Mesenchymal Stem/Stromal Cell-Derived Exosomes Part 3

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6.3.Proliferative Phase

During the proliferative phage, fibroblasts from surrounding normal tissues migrate into the injured site. These fibroblasts produce various matrix proteins including collagen I and Ⅲ to strengthen the newly formed scar tissue. MSC-exosomes affect these dermal fibroblasts to promote migration and proliferation and produce collagen, elastin, and fibronectin: (1) human ASC-EVs or ASC-exosomes induced migration and proliferation of dermal fibroblasts or keratinocytes in vitro[234,235];(2)human ASC-exosomes induced collagen I/II and elastin in HDFs, and they enhanced cutaneous wound healing in mice [234,235];(3)human fetal dermal(FD)-MSC-exosomes induced the expression of collagen I/III, elastin, and fibronectin mRNAs by activating the Notch pathway through delivering Jagged 1 protein [236]; and(4)human UC-MSC-exosomes were shown to contain Wnt4 and accelerated reepithelialization of burn skin in rats[237]. The wound healing effects were inhibited when the Wnt4 expression in UC-MSC-exosomes was knock-downed by siRNA.Furthermore, human MSC-exosomes were reported to induce proliferation and migration of fibroblasts in vitro from diabetic wound patients [250]. The positive effects of MSC-exosomes on keratinocytes were also reported as follows:(1)human UC-MSC-exosomesprotectthe immortalized human keratinocytes HaCaTfrom heat-induced apoptosis by activating the AKT pathway [237]; and(2)human WJ-MSC-and MSC-exosomes increased the secretion of collagen in HaCaT [251].

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As mentioned above, angiogenesis is of importance to support the oxygen needed for the proliferation of fibroblasts or other cells in the injured site [229]. It has been also reported that MSC-exosomes induce the angiogenic activity of endothelial cells. Human ASC-exosomes induced tube formation of HUVECs by delivery of miR-125a, which suppresses the expression of angiogenic inhibitor delta-like 4 (DLL4) [252]. Human BM-MSC-EVs or rat BM-MSC-exosomes were also reported to enhance angiogenesis in stroke mice [118]or in rats with renal IR injury [110], respectively. Exosomes from human endometrial MSCs were reported to increase proliferation, migration, and angiogenesis of HUVECs with increased expression levels of angiogenic markers including Tie2, angiopoietin 1(Angl), Ang2, and vascular endothelial growth factor (VEGF) [253]. In addition, the following pro-angiogenic effects of MSC-exosomes have been confirmed in vivo:(1)human umbilical cord blood (UCB)-MSC-exosomes with thrombin preconditioning accelerated cutaneous wound healing in rats with full-thickness wounds. Human UCB-MSC-exosomes increased the angiogenic factors such as angiogenin(Ang), Angl, hepatocyte growth factor(HGF), and VEGF, while reducing TNF-α and IL-6 [238]; (2) human UC-MSC-exosomes enhanced angiogenesis in rats through the Wnt4/β-catenin pathway. lost empire cistanche The pro-angiogenic effects of human UC-MSC-exosomes were abolished when the Wnt4 expression was knock-downed by shRNA [239]; and (3) human MSC-exosomes accelerated both the formation and maturation of new vessels in the wound sites with unknown mechanism [241].

6.4.Remodeling Phase

MSC-exosomes might be beneficial to further reduce scar formation. Uncontrolled accumulation of myofibroblasts in the wound sites causes scar formation. Recently, human UC-MSC-exosomes have been reported to reduce scar formation by inhibiting the accumulation of myofibroblasts in mice [242]. A variety of proteases such as matrix metalloproteinases (MMPs)are necessary for all phases of the cutaneous wound healing process [254]. During the remodeling phase, the controlled release of MMPs by fibroblasts, macrophages, epidermal cells, and endothelial cells contributes to degrading the majority of collagen llI fibers [255]. Regulation of extracellular matrix remodeling by ASC-exosomes has been reported [235]. In this study, it was demonstrated that ASC-exosomes promoted scarless cutaneous wound repair by regulating the ratios of collagen I-to-collagen IⅢ, TGF-β3-to-TGF-β1, and MMP3-to-MMP1.

