The nerve tissue has a limited ability to spontaneously regenerate following neurodegenerative diseases or traumatic injuries, requiring innovative approaches for tissue regeneration. In this regard, researchers are always looking for an appropriate treatment of neurodegenerative diseases that characterized by degeneration and loss of neurons and glia. Up to now, some drugs and surgeries have been applied to manage the symptoms but there is no definitive treatment for neurodegenerative disorders(1). Due to the side effects of current therapeutic strategies, now-a-days stem cell therapy have been considered as a potential regenerative strategy for patients with neurologic disorders(2).
The stem cells technology has received considerable attention in recent years due to their potential for cellular and noncellular treatments(3). Stem cells have the ability to continuously renew themselves and can differentiate into many cell types(2). There are three types of stem cells that have therapeutic application: embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs)(4). There are some limitations of the use of ESCs and iPSCs such as ethical complications, genetic instability and tumorigenicity(2). ASCs are found in many of the tissues and organs such as adipose tissue, bone marrow and amniotic fluid. However, ASCs have insufficient renewal and differentiation properties compared to other stem cells(2) but these cells are immunocompatible and can produce large number of cells by in vitro cultures(4).
Recently among ASCs, mesenchymal stem cells (MSCs) are more considered due to easily accessible and great potential for regenerative medicine. MSCs are non-hematopoietic, fibroblast-like and plastic-adherent cells that form a heterogeneous population. This heterogeneity is determined with their differentiation capability, morphological differences and rate of proliferation(3). Human MSCs are identified through three criteria: 1) expression of CD73, CD90, and CD105, and the lack of expression of hematopoietic markers such as CD11b, CD34, CD45, CD79, CD19, and HLA-DR, 2) in vitro differentiation to osteoblasts, chondrocytes, and adipocytes and, 3) plastic adherence(3).
Adipose tissue is one of ideal sources for MSCs as large amount of tissue is easy to obtain with the least discomfort for patient and gives a high number of stem cells compared to bone marrow. Human adipose stem cells (hADSCs) have also nonmesodermal lineage differentiation potential such as neuronal lineage(4). Pervious data indicated that hADSCs were used in some degenerative diseases treatment such as osteogenesis imperfecta, dental structure degeneration, heart infarction and ischemic brain stroke(5).
Although, MSCs were widely applied for cell therapy, but there are some concerns about side effects of systemic application, such as favoring tumor growth and the possibility of MSCs differentiation into mesodermal lineages in inappropriate locations.
Based on literature, the therapeutic effects of MSCs work in two ways: the cell mediated effects (direct differentiation of MSCs) and paracrine mediated effects through soluble factors(6). On the other hand, recent experiments have indicated that the beneficial effects of MSCs are mediated mostly by paracrine secretion (MSCs secretome)(7, 8).
MSCs secretome includes miRNAs, growth factors and extracellular vesicles (EVs). Recently, the EVs were more attractive for non-cellular therapy. These EVs are categorized into microvesicles (100-1000 nm), Golgi vesicles or apoptotic bodies (50-5000 nm), and nanovesicles or exosomes (40-100 nm). This category is based on their size, morphology, origin, and mode of release(3, 7).
Stem cells, especially MSCs, use EVs to transfer growth, transcription, and anti-inflammatory factors to target cells. These factors have crucial rules for tissue regeneration and differentiation in local and distant tissues(3). The exosomes are currently being known as important mediators for cell-cell interaction and released by many types of cells such as T cells, B cells, neurons and stem cells(9) and exist in body fluids including urine, blood and cerebrospinal fluid(10).
Exosomes are surrounded by a phospholipid bilayer. The molecular signatures of exosomes are unique to each cell type and include proteins, lipids, coding and non-coding RNAs. RNA molecules within exosomes are the main messengers of the responses. For instance, coding mRNAs are functionally translated at recipient cells and microRNAs (miRNA) regulate gene expression following that mediate cell–cell communication. Therefore, they can induce the phenotypic modifications and modulate the microenvironment(10, 11).
Exosomes are derived from lipid-raft microdomains. The formation of exosomes follows the endocytic-exocytic pathway, that contains early and late endosomes, lysosomes, and multivesicular bodies (MVBs(. Exosomes originate from inward budding of late endosomes and subsequently form MVBs. Following the fusion of MVBs with the plasma membrane, exosomes are released outside the cell (3, 12).
MSC-exosomes are able to induce expression of cell cycle genes, growth factors like NGF(11), and neurotransmission, immune modulation, and differentiation(9). Furthermore, long half-life in circulation, low immunogenicity, and ability to cross the brain-blood barrier (BBB), make exosomes ideal tool for therapeutic applications(10).
Transferring miRNAs by MSC-exosomes accelerates neural plasticity and functional recovery and also increases neuronal differentiation of neural progenitors. As well as MSC-exosomes have anti-apoptotic effects. Taken together, these data clarify that exosomes have potential to protect against neuronal diseases(12).
Also, exosome-based, cell-free therapies reduce the complications of cell-based therapies and improve patients’ outcomes. The therapeutic advantages of exosomes can be attributed to promotion of endogenous repair signals in the injured tissue and immune regulation(12). Although the cell-free therapy by exosomes has a bright prospect, there are still some limitations and disadvantages to be investigated into. One of them is that they are static and more cannot be produced in vivo as may be possible when transplanting the cell itself. Further research is needed to explore the relationship between dosage, injection frequency, and the long-term therapeutic effect, and whether single or multiple administrations will have a negative effect, which is very important for the correct use of exosomes to treatment (13, 14).
PC12 cells are pheochromocytoma cell line of the rat adrenal medulla that are accepted as a classic in vitro model for neuronal differentiation, neurosecretion, and neuronal injury studies(15). After treatment with nerve growth factor (NGF), PC12 cells undergo neural-specific changes such as producing lengthy neurite extensions and increase of cholinergic receptors expression so differentiate into neurons. In results, these cells represent many neural markers(16).
In this study, we attempted to purify exosomes from supernatant of hADSCs and evaluate the effect of these exosomes on PC12 cells survival. Also, we determined effect of exosomes on neuronal differentiation of these cells by assessment of gene expression and representing neural markers.