Of note, studies described herein include, but are not limited to, PSCs and derivatives. Indeed, EVs from cells with well-described paracrine actions, for example, MSCs, have been shown to exert beneficial effects in various applications. Depending on the disease and affected tissue, the therapeutic potential of EVs should therefore be examined from a variety of cell sources.
3.3.1. Tissue Restoration in the Eye
Therapeutic effects of EVs have been evaluated for RP, AMD, and following injuries to the eye. Most of these studies focused on the potential of EVs to limit inflammation that would otherwise trigger the death of retinal cells [
147].
As previously mentioned, different studies have started to reveal that the presence of cells might not be required to generate a beneficial effect. As an example, subretinal implantation of human neural progenitor cells preserved the vision of a model of RP through a paracrine effect [
148]. The same effect is elicited by human fetal retinal progenitor cells in rats modeling RP and human patients [
149,
150]. As these transplantations were performed before PR degeneration, a replacement of dead cells was clearly not expected. Building on this, EVs derived from neural progenitor cells were injected subretinally in rats modeling RP before vision loss [
151]. Following a single injection, the visual function and PR survival was temporarily improved (up to 28 days post-surgery). EVs were mostly internalized by Iba1+ microglial cells that had migrated from the inner retina to the subretinal space. EVs induced the downregulation of pro-inflammatory cytokines and inhibited microglia, whose suppressed activation is involved in PR survival in RP [
152].
A choroidal neovascularization (CNV) is characteristic in wet AMD and can be induced in vivo with laser injuries in rodent (laser-induced CNV model). Human umbilical cord blood MSC (hUCMSC)-derived EVs injected once intravitreally were sufficient to reduce vessel leakage and the development of CNV via downregulation of
VEGF-A [
153]. Preservation of retinal functions and suppression of inflammation is equivalent when MSCs or their EVs are injected intravitreally [
154]. In the same vein, retinal astroglial cell-derived EVs inhibited laser-induced CNV in mice when injected daily through the subtenon route for 7 days [
155]. Interestingly, injections of EVs derived from RPE cells did not recapitulate these results [
155]. Retinal neovascularization was also observed following oxygen-induced retinopathy in mice. The injection of EVs derived from hMSCs cultured under hypoxic conditions also reduced neovascularization [
156].
Therefore, EVs are able to modulate retinal degeneration via the inflammatory response. This function is similar to transplanted cells, suggesting that the action of transplanted cells is likely through a paracrine effect (i.e., MSCs, retinal astroglial cells, human neural progenitors). An important point raised by these studies is that EVs or their parental cells need to be delivered at an early stage before complete degeneration. As EVs may not need complex surgeries or immunosuppression, it could be envisioned as a first line of treatment to support retinal survival and delay the requirement for a cell-based intervention. Future preclinical studies need however to determine a delivery route that allows repeated injections for long-term efficiency.
In the context of an advanced RPE cell degeneration as in late AMD, EVs may not be sufficient to recapitulate all RPE functions in order to preserve surrounding retina. Thus, endogenous RPE may be replaced through cell therapy (
Figure 3A). A patch of RPE cells could be proposed as an ideal therapeutic substrate, using supporting scaffolds made of polymers or of biological composition. As EVs modulate inflammation during retinal degeneration, it should be determined whether a combined approach of RPE transplantation and EV therapy may improve visual outcomes. This is particularly true as RPE-derived EVs are not able to reduce the CNV when compared to EVs derived from paracrine-acting cells [
155]. Such combined approaches may be of interest for multi-factorial diseases like AMD and may also preserve grafted cells from degeneration (
Figure 3B).
