Diseases and Disorders

Cell-Free Approach to Treat Peripheral Nerve Injury

Shruti Mandal


Introduction

Peripheral nerve injury (PNI) is caused by nerve damage in the spinal cord and outside the brain. The resulting outcome is poor functionality such as muscle atrophy. The repair of PNI is quite complex due to difficulties in axonal regeneration since it involves a change in Schwann cell phenotypes, activation of macrophages, and the reconstruction of the vascular network. Approaches with mesenchymal stem cells (MSCs) prove to be effective but consist of several risks and ethical issues. In this article, we discuss the cell-free approach with MSC- derived exosomes as an alternative to PNI regeneration and summarise how they benefit the neurite outgrowth with their cellular interactions.

 

Peripheral Nerve Injury (PNI)

 Peripheral nerves send messages from our brain and spinal cord to the rest of our body, helping us perform actions, such as sensing if our hands are cold, moving our muscles so that we can pick up things, etc. The damage in the spinal cord results in poor functionality such as muscle atrophy (difficult or impossible to move muscles). Peripheral nerves are made of fibers called axons with insulation by tissues [1][2]. The peripheral system is composed of neuronal cells, glial cells, and stromal cells. PNI results in nerve gaps and creates difficulties for axonal regeneration.  

Approaches to addressing the difficulties include treatment with Schwann cells or stem cells which provide an appropriate microenvironment for neuronal regeneration [3]. Several factors are involved in the regeneration process after nerve injuries, such as the activity of macrophages and Schwann cells, vascular regeneration, and inflammatory reaction [4][5][6].

 

Mesenchymal Stem Cells in PNI Therapeutics

 Mesenchymal stem cells (MSCs), a subset of multipotent adult stem cells, have been considered as potential regenerative medicine due to their therapeutic ability of immunomodulation, paracrine secretion, differentiation to multiple cell lineages, and enhancement of cell viability which leads to regeneration of damaged tissues [7]. They are also known to be immune-evasive, meaning, the chances of immunological rejections after MSC transplantation is low.

Transplantation of MSCs in the case of PNI showed positive results as the MSCs got differentiated into a type of glial cell in the peripheral nervous system [8][9]. Researchers found that the transplanted MSCs promote axonal outgrowth, recover denervated muscle atrophy, and formation of myelin, indicating the indirect regeneration of endogenous Schwann cells (SCs) through their cellular paracrine mechanism [10].

 

Disadvantages to MSC-Based Therapy

While developing therapies for clinical usages, all desirable effects must be taken into consideration. MSCs do have encouraging effects, but there are still some doubts with regards to their safety. 

To use MSCs clinically, they must be cultivated in vitro (in a controlled artificial environment such as a test tube or Petri dish) because of the limited supply of MSCs in tissues. But the cultivated MSCs can be biologically different from freshly extracted MSCs which may cause adverse reactions. Studies show that even after transplantation, the number of stem cells engrafted within the target is very low. Also,  the differentiation of the engrafted MSCs into desirable cells in vivo is controversial [11]. Further, MSCs therapeutic action is influenced by the addition or removal of electrical, chemical, or mechanical stimuli. These stimuli can modify the production of different paracrine factors released by the MSCs, in turn changing their effects [12][13][14][15]. The MSC-secretome is influenced by micro-environment characteristics, thus conditioning the therapeutic response.  Nevertheless, when MSCs are present in the proinflammatory or hypoxic microenvironment of the damaged tissues, they secrete more of their therapeutic paracrine factors, but do not guarantee a curative impact in all cases and may also result in deterioration such as tumor development [16]. The heterogeneity of MSCs, instability of their cellular phenotype, ethical issues (new embryonic stem cell lines are derived from a frozen embryo) related to them, high cost, their transport, and storage are some other inherent disadvantages related to their use in therapies [17][18][19][20][21]. Therefore, a cell-free approach for PNI with similar but better efficiency than MSCs is needed to be developed.

The reason behind the therapeutic effects of MSCs is the biological factors released by them, collectively named secretome and extracellular vesicles (EV). The secretome composition is based on parent stem cells and their culture condition [22]. In selected animal disease models, the MSC supernatant EVs showed similar therapeutic benefits to MSCs, confirming their role in cell-free therapies [21].

