Mesenchymal Stem Cell-Derived Exosomes as a New Possible Therapeutic Strategy for Parkinson’s Disease

Abstract

Mesenchymal stem cell-derived exosomes (MSC-Exos) are nano-sized extracellular vesicles that have low immunogenicity and the ability to transfer the effective substances enriched in stem cells freely and reported experimental studies have demonstrated MSC-Exos have effects on some diseases. As the second most predominant neurodegenerative disease worldwide, Parkinson’s disease (PD) is characterized by severe progressive motor dysfunction caused by loss of dopaminergic neurons (DAn) and dopamine depletion. Since MSC-Exos serve as a beneficial promoter of neuroprotection and neurodifferentiation, in this article, we will summarize the application of MSC-Exos in PD treatment and the possible therapeutic mechanisms, especially the role of microRNAs included in MSC-Exos in the cellular and molecular basis of PD, and discuss the potential application prospects against PD.

1. Introduction

Parkinson’s disease (PD), described in 1817 by James Parkinson, is an age-related neurodegenerative disorder. As older people in the world are growing, neurodegenerative diseases will become the second leading cause of death. Especially PD, with a prevalence of 1–2% [1, 2] among aging people [3], affects over 10 million people. Patients with PD have clinical features including motor symptoms, such as rigidity, resting tremor, bradykinesia, akinesia, postural and gait instability, and nonmotor symptoms, like sleep disorders, depression, dementia and peripheral injuries. The primary pathology of this disease is the degeneration of dopaminergic neurons (DAn) in the substantia nigra (SN), which is also associated with the Lewy bodies (LB; protein aggregates of α-synuclein) accumulation, and the decrease of dopamine production in the brain [4, 5]. Although there are some diagnostic test scales and some functional measurements like Positron emission tomography (PET) scan and MRI for PD, recently, the diagnosis of PD mainly still depends on clinical judgment [6]. Therapies to increase DA levels mainly include deep brain stimulation (DBS) and pharmacological treatments based on DA substitutes such as Levodopa preparations, dopamine agonists, monoamine oxidase-B (MAO-B) and catechol-omethyltransferase (COMT) inhibitors [7, 8]. However, these treatments can only relieve symptoms in the early stages but have little effect on the progression of PD, that is to say, they cannot cure or prevent the process and even cause adverse reactions involving involuntary motor action that may affect the quality of their life.

Among all the stem cell-based treatments, mesenchymal stem cells (MSCs)-based therapy may be the most encouraging therapeutic strategy against PD. MSCs can be easily isolated from widespread sources throughout the body, including bone marrow, adipose tissue, umbilical cord Wharton’s Jelly, peripheral blood, brain and dental pulp [9, 10, 11, 12] and can also differentiate into at least osteoblasts, adipocytes and chondroblasts, with low immunogenicity and strong regeneration potential. Not only in experiments for rodent and nonhuman primate PD models but also in some clinical trials for mild to moderate PD patients [13], thanks to the secretion of neurotrophins, growth factors, and regulatory factors released [14, 15, 16], MSCs have been shown the great effects on the progression of PD. Recently, these effects have been ascribed to the products released into the extracellular milieu by MSCs, named extracellular vesicles (EVs). Exosomes, the subgroup with the smallest size of EVs [17], have been found in various body fluids or some tissues, affecting cell-to-cell communication [18] and effects of exosomes derived from MSCs on neurological disorders, especially neurodegenerative diseases, have been reported. Thus, in this chapter, we will review the therapeutic potential and the possible mechanisms of exosomes derived from MSCs in treating PD.

2. Exosomes

Exosomes were first discovered in maturing sheep reticulocytes as relatively uniform-sized particles in 1983 [19]. Later studies have shown that exosomes can be secreted by all cells in the body and most cell lines such as tumor cells, immune cells, neurons, stem cells, epithelial and endothelial cells [20], extracted from all body fluids involving blood, urine, cerebrospinal fluid, breast milk, amniotic and synovial fluid, ascites, pleural effusions and the cell culture supernatant [21, 22]. According to the criteria made by the International Society for Extracellular Vesicles (ISEV), exosomes are one type of EVs based on their size, origin and cargo. Therefore, they also meet the evaluation conditions of EVs [23, 24]: isolated from the conditioned cell culture or body fluids, have at least three different categories in the reparation-cytosolic proteins, transmembrane or lipid-bound extracellular proteins, verified by at least two different technologies in imaging and size measurements.