Another potential EV therapy is related to PR transplantation studies for RP. Early studies showed the feasibility of transplanting post-mitotic PR precursors that achieved some degree of integration into the host mouse retina, ultimately resulting in partial visual function recovery [
42,
157,
158,
159]. The vast majority of transplanted PR precursors were later found to remain in the subretinal space where they engaged in a process of material transfer of functional proteins with host PRs [
43,
44,
45,
160,
161]. The precise mechanism of this material exchange between PRs remains elusive but preclinical studies clearly demonstrated that this exchange occurs only with donor PR precursors and not with other cell types [
7,
160]. It could involve direct donor/host plasma membrane fusion or other methods of intercellular trafficking including EVs [
162]. Therefore, it should be determined whether EVs secreted from PR precursors could recapitulate partial visual recovery obtained with PR precursors. If similar results were obtained, it would be an attractive strategy to preserve endogenous PRs without the need of cell grafting and all associated constraints (i.e., surgery, immunosuppression). When PRs have already degenerated, replacement strategies with PRs susceptible to integrate into the host retina will still be required. As discussed earlier, micro-structured scaffolds and/or addition of RPE cells could improve integration and structuration.
Taking advantage of EV ability to carry nucleic acids, they could be used as vehicles in gene therapy strategies for RP [
9]. As an example, EV-associated AAV2 vector was delivered to the retina and outperformed conventional AAV2 [
163]. Interestingly, these EV-AAV2 served as a robust gene delivery tool in the inner nuclear/outer plexiform and the outer nuclear layer, targeting retinal ganglion cells, bipolar cells, Müller cells as well as PRs [
163]. The natural ability of EVs to deliver bioactive nucleic acids to multiple layers in the inner retina suggests that cell-free EV therapies may also benefit other traumatic or neurodegenerative ocular diseases [
164].
3.3.2. Cutaneous Wound Healing
Major skin injuries, resulting from extensive burns, infection or trauma, require medical interventions to heal properly [
54]. Therapeutic strategies aim at facilitating the 4 phases of cutaneous wound healing–homeostasis, inflammation, proliferation, remodeling-to accelerate wound repair and regeneration [
165]. Molecular and cellular events in these phases are tightly coordinated and many cell types interact with each other in a highly coordinated sequence to restore the damaged tissue [
166].
EVs hold the potential to promote all phases of wound healing and facilitate skin regeneration (reviewed in [
166,
167]). Transition from inflammatory to proliferative phase is a key step for successful wound healing. During the early stages of inflammation, the vast majority of macrophages differentiate towards a pro-inflammatory (M1) phenotype. As the wound matures, the ratio switches to an M2 phenotype that promotes tissue remodeling and wound healing [
168]. EVs obtained from lipopolysaccharide-preconditioned MSCs could convert M1 macrophage polarization to an M2 phenotype, which alleviated inflammation, and enhanced diabetic cutaneous wound healing in rats by shuttling let-7b miRNA [
169]. Similarly, MSC-derived EVs promoted cutaneous wound healing in mice by regulating macrophage polarization through miR-223 [
170]. In line with these results, hUCMSCs significantly decreased the number of inflammatory cells and pro-inflammatory cytokines TNF-a, IL-1, IL-6 levels while increasing the production of the anti-inflammatory cytokine IL-10 in wounds of severe burn rats [
171]. The same team later found that miR-181c expression in hUCMSC-derived EVs reduced burn-induced excessive inflammation by downregulating the TLR4 signaling pathway [
172].
During the proliferative phase, re-epithelization, wound contraction and angiogenesis are essential processes for the restoration of normal tissue architecture. Early recruitment of resident keratinocytes and fibroblasts is particularly important as abnormalities in the intercellular epidermal-dermal crosstalk impairs the skin repair efficiency [
173]. In this context, EVs from both fetal and adult stem cell sources can improve migration and proliferation of both fibroblasts and keratinocytes [
174,
175,
176,
177,
178,
179,
180,
181,
182,
183]. EVs from hUCMSCs and MSCs activate signaling pathways important in wound healing, including RAC-alpha serine/threonine-protein kinase (AKT) pathway [
174,
177,
184] and Notch signaling [
182]. Increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 [
175,
180,
184] and inhibition of phosphatase and tensin homolog (PTEN) [
184] have also been reported. Additionally, an increased extracellular matrix (ECM) deposition by fibroblasts has been observed, facilitating wound contraction. Indeed, fibroblasts increased collagen I and III production following systemic administration of MSC-derived EVs at wound sites in a mice full-thickness wound model [
176]. Similar results were obtained with EVs derived from hiPSC-MSCs [
185] and hUCMSCs [
186]. Human adipose MSCs-derived EVs also prevented the differentiation of fibroblasts into myofibroblasts, increased the ratio of transforming growth factor-β3 (TGF-β3) to TGF-β1 and upregulated the matrix metalloproteinases-3 (
MMP3) expression of skin dermal fibroblasts through the activation of ERK/MAPK pathway [
187]. As such, EVs could be used to promote extracellular matrix remodeling and reduce scar formation.