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6.5. Proteolytic Environment

Uncontrolled protease activities are known to be associated with impaired wound healing [256]. Additionally, prolonged high levels of protease activities have been suggested to be associated with delayed wound healing in chronic wounds [254-257]. In fact, elevated levels and activities of collagenase (MMP-1 and MMP-8) and gelatinases (MMP-2 and MMP-9) are characteristics of chronic wounds [255]. This highly proteolytic environment is not favorable for advanced biologicals such as growth factors [258]. In fact, the use of platelet-derived growth factor (PDGF)for the treatment of chronic wounds has been reported with modest effect [259]. Based on this, a clinical study for the treatment of chronic wounds with a combination of topical growth factors and proteinase inhibitors was recently initiated [260. The proteolytic environment of chronic wounds might be also unfavorable for the treatment of MSC exosomes since the surface proteins on the exosomes are susceptible to proteolysis, which may change the interaction between exosomes and recipient cells [261]. micronized purified flavonoid fraction 1000 mg uses Therefore, a protease-resistant formulation of MSC-exosomes would be necessary for maximum efficacy, especially for topical applications, as reported for PDGF [262,263]. Recently, human gingival MSC (GMSC)-exosomes with chitosan/silk hydrogel showed enhanced wound healing in diabetic rats with appropriate swelling and moisture retention capacity suggested as effects of this hydrogel [242]. This hydrogel may also provide protection of exosomes from proteases in the wound site.

6.6.Animal Models

Most of the animal studies for wound healing with MSC-exosomes have been performed in rodents, except for two studies with rabbits and dogs [232,243] (Table5). However, the structure and physiology of rodent skin do not reflect those of human skin. Pigs are the most optimal preclinical models for wound healing because of the highest similarities between pig and human skin including skin architecture, hair density, and physiology of the wound healing process [264-268]. It is necessary to confirm the effects of MSC-exosomes on cutaneous wound healing in pig models for a better understanding of MoA and clinical applications.

6.7.ASC-Ex0s0m1es

The beneficial effects of fat graft on wound repair are widely accepted, while the underlying mechanism remains unknown [269]. These effects might be related to exosomes from the subcutaneous fat layer. Recently, it has been revealed that human ASC-exosomes induce proliferation and migration of HDFs, and the expression of N-cadherin, cyclin1, PCNA, collagen I/I, and elastin in HDFs in vitro, which results in reduced scar formation in mice by regulating extracellular matrix remodeling [234,235]. No direct evidence that shows an advantage of ASC-exosomes over exosomes from other MSCs is available. ASCs, however, are distinct in immunomodulation compared to BM-MSCs. BM-MSCs enter the wound site through the blood supply to initiate the first phase of wound healing [270]. In the injured site, BM-MSCs prolong and enhance the inflammation by increasing the survival and function of neutrophils [271]. Under hypoxic conditions, which induces the activation of TRL4, BM-MSCs secreted pro-inflammatory factors and decreased the polarization of macrophage from the M1 to M2 phenotype [272,273]. Therefore BM-MSCs in the wound site might not induce the anti-inflammatory M2 macrophages without enough oxygen supply by neovascularization. oteflavonoid On the contrary, the phenotype and secretome of ASCs were largely unaffected by prolonged hypoxia [274], and the CM from ASCs showed better inducing effects of the anti-inflammatory M2 macrophage phenotype than the CM from BM-MSCs [275]. These results suggest that ASC-exosomes might be more beneficial than BM-MSC-exosomes to induce appropriate wound healing processes. In summary, MSC-EVs or MSC-exosomes contribute to each phase of wound healing by inducing M2 polarization and stimulating dermal fibroblasts to produce structural proteins and proteases necessary for remodeling of the extracellular matrix.

7. MSC-Exosome-Induced Hair Growth

Hair follicle cycling is a dynamic and complex process involving alternating phases of rapid growth (anagen), regression(catagen), and quiescence(telogen)[276]. Hair follicles, which reside in the dermal layer of the skin, are made up of various cell types including dermal papilla (DP) cells and outer root sheath(ORS) keratinocytes, each having distinct roles [277]. In addition to these cells, ASCs located in the adipose tissue below the dermis may also affect hair cycling as ASCs differentiate into mature adipocytes and surround hair follicles during the telogen to anagen transition [278]. Although a direct relationship between dermal papilla cells and ASCs has not been elucidated, it can be anticipated that ASCs exert effects on hair growth, as numerous studies have shown that transplantation of ASCs and CM from ASCs enhances the proliferation of DP cells in vitro and promote hair growth in mice and human [279-281]. Indeed, interactions between these cell types through various mediators lead to a transition from the telogen to anagen phase. Activation of the Wnt/ß-catenin signaling is one of the main pathways involved in hair follicle development. Previous studies have shown that dermal Wnt ligands regulate the hair-inducing activity of DP cells by maintaining the anagen phase [282,283]. puritans vitamin c In addition, growth factors such as fibroblast growth factor-5(FGF-5)produced by ORS cells or insulin-like growth factor-1 (IGF-1) produced by DP cells increase the proliferation of hair follicle cells 【284，285】. Thus， the Wnt/ß-catenin signaling and secretion of growth factors are crucial for hair growth.

