Abstract
Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) are the primary effective source in stem cell-dependent regenerative medicine due to their preponderances over direct MSC implantation. An increasing number of studies have been carried out on MSC-sEV derived from different types of cells, and their function of accelerating tissue repair was proved. However, only a few researches were able to demonstrate the functional cargoes in MSC-sEV or their mechanisms in promoting tissue recovery. In this review, we present current achievements in discovering MSC-sEV-carried RNAs and proteins as promoters in tissue regeneration. Their therapeutic function includes modulating immune reactivity, promoting angiogenesis, and accelerating cell proliferation and migration through orchestrates of cell signaling pathways.
Introduction
Recently, mesenchymal stem cell-derived exosomes (MSC-Exo) and extracellular vesicles (MSC-EV) have become one of the most attractive research targets in regeneration medicine [1]. With the illustration of MSC-Exo and MSC-EV skill, researches began to focus on the functional cargoes, such as RNA and protein, that took part in promoting tissue repair (Tables 1 and 2). Considering the nonuniform standard for isolation, identification, and definition of exosomes and extracellular vesicles, in this review, we adopted the term “small extracellular vesicles (MSC-sEV)” to define the extracellular vesicles derived from MSC with diameters <200 nm to avoid ambiguity.
Mesenchymal Stem Cell-Derived Small Extracellular Vesicles RNAs Related to Tissue Repair
Ang-1, angiopoietin-1; DRP1, dynamin-related protein 1; IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; mRNA, messenger RNA; MSC, mesenchymal stem cells; sEV, small extracellular vesicles; TGF-β2, transforming growth factor-beta 2.
Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Proteins Related to Tissue Repair
BDNF, brain-derived neurotrophic factor; CCR2, C-C motif chemokine receptor-2; FGF-1, fibroblast growth factor-1; GDNF, glial cell-derived neurotrophic factor; GPX1, glutathione peroxidase 1; NGF, nerve growth factor; PEDF, pigment epithelium-derived factor; Runx1, runt-related transcription factor 1; SDF-1a, stromal-derived factor 1a; S1P, sphinganine-1-phosphate; SK1, sphingosine kinase 1; SP1, specificity protein 1; VEGF, vascular endothelial growth factor; YAP, yes-associated protein.
Recent reviews have characterized sEV cargoes and their roles in regeneration, but only focused on organs or diseases within a specific system. To attain a broader significance, our review summarized all MSC-sEV regeneration-related functional cargoes and demonstrated the way they worked. It provides a quick view of a specific-cell-derived MSC-sEV, including their carrying cargo, target tissue, and corresponding cell signaling pathway behind tissue repair (Tables 1 and 2). To a certain extent, it will facilitate researchers to explore the gap to fill and the brand-new factors in MSC-sEV [2] and develop engineered sEV to acquire more powerful pro-regeneration skills [3,4]. After searching in PubMed (National Center for Biotechnology Information, National Institutes of Health) on March 17, 2019, we enrolled 39 researches (Tables 1 and 2) from 248 results by manually excluding reviews and articles without English full text as well as phenotype studies with no mechanism exploration.
Mesenchymal Stem Cells
MSC, also named as mesenchymal stromal cells or multipotent stromal cells, are nonhematopoietic multipotent adult stem cells that exist in several types of tissues and cells, including bone marrow, adipose, umbilical cord, and synovium. MSC are of interest to clinical and basic researchers for their potential application in multiple fields, among which, regeneration medicine is the most prominent one [5]. MSC release regulatory substances and extracellular vesicles by paracrine, which contain regulatory noncoding RNAs, messenger RNAs (mRNAs), lipids, and proteins [6]. Furthermore, both clinical trials and studies in vitro and in vivo have proved the feasibility of autologous and allogeneic MSC transplantation as therapies for tissue injuries [7].