Transplantation of cellular skin substitutes have shown considerable potential to treat both acute and chronic wounds [
58]. Complex multicellular 3D models are being developed with the goal of making engineered tissues similar to their natural counterpart (
Figure 3A; reviewed in [
59]). However, there is still an urgent need for improving the vascularization of these substitutes to prevent necrosis and provide better long-term function and integration in clinical practice. This is doubly important as patients with chronic skin wounds usually present defects in the angiogenesis process, which consequently leads to delayed wound healing. One possibility would be the use of pre-vascularized skin substitutes that combine dermal fibroblasts, endothelial cells, and epidermal keratinocytes [
63,
65,
66,
188]. An alternative strategy would be to supplement dermo-epidermal skin grafts with EVs conveying pro-angiogenic signals to activate tissue-resident endothelial progenitor cells (
Figure 3B). Indeed, exogenous EVs were shown to promote local angiogenesis in murine models of wound healing [
185,
189,
190]. For example, EVs derived from hUCMSC, hiPSC-MSC and human urine-derived stem cells (USC) enhanced in vitro endothelial cell proliferation, migration, and tube formation [
178,
185,
191]. HUCMSC-derived EVs promoted angiogenesis in vivo to repair deep second-degree burn injury by delivering Wnt4 and activating Wnt/B-catenin signaling in endothelial cells [
191]. EVs derived from human umbilical cord blood enriched in miR-21-3p promoted the proliferation and migration of fibroblasts as well as enhanced the angiogenic activities of endothelial cells in a full-thickness skin wound mice model, thus accelerating re-epithelialization and cutaneous wound healing [
184]. Similarly, EVs derived from hiPSC-MSC applied to wound sites in a full-thickness skin defect rat model promoted not only the generation of newly formed vessels, but also accelerated their maturation [
185]. In another study, human USC-derived EVs markedly enhanced the generation of newly formed blood vessels in diabetic mice, in part via the transfer of pro-angiogenic protein deleted in malignant brain tumors 1 (DMBT1) [
178]. Alternatively, EVs derived from hESC facilitated pressure ulcer healing by reducing endothelial senescence and promoting local angiogenesis at wound site in aged mice [
190].
At present, one of the main obstacles in the treatment of skin wounds is achieving healing over time, particularly in patients with underlying skin disorders. Biomaterial-based wound dressings could be loaded with EVs to achieve sustained release to the wound sites [
192,
193]. For instance, Tao et al. used the polymer chitosan to prolong delivery of EVs derived from miR-126-3p-overexpressing synovium MSCs to diabetic wounds [
193]. They tested this system in a diabetic rat model and found that it increased formation of granulation tissue, which provides a scaffold for the assembly of neighboring cells at wound margins, along with angiogenesis [
193].
Overall, all these proofs-of-concept experiments raised considerable interest of EVs for skin repair. Of interest, their delivery to skin wounds is relatively simple due to easy access and could be sustained over time by the use of biomaterial or repeated topical applications. However, additional preclinical studies are needed to evaluate the synergic effects of combined acellular and cellular strategies.