For the size, while the other two subtypes, microvesicles and apoptotic bodies, with a diameter of 50–1000 nm and 50–2000 nm, respectively, the exosomes have the smallest range of 30–150 nm [25]. For the origin, exosomes formed inside the multivesicular bodies (MVBs) and released by the fusion of MVBs to the plasma membrane, while the microvesicles formed directly by budding from the membrane and the apoptotic bodies are the products of apoptosis. For the cargo inside, exosomes carry a variety of molecules like oncoproteins, cytoskeleton protein, enzymes, hormones, lipids, steroids, sugars, signaling molecules, cytokines, growth factors and genetic material including small RNA, mRNA, microRNAs, long noncoding RNA, ribosomal RNA (rRNA), transfer RNA (tRNA) and DNA fragments that are contributing to their functions [26, 27]. The contents of exosomes differ depending on the source types and physiological or pathological state of the donor cells.

Structurally, exosomes present a lipid, to be precise, a phospholipid bilayer membrane, which can keep the membrane stable [28]. Containing the internal proteins and nucleic acids mentioned above inside the exosomes, there are some membrane proteins and lipids on the membrane. The membrane proteins, containing membrane transport and fusion-related proteins (e.g., annexins, RABs, flotillins, ARFs and GTPases), antigen presentation-related proteins (MHC-I, MHC-II), adhesion molecules (MFGE8 and integrins), ESCRT complex (Alix, Tsg101), tetraspanins (CD9, CD63, CD81 and CD82) and other transmembrane proteins (e.g., PGRL, LAMP1, LAMP2 and TfR), participate in cell transporting, adhesion and mediate T cell activation [29, 30, 31, 32]. The lipids includs phosphatidylserine, which has great flexibility and plays an important role in the budding and merging from the donor cell [33, 34], and ceramide, phosphatidic acid, diglycerides, ceramides are, which are not only contained in exosome biogenesis, but also in packaging and transporting the substances into the exosomes [35, 36], and other lipids like sphingomyelin, cholesterol, all of which are mainly involved in molecular signal transduction.

3. Treatment of MSCs in PD

Since PD is one of the progressive neurodegenerative disorders, there is not yet any curable therapeutic method for the progression [37]. More and more studies have shown the positive effects of MSCs on treating PD. Direct intracranial administration of MSCs-derived from bone marrow, adipose tissue, or umbilical cord with or without prior differentiation has provided improvement in motor function, striatal dopamine release and dopaminergic neuron survival in rodent models of PD [38]. Although there are still some concerns about the limited crossing of the blood–brain barrier, the effects of venous administration of MSCs on PD have been reported [38]. Our lab has shown that intranasal administration of MSCs-derived from the umblilical cord has similar effects on the MPTP-induced PD model mice through regulating intestinal microorganisms [39]. Moreover, positive effects of MSCs in human PD have also been reported. For example, in a study using autologous MSCs through intravenous and tandem (intranasal + intravenous) injections to 12 patients with PD, a decrease in the severity of motor and nonmotor symptoms (including depression, sleep quality, etc.) in the posttransplant period (1 and 3 months posttransplantation) have been found [40]. In a 12-month single-center open-label dose escalation phase 1 study, 20 subjects with mild or moderate PD accepted a single intravenous infusion of bone marrow-derived MSCs at doses of 1, 3, 6, or 10 × 10⁶ per kilogram body weight, and they were evaluated at 3, 12, 24 and 52 weeks postinfusion. Results have shown that the infusion is safe, well tolerated, and not immunogenic in human PD, and the highest dose seems to be the most effective at 52 weeks [41]. However, there are still challenges with cell sources, the number of homing cells, functional and safety testing, manufacturing and storage, cell survival rate and immunomodulatory effects after implantation in vivo. While MSCs have demonstrated preclinical success in PD during clinical trials, some issues like the clinical-trial design, including cell dose, administration interval time and route of administration, are required to be improved [42, 43].

4. Potential of MSC-derived exosomes in PD

As reviewed above, MSCs do provide benefits to animals or patients with PD, and some reports have provided the idea that the substances released by MSCs may exert protective effects. Indeed, some researchers have started to focus on the use of MSC conditional medium or the MSC-derived secretome, both involving the exosomes, as a cell-free treatment for PD [44]. As previously mentioned, while exosomes are one subtype of EVs, which include the other two major subtypes, microvesicles and apoptotic bodies, the MSC-derived secretome was defined as the composition of EVs and some soluble factors [45, 46], and the MSC-derived conditioned medium (CM) includes all the soluble molecules and vesicular components derived from MSCs [47, 48].