 

Exosomes: A Step Towards Cell-Free Therapeutics

EVs are membrane-bound vesicles secreted by all types of cells and can exhibit diverse intercellular communication bio-functions [23]. Some potential advantages of EVs in therapeutics are (i) the inability to self-replicate, implying no risk of uncontrolled growth, (ii)their non-toxicity, (iii) their smaller sizes, (iv) their limited potential to trigger immune cells, and  (v) their easier storage and transport [24][25][26]. EV is an umbrella term for various types of vesicles: larger apoptotic bodies (50-4000nm), Microvesicles (MVs) (100-1000nm), and exosomes (20-120nm) [27] (See figure 1.).

The bilayer membrane of exosomes provides a controlled internal microenvironment that ensures that their cargo is not degraded and it can move long distances [28]. In addition, they are less immunogenic than MSCs which allows them to overcome immune rejections in a better way.

The advantages and therapeutic similarities of exosomes to MSCs are the reasons for their attraction in treating nerve injury [29][30]. Exosomes are formed by the inward budding of multivesicular bodies (MVBs). They fuse with lysosome or plasma membrane to release the exosomes into extracellular space to mediate cellular communication [31]. Their morphology is homogeneous and is cup-shaped [32]. They are categorized based on the presence of tetraspanins CD9, CD63, CD81, protein Alix, and TSG101 which are involved in the biogenesis of multivesicular bodies. Exosomes also contain other proteins like annexins, heat-shock proteins (Hsp60, Hsp70, Hsp90), clathrin, caveolins, integrins, TfRs, protein; lipids, cholesterol, ceramide, sphingomyelin and low concentration of phosphatidylserine; nucleic acids mRNA, lncRNAs and miRNAs [33].

The miRNAs in exosomes can bind to targeted mRNAs, resulting in mRNA decay or translation dampening and regulates expression [34].

 

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PNI Regeneration With MSC-Derived Exosomes

The Schwann cells (SCs, see figure 2) form the myelin sheath, contributing to axonal development and regeneration. They have a key role in controlling homeostasis in the peripheral nervous system (PNS) [35]. Upon any injury, the SCs are reprogrammed to a repair phenotype which provides biochemical signals and promotes survival of the injured neuron [36].

Organization of myelinating Schwann cells. Schematic organization of myelinating Schwann cells (blue) surrounding an axon (gray); the left cell is shown in longitudinal cross section and the right cell is shown unwrapped. Myelinating Schwann cells are surrounded by a basal lamina (illustrated only on the left), which is in direct contact with the abaxonal membrane. The abaxonal compartment contains the Schwann cell nucleus (SN); it is divided into Cajal bands and periodic appositions that form between the abaxonal membrane and outer turn of compact myelin. The Schwann cell adaxonal membrane is separated from the axonal membrane by the periaxonal space (shown in yellow). Compact myelin is interrupted by Schmidt-Lanterman incisures (SLI), which retain cytoplasm and are enriched in the gap and other junctions; a similar autotypic junctional complex of adherens, tight and gap junctions, forms between the apposed membranes of the paranodal loops. Also shown are the paranodal loops and junctions (red) and the Schwann cell microvilli contacting the axon at the node. The axon diameter is reduced in the region of the node and paranodes. (This figure is adapted, with permission, from an original figure in Salzer 2003; modified in Nave 2010.) 

 

 

Figure 2. Schwann cells form the myelin sheath in the peripheral nervous system [61].

Studies claim that mechanical stimuli can influence the biology of SCs and alter the cargo in MS-SCs-EVs and hence modulate the intercellular communication between neurons and SCs [37][38]. Exosomes decrease the GTPase and RhoA activities and thus help in growth cone collapsing and axon retraction [39]. Studies prove that exosomes derived from glial cells, menstrual MSCs and bone marrow mesenchymal stem cells, adipose-derived stem cells, gingiva-derived mesenchymal stem cells are quite promising in peripheral regeneration [40][41][42].

       

Transport of miRNAs by Exosomes

 MSC-derived exosomes mediate intercellular transport of miRNA or exosomal shuttle RNAs, inducing axonal outgrowth. In the peripheral nervous system, miRNAs can be shuttled by exosomes from Schwann cells to neurons to improve peripheral nerve repair [43][44]. Schwann cell miRNA expression levels are drastically changed following injury, suggesting that local genetics may play an important role in the nerve regeneration process [45][46].