MSC-Derived Small Extracellular Vesicles
Compared with MSC transplantation therapy, extracellular vesicles derived from MSC possess considerable preponderance, such as certain stability, good flexibility, small size, and high cell affinity. With double lipid membranes, they are also able to protect bioactive cargoes from degeneration. Considering limited benefits and potential risks of MSC transplantation therapy [5], a growing number of studies suggest that researches on MSC therapy be concentrated on MSC-derived extracellular vesicles instead of MSC [8]. In accordance with other experts in this field, we believe that rather than “exosomes,” “sEV” tend to be more suitable to describe extracellular vesicles of <150 or 200 nm that originate from endocytic pathway because the purity of these particles cannot be guaranteed in studies nowadays owing to the limitation of isolation methods. Besides, some groups use the term “extracellular vesicles” while they are actually applying the isolation and identification methods of exosomes. Therefore, we prefer to use the term “MSC-sEV” in this review.
MSC-sEV have been reported to function in intercellular communication, microenvironment balance, and immunomodulation [9]. They also play a crucial role in the development of diseases, for instance, anti-inflammation [10] and tissue regeneration [1]. Their effect on promoting regeneration occurs in various systems, including the skin [11,12], heart [13], liver [14], kidney [15], esophagus [16], islet [17], vagina [18], taste bud [19], periodontal tissue [20], ocular tissue [21,22], skeletal muscle [23], tendon and ligament [24,25], bone and cartilage [26,27], and central and peripheral nerves [28,29]. Moreover, recent studies have testified certain proteins and RNAs, such as microRNAs (miRs or miRNAs), long noncoding RNAs (lncRNAs), and mRNAs, as key effective factors in MSC-sEV-dependent tissue repair [18,30,31].
MSC-Derived Small Extracellular Vesicles RNAs
microRNAs
miRNAs are single-strand nonprotein-coding small RNAs that negatively regulate target genes expression by binding to 3′-untranslated regions (3′-UTR) of corresponding mRNAs [32]. Carried by MSC-sEV, miRNAs play important parts in a series of tissue regeneration processes [33 –35] and hold these functions when being internalized by target cells (Table 1).
MSC-sEV miRNA in skin regeneration
Exosomes derived from human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSC-Exo) facilitate promotion of collagen maturity, reduction of scar widths, and acceleration of re-epithelialization and vessels maturation in wound sites [11]. Besides, human umbilical cord-derived mesenchymal stem cell-derived exosomes (hucMSC-Exo) were confirmed to enrich specific miRNAs, such as miR-21, miR-23a, miR-125b, and miR-145 to perform myofibroblast-suppressing and anti-scarring functions during wound healing by inhibiting the transforming growth factor-beta 2 (TGF-β)/SMAD2 pathway [36]. These specific miRNAs in hucMSC-Exo suppressed the activation and expression of TGF-β and SMAD2 and thus reduced α-smooth muscle actin (α-SMA) expression in myofibroblast both in vitro and in vivo [36]. Although miRNAs in MSC-sEV facilitate general skin regeneration progress, their effects on diabetic wound healing under metabolic disorder condition still need to be revealed.
For this purpose, Trinh et al. detected miR-29c and miR-150 in microvesicles (MVs) derived from human nondiabetic adipose tissue-derived mesenchymal stem cells (nAT-MSCs). miR-29c, known as an insulin sensitivity enhancer, along with miR-150, a CXCR4 expression elevator, expedited the wound healing through the mediation of human type 2 diabetic adipose tissue-derived MSC [37]. Moreover, based on a diabetic rat model, Tao et al. found that miRNA-126-overexpressing synovium mesenchymal stem cells (SMSC-126-Exos) were effective in treating cutaneous wounds by activating angiogenesis, promoting collagen maturity, and accelerating re-epithelialization [38]. These studies verified the capacity of miRNAs in MSC-sEV, such as miR-21, miR-23a, miR-29c, miR-125b, miR-126, miR-145, and miR-150 in accelerating skin regeneration in diabetics.
MSC-sEV miRNA in muscle tissue repair
It is already known that both MSC-sEV and the miRNAs they carry could promote skeletal and cardiac muscle's generation [13,23,39,40]. Nakamura et al. confirmed the existence of miR-494 in exosomes secreted from human bone marrow mesenchymal stem cells (BM-MSC-Exo) and confirmed its in vitro participation in C2C12 cell myogenesis and human umbilical vein endothelial cells (HUVEC) cell migration, which allowed BM-MSC-Exo-mediated muscle regeneration through angiogenesis and skeletal myogenesis [41].
