Introduction

The retina is a part of the central nervous system (CNS) which originates from diencephalon. The inner sensory retina and retinal pigment epithelium (RPE) are two layers of it [1, 2]. The association neurons (amacrine and horizontal cells), the conducting neurons (bipolar and ganglion cells), the photoreceptor neurons (cone and rod receptors), and the supporting Müller cells are four cell groups of inner sensory retina whereas the RPE is made up of cuboidal cells which are organized in one layer[1]. The light photons are transformed to electrochemical signals by the retina and projected to the brain via the optic nerve. The whole process gives the organism the ability of vision [3].

Many people suffer from the blindness caused by retinal degenerations around the world. The death of retinal neurons, same as the CNS, is irreversible and causes permanent damage to the retina. Degenerative inherited retinal diseases such as retinitis pigmentosa and age-related macular degeneration (AMD) are important causes of visual disability [1, 3,4,5,6]. The principal reason of retinal degeneration is the loss of photoreceptors, but no effective treatment has been discovered yet [7]. Retina’s structure and anatomical position have made it an ideal tissue for examining new treatment methods such as prosthetic therapy, gene therapy and cell therapy for its neurodegenerative diseases. It is an easily accessible structure of the central nervous system which is quite isolated from the other parts of the body. Researches on cell therapy have become prevalent in recent decades. One of the cell therapy advantages is restricting degeneration via delivering trophic and neuroprotective agents that might inhibit the progression of the visual disease. Another advantage of cell therapy over other methods is the possible differentiation of transplanted cells that might replace the dead cells and restore the function of the tissue [8]. Considering the specifications of stem cells such as their differentiation capacity, multipotency and self-renewal, stem cell therapy has become an important therapeutic approach [1, 3]. Different types of stem cells have been used for retinal differentiation and transplantation including induced pluripotent stem cells (iPSCs), isolated retinal stem cells (RSCs), human embryonic stem cells (hESCs) and mesenchymal stem cells (MSCs) [9, 10]. MSCs do not have the clinical limitations of other stem cells and owing to their immunomodulatory and autologous features, easy isolation and relative abundance, they are more promising choices than other types of stem cells for retinal regeneration [10].

Many studies on regenerative medicine have shown that most of MSCs will be lost in the cell therapy process, this suggests that the main part of tissue regeneration is possibly made by the paracrine factors of the MSCs [11,12,13,14]. One of the main components of MSCs paracrine factors which are highly regarded as tissue regenerators are EVs. The inner components of EVs generally consist of proteins and nucleic acids, especially miRNAs [15]. As new studies have suggested that EVs miRNA content seems to play a more important role in retinal regeneration than other components [12], in this review, we will discuss the potential role of MSCs-derived EVs’ (MSC-EVs) miRNAs as a treatment for retinal diseases.

Mesenchymal stem cells (MSCs)

MSCs are non-hematopoietic stem cells which are derived from various somatic tissues and have the self-renewal capacity. They can be found in different tissues including umbilical cord, embryonic tissues, fetal membranes, dental pulp, adipose tissue, liver, cartilage, skin, breast milk, skeletal muscle, peripheral blood, corneal limbal stroma of the eye and bone marrow [16, 17]. MSCs can migrate to the sites of injury to advance tissue regeneration and suppress the immune reactions by regulating the function of both innate and acquired immune systems [17]. Because of their anti-inflammatory [16], regenerative and immunosuppressive features, they are being used widely in the field of cellular therapy studies nowadays [11]. According to the International Society for Cellular Therapy (ISCT) the minimal requirements of the MSCs are the expression of cell surface markers CD73, CD90 and CD105, and negative expression of CD34, CD45, or CD11b, CD79-α, or CD19, CD14 and HLA-DR markers. The other main requirement is the plastic adherence in standard culture conditions. Moreover, MSCs must be able to differentiate into mesenchymal cells such as chondrocytes, osteoblasts, adipocytes and fibroblasts in vitro [1, 11, 18]. Moreover, researches have shown that MSCs can differentiate into a range of numerous cells such as cardiomyocytes, muscle fibers, renal tubular cells, hepatocytes, pancreatic islands and neurons [11]. So these kinds of cells could be used in many types of tissue regeneration including the retina [12, 16]. For example, Özmert et al. treated 32 patients of retinitis pigmentosa with subtenon space transplantation of Wharton’s jelly mesenchymal stem cells (WJ-MSCs) in a clinical trial. They concluded that the subtenon injection of WJ-MSCs could restrict the disease progression while being completely safe after twelve months of follow-up [19]. Despite the fact that therapeutic use of MSCs was promising, the possible unwanted differentiation of transplanted cells remains a safety issue [20]. Moreover, administration of MSCs for inflammatory bowel disease (IBD) and idiopathic pulmonary fibrosis (IPF) patients who were receiving immunosuppressive drugs shortly before MSC injection caused serious respiratory and gastrointestinal infections, suggesting that applying MSCs in combination or instantly after administering immunosuppressive drugs could be harmful [21].