Dysregulation of hair cycling caused by various factors such as environmental, genetic, hormonal, and aging, results in hair loss [286-288]. Currently, finasteride and minoxidil are the mainstay treatments for alopecia, although they are not fundamental treatments that induce hair growth, not to mention having various side effects associated with them [289,290]. Hair transplantation is frequently utilized as a fundamental treatment of hair loss but it is an invasive procedure and the graft survival rate largely depends on the surgeon [291]. There is a strong unmet need for a minimally invasive treatment that not only retards hair loss but also promotes hair growth.

7.1. The Effects of DP-Exosomes on Hair Cells

As DP cells are the key player in hair follicle cycling as they secrete growth factors, activate the Wnt signaling, and promote differentiation of hair follicle stem cells, it can be anticipated that exosomes derived from DP cells can also modulate hair follicle cycling. Indeed, studies have shown that exosomes derived from DP cells(DP-exosomes) promote hair growth. Cutaneous injection of human DP-exosomes increased the anagen to catagen ratio in mice and stimulated proliferation and ß-catenin expression of ORS cells [292]. Exosomes derived from the 3D culture of human DP cells increased the percentage of Ki67-positive cells in cultured hair follicles and induced hair follicles in mice implanted with human DP spheres by activating the Wnt and bone morphogenic protein (BMP) signaling [293]. A study by Yan et al. identified 34 differentially expressed miRNAs that are involved in the proliferation and differentiation of hair follicle stem cells by goat DP-exosomes [294].

7.2. The Effects of MSC-Exosomes on Hair Growth

Similar to DP-exosomes, MSC-exosomes are also known to carry a myriad of growth factors and Wnt activators in their cargo. For instance, human UC-MSC-exosomes were found to transport Wnt4 and Wntl1 and subsequently activate the Wnt signaling and promote cell proliferation in target cells [237,239,295]. sistanche Therefore, MSC-exosomes are attractive treatment options for hair growth as well. However, to date, there is only one publication reporting the effects of MSC-FVs on hair growth [96l The authors showed that mouse BM-MSC-EVs promoted the proliferation of human DP cells and induced secretion of growth factors such as VEGF and IGF-1, which are essential for hair growth [285,297,298]. In addition, when mice were intradermally injected with BM-MSC-EVs, an increased anagen to telogen ratio was evident in C57BL/6 mice, along with elevated Wnt protein levels in the dorsal skin. These results suggest that MSC-EVs or MSC-exosomes might have the potential to promote hair growth. Further studies will be necessary to elucidate the potential of various MSC-exosomes on hair follicle cycling.

8. Repair and Regeneration of Skin barrier by MSC-Exosomes

The skin is the largest organ in the human body, comprising about 15% of the total body weight, and is well known as the barrier between the external environment and the human body, preventing loss of moisture and protecting the body from UV light, pathogens, chemicals, and mechanical injuries [299]. The skin is composed of three layers: epidermis, dermis, and hypodermis. The epidermis is the outermost layer of skin and functions as the waterproof barrier. The dermis is a layer below the epidermis, consisting of tough connective tissues, hair follicles, sebaceous glands, apocrine glands, lymphatic vessels, blood vessels, and sweat glands. The hypodermis (also known as subcutaneous tissue) is the deepest layer of skin and is composed of fat and connective tissue [300,301].

