acid and 10 nM dexamethasone (all from Sigma-Aldrich). The medium was refreshed every other day. For alkaline phosphatase (ALP) staining, after 10 days the medium was discarded, and the samples were washed with PBS twice and fixed with 4% paraformaldehyde (Sigma-Aldrich). ALP staining was performed with a commercial kit (Beyotime, C3206) according to the manufacturer's protocol. Cells were cultured for 14 or 28 days, and the Alizarin red (Sigma-Aldrich, A5533) staining was performed according to the manufacturer's instructions. Photographs were taken by an inverted optical microscope (Leica, M205FA). The 10% cetylpyridinium chloride was added for quantitative analysis, and the absorbance values were measured at 562 nm.

| Adipogenic differentiation assay
Mesenchymal stem cells were seeded in six-well plates at a density of 5 × 10 5 cells per well. When cells reached 100% confluence, the basal medium was changed into adipogenic medium: α-MEM containing 20% FBS, 0.5 mM isobutylmethylxanthine, 0.5 mM dexamethasone and 60 nM indomethacin (all from Sigma-Aldrich). The medium was refreshed every other day. After induction for 7 or 14 days, Oil Red O (Sigma-Aldrich, O0625) staining was performed to determine lipid droplet formation. Photographs were taken by the inverted optical microscope (Leica, M205FA). The positive area was measured by ImageJ software (NIH). The quantification was studied based on semi-automatic plug-ins, which followed the same operational methods.

| Internalization of exosomes into MSCs in vitro
The MSCs were plated onto dishes and maintained at 37°C over-

| Immunofluorescence
Cells and tissue samples were fixed in 4% paraformaldehyde for 12 hours. The skin tissue samples underwent dehydration with 30% saccharose and were embedded into the optimal temperature

| Skin wound healing model
The mice underwent anaesthesia by an intra-peritoneal injection of pentobarbitone sodium (40 mg/kg). After shaving and cleaning, a full-thickness wound (1 cm in diameter) was created on the dorsal skin. MSCs and MSC-derived exosomes were resuspended in PBS and intra-dermally injected around each wound with four sites, respectively, and the injection was administrated in the layer of dermis. The number of MSCs was 2 × 10 6 (in 100 μL PBS) when they were applied to the wound site, which was calculated by the digital cell counter (Bio-Rad). The concentration of exosomes was 100 μg (in 100 μL PBS) when they were applied to the wound site, which was measured by BCA assay (TIANGEN, PA115). In all experiments, MSCs and exosomes in different groups were used with uniform concentrations. After operation, the wound was covered by surgical dressings (3M, 9546) and the mice were kept individually to ensure that they do not bite each other. The dressing of each mouse was removed uniformly, and then, pictures were taken to observe the healing process. Particularly, we cut the film into a square shape slightly larger than the round wound in the middle of the back and ensure that the film fixed on the back of mouse is completely inaccessible.
The wound area and body weight were measured on day 0 to 14 post-surgery, and the wound healing rate was calculated by ImageJ software (NIH, USA). The mice were sacrificed, and the skin tissues were harvested for the further analysis.

| Histological analysis
To observe the result of wound healing and collagen fibre disposition, the skin tissue samples were fixed in 4% paraformaldehyde for

| Cell migration assay
The endothelial cells (ECs) were seeded in 6-well plates at a density of 5 × 10 5 cells per well. When ECs reached 100% confluence, the scratch was made on the plates by using the sterile pipette tips.
ECs were cultured in special medium (without FBS) and added with different exosomes at 20 μg/mL or co-culture with the pre-treated MSCs. Photographs were taken by an inverted microscope at 0, 12 and 24 hours and evaluated by ImageJ software (NIH, USA).

| Tube formation assay
In vitro capillary network formation was determined by tube for-

| Exosomes inhibition
For experiments requiring exosomal inhibition, firstly, MSCs were pre-treated with exosomes from neonatal serum for 24 hours under basic medium without FBS. After pre-treatment, the fresh FBSfree medium was changed and MSCs were incubated with 20 μM GW4869 (MCE, HY-19363) for 24 hours before exosome isolation.
Finally, the pre-treated MSCs and conditioned medium were collected for follow-up experiments, and the effect of inhibition was measured by BCA assay.