Also, it has been shown that the positive effects of MSC therapy are substantially due to their trophic and immunosuppressive secreted factors and most of the transplanted cells will not differentiate and integrate into retinal tissue [20, 22]. MSCs secrete various trophic factors including FGF-2, IGF-1, BDNF, HGF, VEGF, IGF1, TGF-β1, bFGF and GDNF which attribute to neuronal survival and regeneration [23].

Recent studies have shown that these kinds of cells also release EVs which play an important part in cellular communications that promote tissue regeneration [11, 24].

Extracellular vesicles

EVs are secreted vesicles which are approximately found in all body fluids and the extracellular matrix [3]. They are secreted from all cells to transform vital information as lipids, proteins, mRNAs and small RNAs. EVs’ proteins are mostly a representation of their parent cells; however, the number of certain types of molecules such as cytokines, proteinases, chemokines, cell-specific antigens, cytoplasmic enzymes, signal transduction proteins, heat shock proteins and the ones which are related to cell adhesion and membrane trafficking are higher in the vesicles [25]. EVs include exosomes, microvesicles and apoptotic bodies. They are categorized by the proteins which are located on their surface, the range of their size in nanometer, their inner components and their biogenesis pathway [3].

Exosome formation is via the inward budding of the late endosome membranes which are called multivesicular bodies (MVBs). As the MVBs fuse with cell membrane, they would be released in the extracellular space [26]. The size of exosomes is considered as 30–150 nm [3]. Significant physiological and pathological functions have been attributed to exosomes including antigen presentation, inflammation regulation, immunological responses, angiogenesis processes, neuroprotection, regeneration processes, discarding inessential proteins and diffusing pathogens or oncogenes [27]. Exosomes can regulate the cellular status and their features would change in numerous diseases including cancer [28]. This suggests them as diagnostic and therapeutic tools [15]. For example, Galardi et al. showed that proteins that are characteristically associated with retinoblastoma vitreous seeding (RBVS) invasion and metastasis have been upregulated in RBVS exosomes [29]. Exosomes also have a drug delivery function [25, 30]. Schindler et al. demonstrated that exosomes which are loaded with doxorubicin, an anthracycline antibiotic that is prescribed in the treatment process of many kind of cancers, would be absorbed by cells quickly and their inner doxorubicin would be re-distributed from endosomes to the cytoplasm and nucleus of the recipient cells [31].

Another type of EVs that are formed through the outward budding of cell membrane is microvesicles which their sizes are 100–1000 nm [3]. Microvesicles are also called shedding vesicles, microparticles, shedding bodies, ectosomes and oncosomes. A number of functions are attributed to microvesicles such as intercellular signaling and changing the extracellular environment. They also facilitate cell invasion through cell-independent matrix proteolysis [32]. Microvesicles, same as exosomes, carry mRNA, short interfering RNA (siRNA) and ectopically expressed reporter proteins, but it has been shown that plasmid DNAs, which have reporter functions, could only be transferred to target cells by microvesicles [32, 33]. Researches demonstrated that microvesicles have also crucial roles in stem cell expansion and renewal [34], tumor progression [35, 36], coagulation [37] and inflammation [38].