In an ischemia-reperfusion heart injury model, miR-26a in human MSC-sEV was found to be responsible for cardioprotection. It is reported that human MSC-sEV increased Wnt-signaling-related gap junction protein Cx43 in myocardium by increasing Wnt1 and p-GSK3β/GSK3β and decreasing p-β-catenin/β-catenin via miR-26a and thus stabilized cardiac sodium channel and attenuated arrhythmias [42]. Recently, cardiac stem cells (CSCs) have emerged as an important cell type in cardiac repair, and CSCs could be protected by BM-MSC-Exo through antiapoptosis via the PTEN/PI3K/AKT pathway. Rat BM-MSC-derived exosomal miR-21 was found to be able to protect CSCs from apoptosis through downregulating PTEN, increasing p-AKT, and decreasing cleaved caspase-3 expression [43]. In addition, Zhu et al. testified that exosomal miR-125b-5p derived from hypoxia-conditioned BM-MSC (Hypo-BM-MSC-Exo) could restrain the expression of proapoptotic genes p53 and BAK1 in cardiomyocytes, promoting ischemic cardiac regeneration by ameliorating cardiomyocyte apoptosis [44].
MSC-sEV miRNA in bone and osteochondral regeneration
Exosomes in BM-MSC conditioned medium (BM-MSC-CM) was found to promote fracture healing in a femur fracture model of CD9−/− mice. Since the model produced low level of exosomes, it was deduced that MSC-sEV might play an essential role in fracture healing [26]. Furthermore, according to the findings of Qin et al., BM-MSC-derived EVs might regulate the expression of osteogenic genes and osteoblastic differentiation by inducing the expression of osteogenic genes including alkaline phosphatase, osteocalcin, osteopontin, and runt-related transcription factor 2 (Runx2) through EVs highly enriched miR-196a [45].
Cartilage repair, commonly involved in orthopedics injury, requires more healing time but usually ends up in worse recovery outcomes than bone regeneration. Synovial mesenchymal stem cell-secreted exosomes (SMSC-Exo) could enrich Wnt5a and Wnt5b and enhance chondrocyte proliferation and migration through activating yes-associated protein (YAP) via regulating the Wnt-YAP/TAZ pathway. But, it was accompanied by extracellular matrix (ECM) oversecretion as a side effect due to the decrease in SOX9 expression. To solve this, Tao et al. established an osteoarthritis (OA) rat model and found that SMSC-Exo overexpressing miR-140-5p were able to rescue SOX9 via RalA inhibition. Thus, without disturbing ECM secretion, SMSC-Exo could enhance the migration and proliferation of articular chondrocytes (ACs) [46].
MSC-sEV miRNA in the liver and kidney regeneration
It is known that the liver and kidney are vulnerable to toxicity of chemical reagents, abuse of traditional herbal medicine, and various systemic diseases. Regeneration skill provides an alternative therapeutic method for patients who failed to receive liver or kidney transplantation. Based on the fact that embryonic stem cells (ESC)-MSC-Exo shows protective effects against carbon tetrachloride (CCl4)-induced hepatic injury [14], Hyun et al. proved that miR-125b from chorionic plate-derived mesenchymal stem cells (CP-MSC) could reduce liver fibrosis and contribute to hepatic regeneration by downregulating the expression of profibrotic genes, vimentin, and matrix metalloproteinase 9 (MMP9) and suppressing the expression of Hedgehog signaling molecules, including shh, smo, gli2, and gli3 [47].