| Statistical analysis
Data were expressed as mean ± SD, as indicated. Comparisons between two groups were performed by Student's t test, and multiple group comparisons were performed by one-way ANOVA. Bonferroni correction was used when multiple comparisons were performed. P values <.05 were considered statistically significant. Graphs and statistical analysis were performed by using GraphPad Prism (GraphPad Software, 7.0) and SPSS software (IBM, 19.0).

| Isolation and characterization of MSCs
The purified MSCs were successfully obtained from the murine bone marrow. They were propagated on a standard dish in vitro and exhibited fibroblast-like morphology. The cells exhibited the characteristic pattern of mesenchymal stem cell surface markers, including CD29, CD90, CD105, CD146 and Sca-1, whereas the hematopoietic markers CD34, CD11b and CD45 were negative ( Figure 1A,B). When the MSCs were cultured at a low density, they formed adherent clonogenic cell clusters ( Figure 1C). To investigate the differentiation potential of the MSCs, they were cultured in osteogenic differentiation medium. ALP staining was performed after 10-day induction ( Figure 1D), and mineralized nodules were stained with Alizarin Red after 28-day induction ( Figure 1E). After culturing in adipogenesis inducing medium for 14 days, the MSCs were found to form lipid droplets, as confirmed by Oil Red O staining ( Figure 1F).

| Characterization of the exosomes from serum of neonatal and adult mice
As shown in the results, transmission electron microscopy revealed the vesicles from serum in the size range of ~100 nm (Figure 2A). It was observed that there was a significant red fluorescence signal increasing in the time-and concentration-dependent way as we expected ( Figure 2D,E). To verify the reliable internalization of exosomes by MSCs, confocal microscope measurements varying positions on the z-axis with higher spatial resolution were also carried out ( Figure S3). Therefore, these data demonstrated that little difference of morphology, size distribution and expression of surface markers from exosomes of neonatal and adult serum, and the exosomes could be uptaken by MSCs in the time-and concentrationdependent way.  Figure S4).

| Educated MSCs promoted cutaneous wound healing after transplantation
In order to verify the therapeutic efficacy of MSCs that were edu-  Figure 4G,H). These data demonstrated that educated MSCs accelerated the cutaneous wound healing rate and enhanced the cutaneous regenerative quality by promoting angiogenesis.

| The educated MSC-derived exosomes promoted cutaneous wound healing
To further investigate the therapeutic effect of educated MSCs, we tested whether the exosomes derived from the educated MSCs could also have the same result. We locally administrated exosomes in cutaneous wound healing model by using the same methods with MSC therapy ( Figure 5A). Exosomes were intra-dermally injected into the wound site, and PBS was also used as the negative control.
Photographs of wound area were taken at three different timepoints during the wound healing process, and the wound healing rate was also calculated ( Figure 5B,C). As we expected, the group of exosomes derived from MSC NS-Exo (NM-Exo) healed the fastest among the three groups, while no significant differences were found in the body weight of all groups ( Figure 5D). We then collected the skin samples at day blast will lead to scar hyperplasia and fibrosis, which is undesirable in cutaneous wound healing process. 27,28 Therefore, we also performed the immunofluorescence staining of α-SMA on the samples of different groups and found that the fluorescence intensity in the NM-Exo group was obviously lower than the other two groups, which also demonstrated that the application of NM-Exo could inhibit the hypertrophic scar formation in wound healing process ( Figure S6).
Moreover, the expression level of CD31 by immunofluorescence staining showed the better angiogenesis in NM-Exo-treated group than AM-Exo ( Figure 5G,H). These data demonstrated using NM-Exo could accelerate the cutaneous wound healing and enhance the intensity of angiogenesis more powerfully.