Apoptotic bodies are formed via the membrane blebbing of apoptotic cells. Their usual size is more than 1000 nm [39]. As far as we know to date, no therapeutic effect of apoptotic bodies has been seen in eye diseases [3]. However, exosomes have noteworthy therapeutic effects against many diseases including neurologic ones [40,41,42]. MSC-derived exosomes’ (MSC-Exo) neuroprotective effect was also discovered in retinal cell injuries such as retinal cell degeneration, refractory macular holes, retinal detachment and optic nerve injury. MSC-Exos could reduce cell apoptosis and restrict the area of the injury in these diseases [27].

The main reason that why the EVs have become a research interest is their inner load which contain mRNAs, miRNAs, lipids and proteins. EVs’ cell signaling task is done by these components [3]. Many studies have shown that mRNAs and miRNAs play important roles in this task. While mRNAs can induce translation of new proteins in target cells, miRNAs can regulate the expression of genes [43, 44]. EVs’ multiple therapeutic effects are done by entering mRNAs, miRNAs and proteins into target cells [3]. MSC-EVs express adhesion molecules such as CD29, CD73 and CD44 which allow them to adhere to the damaged and inflamed sites of tissues [21]. Considering the source of EVs, their inner components vary. The two other factors which also influence the inner cargo and subsequently the therapeutic effects of exosomes are the source cell passages and its phase of differentiation [3]. It has been shown that the neuroprotective efficacy of MSC-Exos reduces with raising cell passages [45]. It has also been indicated that exosomes’ cargos vary at different stages of their source cell differentiation. For instance, exosomal miRNAs were differentially expressed in distinct stages of BMSCs osteogenic differentiation [46]. The composition of EVs’ cargos is not just a sample of the cytoplasm of their cell of origin. Studies demonstrated that some proteins, mRNAs, miRNAs and transfer RNAs are more abundant in EVs than the cytoplasm of their original cells [47,48,49].

Ocular therapies which are based on EVs have many advantages over cell-based therapies. Retina MSC-based therapy has incurred safety concerns. For example, a report showed that three patients with AMD who underwent intravitreal injection of adipose-derived MSCs, became blind because of the hemorrhage and retinal detachment [50]. One explanation for these pathologies is the adherence of transplanted MSCs to the inner limiting membrane of retina that would make an epiretinal membrane [51,52,53]. Another explanation would be the possible result of undesired differentiation of transplanted MSCs [20]. Other complications of cell therapy are the lack of information of the rate of cell death and cell division after administration [54]. Moreover, an important downside of cell therapy in retina is that the transplanted cells would not become integrated into the retina efficiently [13, 55]. The occasionally cell integration will be done through the digestion of inner limiting membrane and retinal glial activity modulation that might damage the retina themselves [22]. Since many studies have shown that keeping the therapeutic benefits of cell therapy, the EV therapy would avoid most of the above complications and also some EVs can cross the inner limiting membrane freely, it would be a better choice than cell therapy [12, 15].

miRNAs

miRNAs are a subdivision of evolutionary conserved long non-coding RNAs with approximately 22 nucleotides and a post-transcriptive repressive influence on gene expression [56,57,58]. First step in the biogenesis of miRNAs is the production of partially complementary primary RNA transcripts (pri-miRNA) mostly by RNA polymerase II and sometimes by RNA polymerase III. miRNAs will derive from these structures. Pri-miRNAs become hairpin structures by self-annealing. Then, the miRNA processing complex, which is made of Drosha ribonuclease and the DiGeorge Critical Region 8 (Dgcr8) proteins, will make a cut in the hairpin structure at the end of 11 base pairs (bp) from the foundation of the hairpin stem [59]. A seventy nucleotide sequence called precursor miRNA (pre-miRNA) will be released as a result [56]. The pre-miRNA is transferred to the cytoplasm by Exportin-5. Then, the Dicer endoribonuclease will attach to the pre-miRNA and cleave it to release a ~ 22 nucleotide long double strand RNA named miRNA* duplex. Since the pre-miRNA itself has a 5′ phosphate at one end and a 3′ two-nucleotides’ overhang at the other end, the dicer cleavage makes one phosphate at the 5′ end of each new strand, and a two-nucleotides’ overhang at the 3′ end of each new strand. Afterward, the miRNA* duplex will be incorporated into the Argonaute protein (Ago) which is a part of the RNA-induced silencing complex (RISC) and one strand will be removed. The remaining strand that is connected to RISC will attach partially to target mRNAs and repress their translation or induce degradation (Fig. 1). One miRNA can bind to myriads of target mRNAs [56, 60, 61].