Besides, MSC-sEV could protect renal proximal tubular epithelial cells (PTECs) from ATP depletion injury by inhibiting PTECs apoptosis. During this process, miRNAs, such as miR-148b, miR-410, miR-495, and miR-548c were transferred to renal PTECs via MSC-sEV. They were revealed to downregulate ischemia–reperfusion injury-induced Src homology two domain containing transforming protein 1 (SHC1), SMAD4, caspase-3, and caspase-7 gene expression [48]. A regulatory miRNA–mRNA network was detected to be involved in the recovery of cisplatin-induced kidney injury where mice adipose tissue-derived mesenchymal stem cell-secreted microvesicles (AD-MSC-MVs), miR-880, miR-141, miR-377, and miR-21, took effect [49]. Furthermore, miR-21 and miR-377 inhibited cell death (reduction of caspase-3 and 7-AAD expression) and suppressed intracellular anion superoxide (O2 −) level, rescuing the cisplatin-induced overexpression of Ulk2 and Cul1 [49]. Besides, in acute renal ischemia reperfusion injury, human Wharton Jelly mesenchymal stem cell-derived sEV (hWJMSC-sEV) were found to reverse the decreased expression of miR-30b/c/d and inhibit apoptosis of renal tubular epithelial cells by alleviating mitochondrial fission through blocking the activation of dynamin-related protein 1 (DRP1), which facilitated the renal function recovery [50]. Moreover, through directly repressed p15 and p19 via 3′-UTR, hucMSC-sEV-derived miR-451a enhanced the proliferation and viability of hyperglycosis- and hyperuricemia-induced human renal PTECs. In this way, miR-451a reversed epithelial mesenchymal transformation and promoted blocked cell cycle in diabetic nephropathy with hyperuricemia. Therefore, hucMSC-sEV-derived miR-451a was crucial for kidney protection against morphologic and functional injury by reducing renal fibrosis and improving cell proliferation and viability [51].
MSC-sEV miRNA in the brain and spinal cord regeneration
Previous studies have shown that sEV were of high flexibility and small size, enabling sEV derived from stem cells to cross biological membranes such as blood–brain barrier. Meanwhile, their bi-lipid membrane protects small RNA cargo from degradation during the period of being transferred to target cells [52]. Furthermore, it was revealed that exosomal miR-133b delivered from multipotent MSC to neural cells might accelerate neurite outgrowth through downregulation of RhoA protein expression [53]. Recently, the same research group has reported that miR-133b delivered from miR-133b-overexpressing rat BM-MSC also preserved neuron and promoted axons repair via increasing the phosphorylation of ERK1/2, STAT3, and CREB, and decreasing the expression of RhoA protein in spinal cord injury [54].
MSC-sEV miRNA in vagina regeneration
Vaginoplasty is recommended when patients have vaginal hypoplasia, and vaginal dilation treatment is unsuccessful or unsuitable. However, it requires a long-term wearing of vaginal mold after the procedure, causing inconvenience and pains to patients [55]. Therefore, we devoted to find an alternative therapy to alleviate the discomfort and we observed the therapeutic properties of hucMSC transplantation in a Sprague–Dawley rat model after vaginoplasty [56]. Moreover, our recent work illustrated that hucMSC-sEV could stimulate vaginal epithelium growth through specifically enriched miRNAs, including miR-21, miR-100, miR-143, miR-146a, and miR-221, by inhibiting cell apoptosis and promoting cell cycle through modulation of relative signaling pathways, such as Wnt, PI3K, and p53 [18]. The expression level of mRNAs, which were co-targeted by enriched miRNA and signaling repressors such as ANDP, were detected to change in hucMSC-sEV-treated vaginal epithelium [18].
Long noncoding RNA
RNAs longer than 200 nucleotides (nt) and never translated into proteins are called lncRNAs. They are usually equipped with a 3′-UAU triple-helix mature structure [57].
Only one research was found in our research that illustrated the regeneration function of MSC-sEV-derived lncRNA (Table 1). Liu et al. observed regulation of chondrogenic genes in chondrocytes by human MSC-Exo, including overturning interleukin (IL)-1β-induced upregulation of MMP13 and Runx2, and downregulating aggrecan and type II collagen alpha 1 (Col2a1). Moreover, they viewed a remarkable enrichment of lncRNA KLF3-AS1 in human MSC-Exo and verified lncRNA KLF3-AS1's significance for ameliorating IL-1β-induced cartilage injury by suppressing chondrocytes apoptosis during cartilage repair [31].