3.3.3. Heart
EVs have been investigated as promising therapeutic options for various cardiac diseases such as ischemic heart diseases and myocardial infarctions. One of the main objectives is to promote vascular repair mechanisms to reduce myocardial injury that would lead to cell death and therefore improve cardiac functions.
hESC-derived MSC conditioned medium (hESC-MSC-CoM), collected with clinically compatible processes, were shown to contain factors susceptible to modulate cardiovascular-related pathways [
194]. Administration of hESC-MSC-CoM recapitulated the benefit of hESC-MSC injections in a context of post-myocardial infarction [
195]. Indeed, in a porcine model of myocardial infarction, hESC-MSC-CoM intravenous treatment for 7 days enhanced capillary density, reduced the myocardial infarct size and preserved systolic and diastolic functions [
195]. In addition, hESC-MSC-CoM reduced myocardial apoptosis and oxidative stress in another porcine model of ischemia and reperfusion injury [
196]. This hESC-MSC-CoM contained large particles of 50–100 nm that were purified and characterized as EVs [
197]. hESC-MSC-derived EVs similarly diminished the infarct size in an ex vivo mouse model of myocardial ischemia and reperfusion injury.
It was also proposed that ESC-derived EVs could stimulate endogenous myocardial regeneration [
198]. Their delivery via an intramyorcardial route following mouse myocardial infarction stimulated endogenous repair (i.e., revascularization, cardiomyocyte proliferation/survival and reduced fibrosis). Interestingly, fibroblasts-derived EVs did not improve cardiac functions when compared to ESC-derived EVs in this model, highlighting differences between EV sources.
The functionality of cells from the cardiac lineage is superior to MSCs in the different heart disease models [
199,
200]. Therefore, EVs derived from these cells might achieve the most efficient heart recovery. Indeed, hPSC-CM-derived EVs recapitulated the therapeutic effects of their parental cells in the mouse model of chronic heart failure [
201]. In this study, EVs or their parental cells were delivered once intramyocardially. Gene expression profiling identified 927 genes similarly upregulated in hearts treated with hPSC-CM-derived EVs and their parental cells as compared to control. The majority of enriched biological processes associated with these genes were predicted to improve heart regeneration and decrease fibrosis [
201]. A recent study further highlighted the importance of determining which cellular source is the best candidate to produce therapeutic EVs. While both hPSC- and hPSC-CM-derived EVs protected CMs from hypoxia in vitro, only hPSC-CM-derived EVs completely improved the hypoxia-induced phenotype [
202].
In order to maintain a sustained delivery, hPSC-CM-derived EVs were loaded into a collagen-based hydrogel patch [
202]. Such system allows the release of EVs during 21 days in vitro. Patches loaded with EVs were implanted directly into the myocardium following an ischemic insult in a rat model of acute myocardial infarction. Rats recovered with this treatment, with improved heart contractile function and a reduction of the infarct size [
202].
Overall, recent results indicate that EVs recapitulate the beneficial effects of their parental cells in the treatment of heart diseases (
Figure 3B). Importantly, overall complexity associated with cell manufacturing, graft survival and patient immunosuppression are bypassed by this strategy. Future studies are nevertheless required to validate sustained EV release in large animal models as well as reproducibility across hPSC-CM-derived EV production protocols.
3.3.4. Skeletal Muscle
Severe muscle injuries and genetic defects like muscular dystrophies cause myofiber death. Spontaneous reparation to regenerate skeletal myofibers do occur but are insufficient [
203]. A central goal of therapeutic approaches is to re-establish the muscle structural integrity and functionality by re-populating the satellite cell niche, promoting vascularization while inhibiting fibrosis formation, and stimulating the formation of contractile muscle fibers [
100]. Several cellular candidates with myogenic or non-myogenic origins have been proposed for skeletal muscle regeneration, and their transplantation has been a widely investigated therapeutic strategy [
100]. However, the massive donor cell death and cellular dispersion observed after delivery of cells via injection limit their therapeutic potential. Cell therapy products are still a long way from being able to reconstruct the muscle architecture, let alone to reconstruct it with nerve and sufficient vascularization. Overall, cell therapy could be considered for small muscles but is difficult to implement for diseases affecting all body muscles.