Fig. 1
figure 1

MiRNA synthesis pathway. Biogenesis of miRNA begins with transcription of a miRNA gene (Canonical pathway) or the intron region of a protein-coding gene (Mirtron pathway) mainly by RNA polymerase II, and sometimes by RNA polymerase III in the nucleus. Canonical pathway: The sequences from miRNA genes transcription self-anneal and make hairpin-like structures called primary miRNAs (pri-miRNAs). Pri-miRNAs are being cut by DGCR8/Drosha complex and become pre-miRNAs. Mirtron pathway: Pre-miRNAs which are the result of intron regions of protein-coding genes are not dependent on Drosha complex. They are divided by spliceosome from the primary transcript of mRNAs. Then, they will self-anneal and become pre-miRNAs directly. All Pre-miRNAs from both pathways leave the nucleus and enter the cytoplasm by Exportin-5. There, the pre-miRNAs are cleaved by the Dicer/TRBP complex, yielding an about 22 nucleotides long miRNA: miRNA* duplex molecule. Then, this molecule will be loaded into the Argonaute (Ago) part of RNA-induced silencing complex (RISC). After discarding one of the strands, the other one will remain in the RISC and binds to 3’ untranslated regions of target mRNAs. miRNAs binding to target mRNAs lead to their translational repression, deadenylation and cleavage

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miRNA nomenclature is based on an annotation system which was introduced by Ambros et al. [62]. In brief, miRNA genes are numbered by the sequence of their discovery. Identical or nearly identical miRNAs from different species get the same number. A miRNA number is always accompanied by a prefix: mir or miR. The pre-miRNA is shown by “mir” prefix and the mature miRNA is preceded by “miR.” They are followed by a dash and then the number comes (e.g., mir-25 and miR-25). Identical mature miRNAs with one or two different nucleotides in their sequences are distinct by a lower case letter (e.g., miR-36a and miR-36b). A dash and a number suffix will be added to the names of pre-miRNAs that make identical mature miRNAs despite locating on different loci of the genome (e.g., mir-42a-1 and mir-42a-2 produce an identical mature miRNA, miR-42a). In the miRNA formation process, a miRNA duplex will be cleaved to two different mature miRNA strands: the one that comes from the 5′ arm is shown by 5p (e.g., miR-146b-5p) and the one from the 3′ arm by 3p (e.g., miR-146b-3p). Having said that, if the relative level of cell abundance of same miRNAs’ two strands is known, the arm with the lower expression will get an asterisk following the number (for instance miR-9 is more abundant than miR-9*). miRNA names can also indicate the species of origin by a three-letter prefix: for example, “hsa” stands for Homo sapiens in hsa-miR-132 and “rno” for Rattus norvegicus in rno-miR-125 [62, 63].

Defects in miRNAs synthesis can make serious problems in the development process and is related to pathologies including inherited genetic disorders, diabetes, cancers, heart failure and neurodegenerative diseases. miRNAs maintain the healthy condition of gene networks and modulate the ups and downs of gene expression in developed tissues [56]. As well as other tissues, miRNAs play important roles in retina and some of them are more enriched in retinal cells (Fig. 2) [64]. Many studies showed their role in the function and survival of different retinal cells such as photoreceptors or Müller glias [65, 66]. Here, we discuss retinal cell miRNAs (Table 1) similarities with MSCs-EVs’ miRNAs (Table 2) and their possible therapeutic effects on retinal diseases.