Messenger RNA
Apart from noncoding RNAs, mRNAs were shuttled to injured cells and exerted protection effects through modulating the expression of transcription factors, angiogenesis, and adipogenesis-related genes [58].
Soluble insulin-like growth factor-1 (IGF-1) released by human BM-MSC at the site of injury was important for acute kidney recovery [59]. Similar to other growth factors, IGF-1 relied on corresponding receptor IGF-1R in the target cells to function. Tomasoni et al. found that BM-MSC might stimulate proliferation of damaged renal cells through IGF-1 by BM-MSC-sEV. And this effect could be enhanced by transferring IGF-1R mRNA to cisplatin-damaged proximal tubular cells via elevating their sensitivity to IGF-1 [60]. Human BM-MSC-sEV also attenuated acute lung injury (ALI) by delivered mRNA of angiopoietin-1 (Ang-1), a vascular stabilizing factor. Ang-1 mRNA in sEV was proved to reduce neutrophil counts, decrease the levels of macrophage inflammatory protein (MIP-2) and tumor necrosis factor-α (TNF-α), increase IL-10 level, and maintain the integrity of microvascular endothelial cells (ECs) in a lipopolysaccharide (LPS)-induced ALI model [61].
MSC-sEV Proteins
Wnt family
For sEV-carried Wnt protein and activators of Wnt/β-catenin signaling pathway being essential for tissue repair, Wnt/β-catenin signaling pathway has become a promising and attractive target for disease therapy, especially in regeneration medicine [62,63].
In human primary osteoblastic cells, inhibiting Wnt signaling leads to a significant decrease in osteogenic gene expression, and it can be rescued by Wnt3a from adipose tissue-derived MSC-derived exosomes (ASC-Exo) [64]. Additionally, Wnt5a and Wnt5b, carried by synovial MSC-Exo, are able to activate YAP via Wnt signaling pathway, maintaining proliferation and migration of chondrocytes in OA [46].
Besides, MSC-sEV-carried Wnt proteins promote repair in skin regeneration. Zhang et al. reported that hucMSC-exosomal Wnt4 encouraged β-catenin nuclear translocation and thus enhanced proliferation and migration of skin cells during cutaneous regeneration [30]. Data showed that MSC-sEV delivered cytokines, such as IL-6, IL-8, vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), granulocyte-colony stimulating factors (G-CSF), and platelet-derived growth factor BB (PDGF-BB), activating AKT signaling in promoting wound healing. And, they confirmed that both in vitro and in vivo, independent activation of AKT and Wnt/β-catenin signaling within this MSC-sEV mediated wound regeneration process [30]. Furthermore, this process was orchestrated by hucMSC-sEV-derived 14-3-3ζ, which adjusted the binding of YAP and p-LATS [65]. HucMSC-sEV-derived 14-3-3ζ was found to induce phosphorylation of YAP by elevating p-LATS-YAP interaction only under the condition of high cell density in vitro. Moreover, hucMSC-sEV restricted unfavorable cell proliferation and excessive collagen deposition in vivo via YAP Ser127 phosphorylation induced by transported 14-3-3ζ protein [65]. HucMSC-sEV-delivered 14-3-3ζ also induced autophagy and prevented cisplatin-induced nephrotoxicity by inhibiting cell apoptosis and promoting proliferation [66]. The mechanism behind autophagy activation was interaction between 14-3-3ζ and autophagy-related protein 16L (ATG16L), which may accelerate the formation of autolysosomes and autophagosomes [67]. HucMSC was also found to accelerate skin repair through exosomal Wnt protein, Wnt11, by elevating stemness of itself through sEV autocrine signaling. Exosomal Wnt11 was verified to induce the expression of stemness transcription factors, including Nanog, Sox2, Sal4, and Oct4, via activating Wnt/β-catenin signaling by promoting expression and nuclear translocation of β-catenin in hucMSC [68].
These findings strongly suggest that Wnt family proteins in MSC-sEV are highly associated with repair and regeneration of tissue, including the skin, kidney, cartilage, and bone (Table 2).