Research says that MSC-derived exosomes enriched with miR-133b  stimulate the release of exosomes from astrocytes, therefore enhancing neural plasticity and neurite outgrowth [44]. Briefly, exosomes target neural cells by transmitting their miRNAs to contribute. Bucan et al. noted that MSC exosomes are enriched with neurotrophic factors related to nerve repair, such as fibroblast growth factor-1, Insulin-like growth factors-1, and nerve growth factor [47].

 

MSC-Derived Exosomes Modulate Neuroinflammation

Inflammation is a factor in peripheral nerve regeneration,  and many studies have shown that inflammatory reactions and cytokines are key facilitators of the process of regeneration [40][48].

Going forward into the details, axonal regeneration after PNI is not completely mediated by SCs but also by macrophages. A majority of immune cells, such as phagocytic neutrophils and macrophages, migrate to the damaged site after peripheral nerve injury [49].  The release of chemokines and proinflammatory cytokines by dedifferentiated Schwann cells leads to a neuroinflammatory response. Neuroinflammation plays a crucial role in circulating the macrophages [50]. It should be noted, however, that excessive inflammation can hinder nerve regeneration [51]. MSC exosomes are major immunomodulatory mediators as they have fewer membrane-bound proteins compared to parental cells [52].

Exosomes harvested from stem cells can mediate the transmission of anti-inflammatory RNAs to injury sites and potentially orchestrate the resolution of the inflammatory responses to better facilitate healing processes [53]. MSC-derived exosomes can also influence brain remodeling by regulating the immune reaction [54].  Zhang noted, exosomes induced regulatory T-cells (a subset of CD4+ T cells) and, thus, induced immune tolerance [55].

Recent studies have shown that MSC-derived exosomes can deliver miRNA to regulate inflammation in cardiac, wound, and bone repair [56]. Another study indicated that exosomes can regulate the plasticity of macrophages to facilitate polarization into anti-inflammatory phenotypes, thereby reducing the production of proinflammatory cytokines [48].

 

MSC-Derived Exosomes in Vascular Regeneration

By maintaining vascular integrity, a regenerative microenvironment can be attained, beneficial in nerve repairs.  Interestingly, MSC exosomes act as mediators of communication within the vascular cells for the improvement of blood vessels after nerve injuries by promoting endogenous angiogenesis and neurogenesis in rats [44]. Xu et al. showed that neurons transfer miR-132 to endothelial cells via exosomes to maintain brain vascular integrity [57]. 

MSC exosomes can induce angiogenesis, and decrease neurologic deficits, thus providing new opportunities for PNI repair. Neurons and MSCs can promote angiogenesis through exosome-mediated cell-to-cell communication in the nervous system, which again proves that exosomes are a valuable tool for peripheral nerve regeneration. Exosomes can be a promising tool for transporting drugs, proteins, and lipids, genetic material to axons by their cargo, and also help in biological function regulations in the target site [52]. Another positive aspect is their easy storage and transport without the involvement of any toxic cryopreservatives [58].

But, there are major clampdowns in using exosomes including unavailability of standardized isolation, purification, and quantification of the exosomes. The effective administrative route besides traditional drug therapy for PNI repair is not yet determined explicitly. In addition, the molecular mechanism for nerve regeneration after a nerve injury by exosomes is also not yet elucidated [59].

 

Conclusion

Exosomes are the main regulator of paracrine mechanisms. The use of exosomes in the cell-free approach for treating peripheral nerve injury holds to be effective and eliminate several disadvantages of MSC-based stem cell therapy.  Exosomes can transmit genetic materials, neurotrophic factors, and proteins to axons and restore the homeostasis of the microenvironment. The intercellular communications within the peripheral nerve microenvironment by exosomes is the key to promote recovery. Evidence shows that miRNA containing exosomes from differentiated MSCs can also directly enhance axonal regeneration, or indirectly promote recovery by regulating the inflammatory response and vascular regeneration to promote nerve repair. The field is still advancing and exosomes can be used increasingly to develop new clinical therapies for peripheral nerve repair.


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Shruti Mandal

Shruti Mandal


Undergrad from Indian Institute of Science Education and Research, Kolkata, India.