Fig. 2
figure 2

MiRNAs enriched in retinal cells which are also present in MSC-EVs

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Table 1 miRNAs of retina
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Table 2 miRNAs of MSC-EVs
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miRNAs of EVs

Literatures have shown different procedures of loading miRNAs into EVs. Some studies demonstrated that when MVBs bind to plasma membrane and EVs are made, RISC complex is associated with them [67, 68]. Other studies which concluded that RISC or Argonaute2 (Ago2) is not present in EVs indicated that packing miRNAs takes place by a type of ubiquitous proteins called heterogeneous nuclear ribonucleoproteins (hnRNP) [69]. Some motifs of miRNAs either alone or associated with proteins such as Ago2, Alix and MEX3C can be detected by and attached to hnRNP [70]. For instance, the loading of GGAG motif of miRNAs into EVs is controlled by the attached nuclear hnRNPA2B1 (ribonucleoprotein A2B1) [71].

Other proteins such as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP) detect miRNAs’ motifs which bind to the GGCU motif [72]. As a study showed that the mutation in Alix protein diminishes miRNAs levels in EVs, it can be concluded that this protein is also important in packing miRNAs into EVs [61, 73].

EVs inner cargos enter the target cells by two methods: endocytosis and fusion [70]. EVs are mainly taken up by endocytosis, according to previous studies [74,75,76,77]. Clathrin-dependent endocytosis and clathrin-independent pathways that are mediated by caveolin, phagocytosis, macropinocytosis and lipid raft-mediated uptake are different types of this mechanism [74]. Considering the cell types and components of EVs, a group of them may be absorbed by more than one mechanism[78]. The direct fusion of EVs’ membrane with cell membrane is the second mechanism of EVs entering into the target cells [79]. It was reported that spontaneous transfer of EVs took place between dendritic cells by fusion and release of the inner cargo into the cytoplasmic matrix [75].

Many literatures demonstrated that EVs miRNAs may affect target cells. Valadi et al. made the first report on evident transfer and function of mRNAs and miRNAs of EVs. They found new mouse proteins in the target cells after conveying the cargo of mouse EVs to human mast cells [44].

In addition, Song et al. indicated the transfer of functional miRNAs of MSC-EVs. After treating MSCs with IL-1β, the expression of miR-146a increased. Then, miR-146a was packaged into EVs selectively. As a result of co-culturing the MSC-EVs with macrophages, the level of miR-146a in macrophages had been raised which led to M2 polarization [80].

Many studies have shown the differences of miRNAs between EVs and their parental MSCs. A research showed that the expression of mir-15 and mir-21 was significantly higher in MSCs than their EVs [81]. Baglio et al. manifested that the miR-34a-5p, miR-34c-5p, miR-15a-5p and miR-136-3p are more represented in MSCs than their EVs and miR-4485, miR-150-5p, miR-6087 and miR-486-5p are enriched in MSC-EVs compared to MSCs [82].

There are differences among MSC-EVs’ miRNAs from various sources. Baglio et al. compared the miRNA contents of EVs derived from bone marrow and adipose MSCs. Most abundant miRNAs of bone marrow-derived MSC-EVs were miR-143-3p, miR-10b-5p, miR-486-5p, miR-22-3p and miR-21-5p, whereas, miR-486-5p, miR-10a-5p, miR-10b-5p, miR-191-5p and miR-222-3p were the most frequent miRNAs of adipose-derived MSC-EVs [82]. 171 miRNAs of hBMSC-EVs were disclosed in another research. While 148 miRNAs constitute 0.03 to 0.7% of the total reads, the 23 most abundant miRNAs made up 79.1% of them [83]. Luther et al. showed that the highest expressed EVs miRNA of mouse bone marrow-derived MSCs is miR-21a-5p which is responsible for MSCs cardioprotection [84]. The variety of miRNA profile among MSC-EVs may suggest that the expression of miRNAs is due to multiple factors and the effects of MSC-EVs may be the result of each miRNA synergistical activity with other elements [70]. MSC-EVs’ miRNAs are provided in Table 2.

MSCs’ miRNAs potential therapeutic effects

Over the last years, the effects of many miRNAs on retinal cells development and function have been revealed and the expression of miRNAs in normal and pathological conditions have been investigated. MSC-EVs contain some miRNAs which their roles in retinal cells’ function and development have been proved, so studying them as therapeutic agents for retinal neurodegenerative diseases has not been overlooked.