PI3K/AKT signaling-related proteins
Cardioprotection mediated by stem cells involves PI3K/AKT signaling pathway. Recent research showed that Akt adenovirus-transfected hucMSC (Akt-hucMSC)-derived exosomes were more effective in myocardial infarction protection with superior efficacy of promoting cardiac angiogenesis [69]. Akt-hucMSC-secreted exosomes expressed higher PDGF-D, which was critical in Akt-hucMSC-derived exosome-mediated blood vessel formation by promoting ECs migration and tube-like structure formation [69]. A near-term work confirmed stromal-derived factor 1 (SDF-1) produced by MSC as a necessary factor in regenerative microenvironment, supporting tissue renewal through the PI3K/AKT signaling pathway [70]. Furthermore, SDF-1 carried by human umbilical cord blood-derived MSC has been verified to benefit ischemic myocardial infarction. By activating the PI3K/AKT signaling pathway via raising the expression of p-PI3K, AKT, p-AKT, mTOR and p-mTOR, it inhibits autophagy and apoptosis of myocardial cells and improves the ability of microvascular formation of ECs [71].
Apart from this, Zhang et al. discovered the expression of CD73 in exosomes derived from human embryonic SC-derived MSC [72]. In cartilage and periodontal repair, exosomal CD73 was found to mediate the activation of AKT and ERK signaling pathways to improve proliferation and migration of osteochondral cell and periodontal ligament (PDL) cell, respectively [25,72]. MSC-Exo activated CD73-mediated adenosine receptor signaling via pro-survival kinases AKT and ERK and thus induced gene expression, including fibroblast growth factor-2 (FGF-2), Bcl-2, PCNA, TGF-β, Survivin, and COL1A1, to promote PDL cell proliferation and migration, which were relevant to periodontal repair [25]. Similarly, during exosome-mediated repair of osteochondral defects, human embryonic SC-derived MSC-Exo were confirmed to induce the phosphorylation of AKT and ERK through CD73-mediated nucleotidase activity [72].
Overall, SDF1 and CD73, along with their related PI3K/AKT signaling activation, might play key roles in tissue regeneration, especially in cardiac, vascular, cartilage, and periodontal growth (Table 2).
NF-κB signaling-related proteins
In hepatic oxidant injury, hucMSC-sEV-transported glutathione peroxidase 1 (GPX1) prevents hepatocytes from oxidative stress and apoptosis by reducing cellular reactive oxygen species and malondialdehyde level in vitro. Besides, GPX1 transferred by hucMSC-sEV also mediated NF-κB P65 phosphorylation, hepatocytes apoptosis and Bcl2 expression, and thus rescued CCl4-induced liver failure in vivo [73].
C-C motif chemokine receptor-2 (CCR2) that was derived from mouse BM-MSC-sEV reduced the concentration and suppressed the function of its ligand, CCL2, leading to recruitment and activation of macrophage, which regulated inflammation and promoted renal ischemia/reperfusion injury repair in vivo. CCR2 of BM-MSC-sEV was also confirmed to inhibit the expression of inflammatory factors such as TNFA, IL6, and 1B. With decrease in phosphorylated NF-κB p65 after binding with CCL2, CCR2 suppressed macrophage migration and activation [74].
Growth factors and receptors
Shah et al. revealed that the subset of EVs originated from MSC in acute respiratory distress syndrome (ARDS) patients comprised transforming growth factor-beta receptor I (TβRI)/Alk5 and Runx1 transcription factor. Besides, MSC-Exo derived from human bone marrow expressed Runx1 transcription factor isoform p66, which stimulated proliferation of LPS-treated pulmonary ECs. Consequently, it was suggested that human bone marrow-derived MSC-Exo had potential therapeutic effect on acute inflammatory lung injury such as ARDS [75].
A series of neural growth factors and neurotrophic factors were detected in adipose-derived MSC-Exo, including FGF-1, glial cell-derived neurotrophic factor (GDNF), IGF-1, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) transcripts. All these signaling proteins were involved in supporting axonal growth and neural survival [76].