8.1.Skin Barrier

The skin barrier is commonly divided into three distinct functional barriers: microbiome, chemical, and physical barriers [302]. The microbiome barrier comprises the outer side of the skin barrier and is composed of diverse microbial communities such as bacteria, fungi, and viruses [295]. The skin microbiome can protect the body against exogenous exposure and invasion of pathogens and can affect immune cell maturation in skin development. It also functions as the skin immune mediator, which cross-talks between skin cells and the skin immune system [302].In some cases, altered microbial states result in skin disease [303]. As an example, the increased abundance of Gemella and Streptococcus species is observed in AD [304]. The chemical barrier provides the acidic surface pH, which is the key factor in the desquamation and regeneration of the skin barrier[303]. It also provides the lipid barrier of ceramides, cholesterol, and free fatty acids, consisting of a molar ratio of 1:1:1 [305]. The lipids prevent loss of moisture from skin and invasion of environmental substances. Furthermore, free fatty acids contribute to the homeostasis of the barrier function, maintaining acidic pH in the skin [306]. In addition, the chemical barrier, especially the biochemical barrier, provides antimicrobial peptides. Antimicrobial peptides are a major factor of the innate immune systems and build the first line of defense against bacteria and viruses [307].

The physical barrier consists of stratum corneum (SC) and tight junction (TJ). The SC is the outermost layer of the epidermis consisting of dead keratinocytes (corneocytes)[308]. Living keratinocytes are transformed into non-living corneocytes during cornification. Cornification is completed by the replacement of the cell membrane with a layer of ceramides covalently linked to the cornified envelope. This ceramide-corneocyte complex in SC contributes to the skin's barrier function [309]. The epidermal TJ not only anchors cells to the neighboring cells but also prevents the escape of moisture between cells[310]. If TJ is damaged, the Langerhans or dendritic cells, which are located below the TJ network, stretch their dendrites to the upper side of TJ and then are activated by allergens and lead to allergic responses [301,311].

Dysfunction and damage of the skin barrier lead to several diseases such as AD [310], psoriasis [310], rosacea [312], and acne vulgaris [313]. Up to now, most of the therapeutic approaches for these diseases have targeted inflammation:(1) dupilumab, a dual inhibitor of IL-4 and IL-13, was recently approved to treat AD[314];(2) monoclonal antibodies inhibiting IL-12, IL-23, or IL-17 are being developed for the treatment of psoriasis [315];(3) a topical drug, ivermectin, for the treatment of mild-to-moderate rosacea has an anti-inflammatory effectJ316); and(4) anti-inflammatory drugs are also used to treat acne vulgaris, although the first-line treatment of acne vulgaris is antibiotics [317]. Moisturizers, used to reduce xerosis or dryness, could prove to be toxic to individuals with compromised skin while being harmless to those with normal skin [318]. Physiologic lipid-based barrier creams, containing three essential lipids including ceramides, cholesterol, and free fatty acids, have been reported to improve barrier function and reduce pruritus as well[318]. However, currently, no treatment option is available to repair or regenerate skin barrier functions.

8.2.The Effects of ASC-Exosomes on Skin Barrier

Recently, human ASC-exosomes have been reported to promote epidermal barrier repair in a mouse AD model109]. Repeated exposures of oxazolone to hairless mice induced AD-like symptoms including inflammation and skin barrier abnormalities [319]. Subcutaneous injection of ASC-exosomes induced restoration of the skin barrier by the production of ceramides and dihydroceramide with long acyl chains in a dose-dependent manner. ASC-exosomes also induced the synthesis of sphingoids including sphingosine and S1P, increased SphK1 activity, and reduced S1P lyase (S1P1) activity in the injured skin. As mentioned previously, the S1P/Sphk1/S1PR axis is of importance for inducing M2 macrophage polarization by ASC-exosomes, which reduce inflammation and promote cutaneous wound healing[94]. Further study will be needed to elucidate the role of M2 macrophage polarization by ASC-exosomes in skin barrier repair. In addition, ASC-exosomes increased the number of epidermal lamellar bodies and the formation of the lamellar layer at the interface of SC and stratum granulosum. Transcriptome analysis of diseased skins revealed that ASC-exosomes reversed the abnormal expression of genes involved in skin barrier maintenance, lipid metabolism, the cell cycle, and inflammatory responses induced by repeated oxazolone exposures. These results suggest that ASC-exosomes could be a promising cell-free treatment for the regeneration of the skin barrier in various diseases with skin barrier defects.