A mounting body of evidence suggests that EVs are actively produced by skeletal muscles cells and contribute to muscle repair and regeneration [
203]. For example, EVs secreted during the differentiation of human skeletal myoblasts (HSkM) into myotubes contain specific biochemical cues that promote and regulate the myogenic differentiation of human adipose-derived stem cells (HASCs) [
204]. Treatment of lacerated muscle sites with these differentiating HSkM-derived EVs led to an improved muscle regeneration with a large number of regenerative myofibers associated to minimal fibrosis compared to the control group [
204]. In addition to the facilitation of myofiber repair, EVs also attenuate excessive ECM deposition for optimal muscle remodeling. In muscular dystrophies and severe muscle injuries, fibrogenic cells are overactivated and hyperproliferate, leading to the substitution of skeletal muscle with nonfunctional fibrotic tissue [
205]. This excessive accumulation of extracellular matrix components not only alters muscle function but also reduces the amount of tissue available for therapy and repair. Establishing new anti-fibrotic therapeutic strategies is one of the major clinical options to improve muscle function in patients. In response to hypertrophic stimuli, satellite cells give rise to myogenic progenitor cells (MPCs) able to secrete EVs containing miR-206, which represses cell collagen expression through ribosomal binding protein 1 (Rrbp1) by neighboring fibroblasts, thus preventing excessive ECM deposition [
206]. Similarly, fibroblasts derived from muscle biopsies of DMD patients secreted exosomes with increased levels of miR199a-5p, causing increased fibrosis in skeletal muscle and surrounding matrix [
207]. These data indicate that EVs could be of interest as potential anti-fibrotic agents.
EVs are also evaluated as potential therapeutic agents to counteract muscle wasting and skeletal muscle dysfunction. Chronic kidney disease (CKD), which ultimately leads to end-stage renal failure, often leads to muscle wasting. Intramuscular injection of EV-encapsulated miR29, previously shown to have anti-fibrotic activity, could attenuate UUO-induced body weight loss and muscle atrophy [
208]. Similar results were obtained after injection of EV-miR26a in CKD mice [
209]. DMD is a heritable myodegenerative disease characterized by the absence of functional dystrophin leading to progressive muscle weakness and degeneration. Recent data suggest that a treatment with EVs from cardiosphere-derived cells (CDCs) originally targeted at DMD cardiomyopathy could potentially benefit both cardiac and skeletal muscle [
210]. CDC-derived EVs injected into the soleus of mdx mouse model of DMD enhanced muscle regeneration, decreased inflammation and fibrosis, allowing complete restoration of contractile forces. More surprisingly, detectable levels of full-length dystrophin were evident in the diaphragm and soleus up to three weeks after systemic CDC-derived EV delivery [
210]. Dystrophin protein and transcript were undetectable in CDC-derived EVs. Moreover, analysis of exon-intron junctions for dystrophins transcripts after CDC-derived EV treatment showed no exon skipping or alternative splicing. However, RNA-seq of CDC-EVs revealed a 144-fold increase in miR-148a. Intramyocardial injection of miR-148a restored expression of dystrophin in mdx hearts 3 weeks after administration, implicating this miRNA as a potential mediator of enhanced full-length dystrophin protein synthesis [
210]. Targeting EV-derived miRNAs appears as a promising strategy to improve muscle function [
211].
Finally, EVs could be used as efficient delivery tools of functional cargoes in vivo to restore expression of missing proteins in patients. For example, Gao et al. demonstrated that systemic administration of exosomes loaded with CP05-conjugated dystrophin splice increased dystrophin protein expression in dystrophin-deficient mdx mice with functional improvements [
212].
It has become clear that EVs enable intercellular signaling that facilitate myofiber regeneration, limit excessive ECM deposits and improve muscle functions. More strikingly, their systemic delivery improves muscle function in diseases like DMD or CDK. Future studies are now required to characterize their biogenesis, compositions and biological activities on recipient cells in both physiological and pathophysiological conditions to determine whether they might be envisioned for therapy [
213].