It is known that MSC release both soluble VEGF and sEV-associated VEGF. Ahn et al. revealed that approximately half of VEGF was released by human umbilical cord blood-derived MSC in sEV-associated form, and these VEGF played more critical role than soluble form VEGF in protecting lung epithelial cells from oxidative injury. They reported that MSC-sEV-associated VEGF ameliorated impaired alveolarization and angiogenesis by attenuating cell apoptosis and suppressed inflammatory cytokines such as IL-1α/1β/6, TNF-α, and ED-1-positive alveolar macrophages [77].
Others: α2-M, pigment epithelium-derived factor, and specificity protein 1
Not only proteins of Wnt, PI3K/AKT, and NF-κB signaling pathways and growth factors as well as their receptors, but other MSC-sEV-carried proteins exert pro-regeneration effects (Table 2).
Exosomes derived from human Wharton's jelly that carried abundant alpha-2-macroglobulin (α2-M) were found to enhance migration, proliferation, and viability of skin cells to accelerate wound healing. Moreover, exogenous α2-M had the same power as exosomal ones in promoting fibroblasts migration, proliferation, and cell viability in vitro [78].
Huang et al. reported that the protective effect of adipose-derived MSC was significantly improved after being modified by pigment epithelium-derived factor (PEDF) in cerebral ischemia–reperfusion injury model. PEDF in modified MSC-sEV inhibited apoptosis by suppressing caspase-3/9-dependent apoptotic pathway and modulated autophagy by regulating expression of autophagy-associated protein, P62 and LC3 [79].
Specificity protein 1 (SP1) is the transcript factor of sphingosine kinase 1 (SK1) and is highly enriched in hiPSC-MSC-sEV. The anti-necroptosis effect of hiPSC-MSC-sEV was revealed to be relied on the activation of SK1-sphinganine-1-phosphate (S1P) signaling pathway via transferred SP1 in renal ischemia–reperfusion injury. sEV-delivered SP1 increased the expression of SK1 and S1P formation through interacting with the binding sequence of SK1 promoter and thus inhibited renal proximal tubular cell necroptosis [80].
Summary
In summary, MSC-sEV-carried miRNAs, lncRNA, mRNAs, and proteins act as promoters in tissue regeneration. The therapeutic function includes modulating immune reactivity, promoting angiogenesis, and accelerating cell proliferation and migration, which is mainly through activation and downregulation of cell signaling pathways such as TGF-β2/SMAD2, Hedgehog, Wnt/β-catenin, PI3K/AKT, and ERK. Among these promoters, a number of miRNAs have been studied, including miR-21, miR-23a, miR-26a, miR-29c, miR-30b/c/d, miR-100, miR-125b, miR-126, miR-133b, miR-140, miR-141, miR-143, miR-145, miR-146a, miR-148b, miR-150, miR-196a, miR-221, miR-377, miR-410, miR-494, miR-495, miR-451a, miR-548c, and miR-880. However, only a few mRNAs, such as IGF-1R and Ang-1 mRNA, and one lncRNA, KLF3-AS1, have been revealed. As for MSC-sEV protein cargoes, positive roles of tissue repair were verified in α2-M, PEDF, SP1, growth factors and receptors, and related proteins of Wnt/β-catenin, PI3K/AKT, and NF-κB signaling pathways.
Although molecules carried by MSC-sEV have been studied in different fields, the mechanism exploration remains superficial. Owing to the limited harvest of MSC-sEV, most of the studies only focus on the targeted cells' phenotypes such as vitality, proliferation, apoptosis, and migration rather than dig detailed mechanisms at a molecular level. Considering clinical application, it would be better to perfect isolation approach and standard of MSC-sEV and develop practicable and economical methods to guarantee the harvest.
As reviewed above, miRNAs, lncRNA, mRNA, and proteins carried by MSC-sEV have been found to be promoters in tissue repair. But, there is a scarcity of studies on other MSC-sEV cargoes, such as circular RNAs, DNAs, and lipids, which are probable to function in tissue and organ regeneration. After all, a comprehensive view on cargo functions in tissue repairs worths further exploration in the future.
Footnotes
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This study was supported by the Chinese National Nature Sciences Foundation (grant no. 91440107, 81471416, and 81771524) and the Strategic Priority Research Program of the Chinese Academy of Sciences (no. XDB19040102).