9. Application of MSC-Exosomes for Regenerative Aesthetics

Physical changes in the skin over time produce psychosocial impacts that significantly affect social interactions [320]. With the global increase of older individuals over 65, there is an expanding demand for repair or rejuvenating products and procedures for aged skin [300,320]. Stem cell conditioned media(CM), mostly from the culture of MSCs, have been used as a skincare product for anti-aging, anti-wrinkle, and skin and hair care [321]. MSC-CM contains beneficial secretomes, including secreted growth factors as well as exosomes. However, MSC-CM also contains unintended ingredients including media components and additives, and cellular waste such as lactate and ammonia, both of which are restricted in cosmetics [322,323]. On the contrary, isolated MSC-exosomes avoid these potentially harmful components. Currently, the tangential flow filtration (TFF)method is recommended as a suitable industrial-scale method to isolate exosomes among various techniques [40,324]. The TFF method can markedly reduce the levels of lactate and ammonia from exosome preparation (Ha et al. unpublished observation). Recently, it has been demonstrated that human ASC-exosomes isolated by the ExoSCRTrM technology, a TFF-based exosome isolation method, are safe, showing no adverse effects in GLP toxicological tests including skin sensitization, in vitro photosensitization, eye, and skin irritation, or acute oral toxicity in accordance with OECD guidelines [325]. In addition, the commercial product ASCETM(the trademark of ExoCoBio), the ASC-exosome isolated by the ExoSCRTTMtechnology, was firstly registered as a cosmetic ingredient in the International Cosmetic Ingredient Dictionary (ICID). The TFF-isolated ASC-exosomes have multiple effects on the skin:(1)inducing regeneration of epidermal skin barrier by increasing synthesis of ceramides, dihydroceramide, sphingosine, and S1P [110];(2) reducing inflammation through downregulation of multiple cytokine levels [20,109,325];(3) reducing the level of TSLP a pruritus-causing cytokine [110];(4) inducing synthesis of collagen and elastin in HDFs [325]; and (5)inducing proliferation of HDFs and HDPs(Ha et al. unpublished observation). Recently, a potential effect of ASC-exosomes on subcutaneous fat has been also suggested. Mouse ASC-exosomes promoted WAT beiging through induction of M2 macrophage polarization in WAT of obese mice [95]. Under the same condition, ASC-exosomes induced the proliferation of ASCs themselves. Further studies are needed to decipher the effects of human ASC-exosomes on subcutaneous fat in normal physiological conditions.

The safety and efficacy of secretomes from different cells were analyzed for skin and wound care products, and it was found that secretome from ASCs is safer and more effective than that from BM-MSCs in many aspects:(1) lack of expression of major histocompatibility complex(MHC)class II on ASCs; (2) induction of higher levels of anti-inflammatory M2 macrophages by ASC-CM than by BM-MSC-CM; and (3)suppression of cancer growth by ASC-exosomes both in vivo and in vitro [326,327]. ASC-exosomes could be a preferable regenerative aesthetic ingredient since an important function of ASCs in the skin is signaling to surround cells to induce the differentiation of dermal fibroblasts and keratinocytes, and activate epidermal stem cells including hair follicles [326]. A pioneering cosmeceutical product, the ASCE+TM lyophilized human ASC-exosomes(ASCE+ is the trademark of ExoCoBio), showed various beneficial effects including anti-inflammation and reduction of downtime after ablative skin treatments such as laser therapies (unpublished observation). Taken together, ASC-exosomes could be a next-generation product for regenerative aesthetics, which affects multiple layers of the skin including the epidermis (keratinocytes), dermis (fibroblast, inflammatory cells, and hair follicle), and potentially the hypodermis (subcutaneous fat)(Figure 1).

10. Conclusions

With the recent burst of research, MSC-exosomes are now widely accepted as next-generation cell-free therapeutics for intractable diseases. Many challenges in the industrialization of exosomes are still out there such as the large-scale culture of MSCs, continuous supply of MSCs with comparable therapeutic effects, and accurate determination of quantity and quality of exosomes. However, technical advances in the MSC cell therapy field, with the expected first marketing approval by the US FDA in the near future [328], are also able to be integrated in the exosome industry soon. The use of immortalized MSCs, with similar functionalities and safety profiles compared to naive MSCs, might be also an alternative strategy for stable production of MSC-exosomes [329,330]. Successful commercialization of MSC-exosomes may provide a completely new therapeutic paradigm for human healthcare.

This article is extracted from Cells 2020, 9, 1157; doi:10.3390/cells9051157 www.mdpi.com/journal/cells