Therapeutic effects of a number of MSC-EVs’ miRNAs on retinal degenerative diseases have been assessed (Fig. 3). For example, Mead and Tomarev showed that by knocking down the Ago2 which plays a critical role in regulating the biological function of miRNA and the consequent reduction of miRNA abundance in exosomes, the BMSC-derived exosomes (BMSC-Exos) had lost their effects in advancing RGC neuroprotection, axon viability/regeneration and RGC functional maintenance [12]. They concluded that while knocking down Ago2 does not have an influence on exosomes’ protein content, the above results demonstrated the dependency of RGC treatment on miRNA in comparison to the protein. BMSC-derived exosomes contain miR-17-92 which can downregulate phosphatase and tensin homolog (PTEN) expression [85]. As PTEN expression is a major suppressor of RGC axonal growth and survival [86, 87], RGC neuroprotection was done probably by miR-17-92 [12]. miR-21 and miR-146a which were identified in exosomes of umbilical cord MSCs and BMSCs, respectively, may be another candidates of RGC protection and survival [12, 88]. In another study, Zhang et al. showed that MSC exosomes containing miR-126 ameliorate the inflammation and promote vascular repair in diabetic retinopathy (DR). They indicated that miR-126 reduces the inflammation in diabetic rats by inhibiting HMGB1 signaling pathway [89].

Fig. 3
figure 3

MSC-EVs’ miRNAs with studied effects on retinal cells. ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment of photoreceptors; OS, outer segment of photoreceptors; RPE, retinal pigment epithelium. General effects of miRNAs on retinal cells: 1differentiaition, 2function, 3survival & apoptosis reduction, 4development & growth, 5reprogramming, 6maturation, 7proliferation, 8protection & maintenance, 9dedifferentiation

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Having knowledge of the similarities between miRNAs that have an effect on retinal cells development and function and the miRNA content of MSC-EVs, we can design research and therapies more effectively and specifically for retinal degenerative diseases. Functions of miRNAs in retina can be divided into different categories. Many of them take part in differentiation process (e.g., miR-204, miR-124, miR-30b, miR-133b, …), a remarkable number in development (e.g., miR-181, miR-126, miR-155, miR-17, …), and a group of them in cell proliferation (e.g., miR-103, miR-124, miR-34a, miR-15b, …). Some of them will decrease cell apoptosis and contribute to cell survival and maintenance (e.g., miR-30, miR-124, miR-22, miR-29a, …) while a few participate in neurons’ connectivity and plasticity (miR-124, miR-133b, miR-132). Moreover, therapeutic effects of a number of miRNAs have been discovered in some of retinal diseases. miR-200b, miR-148a-3p and miR-15a act against DR while miR-361, miR-497 and miR-140 are retinoblastoma tumor suppressors. It had also been reported that miR-222 can prevent the progression of retinal degeneration and miR-124 has therapeutic effects on it. A few miRNAs have various proven functions in retina: for instance, miR-204 plays roles in differentiation, development and decreasing apoptosis whereas miR-124 has effects on differentiation, proliferation, survival of photoreceptors, plasticity and connectivity of neurons and a studied positive effect on retinal degeneration. The data are presented in detail in Table 3.

Table 3 MSC-EVs and retina common miRNAs; their expression, sequences and effects
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Conclusions

miRNAs have complicated functions in retinal health and disease which most of them are yet to be understood. Each miRNA can regulate the whole genetic program of a cell, so knowing their specific effects on different types of cells could be helpful for designing more beneficent studies and therapies. Owing to the fact that a miRNA has many mRNA targets, we should consider that we still don’t know many functions of miRNAs and the procedures of their actions. Although multifunctional miRNAs such as miR-204, miR-124 seem more promising, the timing of their application should be planned more precisely to avoid undesired effects. Besides having other therapeutic agents, MSC-EVs are a great source of miRNAs which make them a good choice for a multifactorial therapy.

Identifying miRNAs that are common between retinal cells and MSC-EVs, with due attention to the role of miRNAs as master regulators, could help us to preserve or restore the state of retinal cells in a more accurate way in retinal degenerative diseases.