It has been demonstrated that human MSCs conditioned medium could increase dopaminergic neurons and decrease the motor and histological symptoms in the transgenic PD model [49] and the similar effects in 6-OHDA PD model rats [50]. In research to compare the effects of BMSCs and their conditioned medium, for the PD model rats made by rotenone, both BMSCs and the medium had effects on the behavioral performances and histological characteristics, and the medium even had better effects than BMSCs [51]. Besides these, conditioned medium from human exfoliated deciduous teeth MSCs (SHED) [52], menstrual blood MSCs, and adipose MSCs [50, 53]. All showed the protective effects in PD rat model induced by rotenone, PD cell model treated by 1-methyl-4-phenylpyridinium (MPP+) and 6-OHDA-lesioned PD rats, respectively, which were related to the decreased neuroinflammation marked by Iba-1 and CD4 levels, induced oxidative stress, increased brain-derived neurotrophic factor (BDNF) and neurotrophin-3 expression.

In a study of the neuroprotective effects comparison between human bone marrow-derived MSC (hBM-MSCs) and secretome derived from them, for the 6-OHDA PD model, the secretome even has better effects on protecting dopaminergic neurons such as neuronal differentiation and survival than hBM-MSCs, and the proteomic analysis showed that there were some factors related to the ubiquitin-proteasome and histone systems [50]. Abnormal aggregation of α-synuclein, the biomarker of PD, could be degraded by matrix metalloproteinases (MMPs) that are contained in MSC-secretome both in vivo and in vitro PD models [54]. In addition, many neuroregulatory products, such as brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), pigment epithelium-derived factor (PEDF), DJ-1, and cystatin-C (Cys-C) contained in MSCs-secretome may be the mediators against PD [50, 55, 56].

As mentioned above, exosomes are included in the conditioned medium or the secretome and could be separated from the medium. Since the MSC-derived exosomes were first isolated from human MSCs-derived from embryonic stem cells (ESC) in 2010 [57], and the effects of the medium or the secretome have been proven, a number of studies have begun to explore their potential for various diseases, especially for the central neural systems diseases such as stroke [58], traumatic brain injury [59], spinal cord injury [60], Alzheimer’s disease [61] and PD, which we highlight in this article. It has been found that in vitro, exosomes but not microvesicles, derived from human dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) exerted their anti-apoptosis ability in the 6-OHDA-induced PD cell model, and the exosomes could reduce 80% of the dopamine neuron apoptosis [62]. After that, the same group reported that the EVs derived from SHEDs could improve motor symptoms and increase the expression of dopamine neurons of 6-OHDA-induced PD model rats in vivo [63].

In the previous study, our group found that the exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) not only reduced the SH-SY5Y apoptosis induced by 6-OHDA in vitro but also relieved the motor disorder of PD rat model induced by 6-OHDA, increased the dopamine levels in striatum and the numbers of dopaminergic neurons in the substania nigra through the taken up of exosomes by SH-SY5Y cells in vitro and the neurons in vivo observed by the PKH labeling. The exosomes have played a role in increasing the autophagy ability of dopaminergic neurons [64]. Then, we further made an observation on the effect of exosome treatment in the different way in vivo and explored the anti-inflammatory effects. We found that the lateral ventricle transplantation of huc-MSCs-derived exosomes had the same effect as tail vein injection, and the exosomes could be absorbed by both dopaminergic neurons and microglia on the lesioned side of the brain in vivo and by the BV2 cells in vitro. The protection of dopaminergic neurons was perhaps mediated by inhibiting microglia activation. In vitro, exosomes reduced secretion of inflammation factors such as interleukin-1β and interleukin-18, prevented the pyroptosis-associated morphology of BV2 cells, and increased the cell viability of SH-SY5Y cells in the neuroinflammatory cell model system [65].

5. Mechanism of exosomes derived from MSCs in the treatment of PD

Compared with the MSC-based treatment and drug therapy, besides the advantages such as cell-free, rare side effects, no undesirable differentiation of transplanted cells, low immunogenicity, compatibility, long circulating half-lives, the most apparent advantage is the ability to cross the blood–brain barrier (BBB) [66]. For various neurological diseases, the most common barrier for the therapeutic stem cells or the drugs is the BBB, which is a selective semipermeable barrier maintaining the central nervous system homeostasis by preventing molecules larger than 400 Da [67, 68, 69]. Therefore, almost all the drug molecules, like recombinant proteins, peptides, antibodies and even genes, short interfering RNAs (siRNAs) cannot cross BBB [70]. Classical methods for the delivery of compounds to pass the BBB include invasive injection, changing the shape, size, surface charge, and ligands type, and using the transporters such as nanoparticles or liposomes or receptors that are highly expressed at the BBB surface, which are considered as the most popular way [71, 72].