| Exosomes released by educated MSCs upregulated the functions of endothelial cell to promote angiogenesis
As we have shown that the exosomes derived from educated MSCs Meanwhile, the aim of our study was to obtain functionally improved exosomes by pre-treating the MSCs. Therefore, to verify the key role of releasing exosomes, we used GW4869 to treat MSC NS-Exo for inhibiting the ability of releasing exosomes ( Figure S7). We established coculture system and collected the conditioned medium from MSC NS-Exo to treat ECs and examined the changes on the abilities of proliferation, migration and tube formation. The results showed that the functions of MSC NS-Exo were blocked after the application of GW4869 in promoting angiogenesis, indicating the exosomes released by the educated MSCs play a key role in therapeutic process ( Figure S8). Moreover, we explored the detail mechanisms underlying angiogenesis activated by exosomes.
Since vascular endothelial growth factor (VEGF) signalling is critical for regulating angiogenesis, we examined the expression of several important proteins in this pathway. [29][30][31] We found the protein expression levels of p-AKT, p-eNOS in ECs increased after application of exosomes, especially in NM-Exo group ( Figure 6G, H). These data revealed that the exosomes released from educated MSCs stimulated angiogenesis and involved in activation of AKT-mediated VEGF signalling pathway.  As a kind of extracellular vesicles compared to chemical compounds, exosomes derived from the living cells can be more biocompatible. Moreover, it is known that the regenerative ability of mammals decreases with ageing. 51 For example, the adult mammalian heart possesses limited potential for repair and regeneration, while in neonatal mice, the heart can regenerate fully without scarring following injury. 52 In the treatment of myocardial infarction, the researchers found that MSCs from younger donors are more efficacious than those from older donors. 53 Previous studies suggest that the circulating environment of young animals has the ability to promote tissue regeneration and reverse age-related impairments, and the molecular signalling pathways which critical to the activation of tissue-specific progenitor cells can be modulated by circulating factors. 54,55 Utilizing a parabiosis model between young and old mice, researchers have demonstrated that the high levels of GDF11 in young blood are beneficial to stem cell functions and skeletal muscle regeneration. 56 However, the role of circulating exosomes in young blood has not been investigated. At present, studies on circulating exosomes mainly focus on their role as biomarkers for disease diagnosis or as the delivery vehicles for therapy. [57][58][59] Recent studies have revealed that circulating exosomes reflect the physiological conditions of the body and can be stimulated by exercise. 60 Hence, the exercise-induced circulating exosomes mediate beneficial biological effects on tissues, such as cardioprotective effects, demonstrating that circulating exosomes enhance physiological functions of the body. 61 Therefore, our study considered the potential role of circulating serum exosomes in neonatal blood and used them to pre-treat MSCs which have not been reported yet. In this study, we compared the differences between the exosomes from neonatal and adult serum. Then, we investigated the biological effect of two exosomes on MSCs and compared the therapeutic ability between these two educated MSCs. We found that the MSCs educated by exosomes from neonatal serum had a better effect on wound healing process.
This finding further underlines the importance of exosomes from young individuals in maintenance of stem cell functions and shows the good effect on pre-treatment of the exosomes, which enriches our understanding of the role of exosomes in maintaining the cell and tissue homeostasis.
In recent years, the paracrine pathway has been recognized, and exosomes have been demonstrated to be the key factor mediating the functions of MSCs. Many studies have confirmed the therapeutic effect of exosomes as a cell-free therapy in multiple tissue injury models. 16 MSC transplantation has some disadvantages and risks, for example, the microenvironment of damaged tissue is not conducive to the survival of MSCs, which resulting in a low retention rate and affecting the therapeutic effect. Exosomes are rapidly emerging as a promising therapeutic platform for cell-free application with the advantages of immune privilege, signalling transfer and efficiency for endocytosis. 62 Recent studies indicated that the therapeutic effects of MSC administration that happened via paracrine mechanism and exosomes can be directly used to enhance the cutaneous wound healing by promoting angiogenesis. 63 Exosomes are smaller, less complex, less immunogenic and more biocompatible than cells, so that application of exosomes is safer.
Meanwhile, the quality of exosomes is more controllable, and the cargos of exosomes are more stable, so that exosomes are more convenient to product, store and transport. Unlike living cells, there is no need to worry about the survival and retention rate. 50 In our study, we found that the exosomes released by educated MSCs had the powerful ability to treat the cutaneous wound healing, which is consistent with the effect of MSC therapy. Moreover, we revealed that exosomes released from MSCs which educated by exosomes from neonatal serum before had a more outstanding performance through enhancing the functions of endothelial cell via regulating the related signalling pathway.
In conclusion, our study shed light on the efficacy of exosomes in tissue repair, which widens the research scope of extracellular vesicles. Meanwhile, our study provides new evidence to expand the possibility of pre-treatment of MSCs as the strategy. In addition, the underlying mechanism through which the upgraded exosomes released by educated MSCs could promote angiogenesis that can be applied in tissue engineering and regenerative medicine by regulating the biological properties of implanted MSCs.