Since the exosomes are nano-sized, protein-embedded and membrane-bound vesicles, they can cross the BBB well, not only from the bloodstream to the brain but also from the brain to the bloodstream, which has been proved that the exosomes derived from stem cells in the brain can be found in the peripheral blood [73]. The ways exosomes interact with the receiving cell described are as follows: 1) to adhere to the receiving cell surface and fuse with it, and release the inclusion into the cell, which probably causes the occurrence of some biological processes; 2) to associate with a cell surface protein G-coupled receptor and then induce signal cascade reactions; 3) through different transcytosis mediated by nonspecific/lipid raft or the receptor or by the macropinocytosis [74, 75, 76]; 4) in some pathological conditions like neurodegenerative diseases, BBB permeability may increase [77]. Among these routes, the key factors would be the surface markers that mediate the interaction of exosomes with receiving cells [74]. After passing BBB, exosomes may release the contents in the receiving cell cytoplasm and induce some related signal transduction, reach the receiving cell plasma membrane as the neoformed exosomes to the adjacent cell, or be degradated by lysosomes [78].

Another important mechanism is related to one of the most common contents of exosomes, miRNA. MiRNAs are a class of noncoding RNAs with a length between 21 and 25 nt. Through binding to the untranslated region (UTR) of mRNA and recruiting the RNA-induced silencing complex (RISC), miRNAs regulate the target genes expression by degradating mRNA or inhibiting the translation [79, 80]. Since miRNAs have been indicated as a potential tool for diagnostics and therapies of various diseases, massive miRNAs expression dysregulation has been shown in PD [81]. The most obvious biomarker of PD, a-synuclein, could be modulated by miR-433 [82], miR-16-1 [83], which binds to the fibroblast growth factor 20 (FGF 20) mRNA and the HSP70 mRNA, respectively, in addition with miR-153, miR-34b/c [84, 85, 86], miRNA-155 [87, 88], miR-7 [89], all of which could increase the a-synuclein level. Considering the related genes, LRRK2, PRKN and PARK7 genes, which play key roles in the pathogenic process of PD, such as formation of LB, mitochondria damage and oxidative stress damage, are correlated with miR-205, miR-34b/c [90], miR-494 and miR-4639-5P [91, 92] levels.

As a novel miRNAs carrier, exosomes derived from MSCs may deliver the miRNAs into the target cells of the brain crossing the BBB. It has been found that miR-133, which is an important factor in DAn development, presents in the exosomes derived from MSCs and could be transferred to the neuronal cells and promote neurite outgrowth [93]. In addition, miR-143, miR-21, miR-17, miR-18a, miR-19a/b, miR-20a and miR-90a that enriched in MSCs-derived exosomes, are able to modulate immune response, neurogenesis, axonal growth and neuronal death [94, 95]. On the basis of the effectiveness of exosomes derived from hucMSCs on PD models, our lab further examined the miRNAs included by high-throughput miRNA sequencing and identified 616 miRNAs (associated with 14,235 target genes) in exosomes. MiR-7, miR-125-5p, miR-122-5p, miR-126-3p and miR-199-3p were the most abundant miRNAs [65]. It has been shown that miR-7 can inhibit NLRP3 inflammasome activation and α-synuclein aggregation, attenuate the death of DAn in the MPTP-induced PD model mouse [89], mimics-miR-124 can promote behavioral improvements and neurogenesis in the 6-OHDA induced PD model mice [95], while the antago-miR-155 and antago-miR-126 [96] are useful to the PD therapy, since miR-155 could lead to neuroinflammation and miR-126 could result in the increased dopamine vulnerability. Despite limited research, and the cellular and molecular mechanisms in how they impact PD regulated by miRNAs are still not entirely clear, the beneficial effects of MSCs-derived exosomes have been demonstrated in the present findings, and new therapeutic approaches based on miRNAs may attract particular attention.

6. Conclusion

MSC-derived exosomes have been proposed as a new strategy for PD due to their characteristics. Their ability to cross BBB, secreting various factors into the receiving cells can improve symptoms and regulate vital biologic processes, such as inhibiting the neuroinflammation, reducing apoptosis, and regulating autophagy. Since miRNAs have gained an important status in PD researches recently for that they can not only affect the onset and the progression of PD, be served as the biomarkers of PD, but also be considered for the treatment of PD transported in the exosomes, therefore, as a natural carrier of miRNA and other effective substances, exosomes derived from MSCs will be a potential clinical therapy for PD patients. However, there are still some issues, like the selection of cell lines, development of isolation technique, improvement of exosomes’ targeting capability, mechanisms of how the miRNAs interact with target cells, exosomal cargo selection process, ways of taken up by cells should be overcome, and further investigations are needed to characterize all the bioactive molecules fully for better use in PD and other degenerative or central nervous system diseases.

Acknowledgments

This work was supported by the Natural Science Foundation of Hebei Province, Nos. H2022423319, H2021423063, 18967728D and the National Natural Science Foundation of China, Nos. 81873230, 82204658.

Conflict of interest

The authors declare no conflict of interest.