Mesenchymal stem cell-derived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration

Abstract

Traumatic brain injury is a serious and complex neurological condition that affects millions of people worldwide. Despite significant advancements in the field of medicine, effective treatments for traumatic brain injury remain limited. Recently, extracellular vesicles released from mesenchymal stem/stromal cells have emerged as a promising novel therapy for traumatic brain injury. Extracellular vesicles are small membrane-bound vesicles that are naturally released by cells, including those in the brain, and can be engineered to contain therapeutic cargo, such as anti-inflammatory molecules, growth factors, and microRNAs. When administered intravenously, extracellular vesicles can cross the blood-brain barrier and deliver their cargos to the site of injury, where they can be taken up by recipient cells and modulate the inflammatory response, promote neuroregeneration, and improve functional outcomes. In preclinical studies, extracellular vesicle-based therapies have shown promising results in promoting recovery after traumatic brain injury, including reducing neuronal damage, improving cognitive function, and enhancing motor recovery. While further research is needed to establish the safety and efficacy of extracellular vesicle-based therapies in humans, extracellular vesicles represent a promising novel approach for the treatment of traumatic brain injury. In this review, we summarize mesenchymal stem/stromal cell-derived extracellular vesicles as a cell-free therapy for traumatic brain injury via neuroprotection and neurorestoration and brain-derived extracellular vesicles as potential biofluid biomarkers in small and large animal models of traumatic brain injury.

Introduction

Traumatic brain injury (TBI) continues to be one of the major causes of death and disability worldwide. Although significant efforts have been made to develop neuroprotective agents administered early after TBI in an attempt to prevent neural cell death or salvage neurons in the injured brain in the past decades, all the preclinical promising neuroprotective treatments have failed to demonstrate therapeutic efficacy in TBI clinical trials and TBI often needs long-term care, with immense socioeconomic and emotional burdens (Narayan et al., 2002; Loane and Faden, 2010; Briones, 2015; Tortella, 2016; Maas et al., 2017; Alves et al., 2019; Ng and Lee, 2019; Shultz et al., 2020; Lerouet et al., 2021). TBI occurs in two phases including primary injury and secondary injury. Primary injury is caused by the initial mechanical force that results in tissue deformation, shearing and tearing of blood vessels and neural cells, and necrosis. The primary injury event triggers a secondary injury cascade including excitatory amino acid neurotoxicity, calcium overload, neuroinflammation, cellular energy deficits, oxidative stress, autophagy, and neural apoptosis (Xiong et al., 2013; Krishnamurthy and Laskowitz, 2016; Ladak et al., 2019; Ng and Lee, 2019). These secondary injuries contribute to further brain damage and functional deficits after TBI. Therefore, therapeutic approaches targeting secondary injury will be beneficial for treatment of TBI. This article will discuss the roles of mesenchymal stem cell (MSC)-derived exosome-mediated neuroprotection and neurorestoration after TBI.

MSCs are pluripotent, non-hematopoietic stem cells with self-renewal capability. MSCs can be isolated from almost all adult tissues, expanded in vitro, and used as cell-based regenerative medicine. MSCs are the most frequently derived from bone marrow, umbilical cord, adipose tissue, and amniotic fluid and have potential therapeutic benefits in many diseases including neurological diseases and injury (Kulus et al., 2021; Pischiutta et al., 2021). The therapeutic effects of MSCs have been extensively investigated, due to their capability of homing to injured sites with immunoregulatory, pro-angiogenic, pro-neurogenic, anti-apoptotic, anti-inflammatory competencies along with fewer ethical issues and fewer risks for tumorigenicity (Miceli et al., 2021). MSCs obtained from donor rats (Mahmood et al., 2001) or humans (Zanier et al., 2011; Kholodenko et al., 2012) selectively target injured brain tissue (homing) after injections and promote functional recovery. It is highly unlikely that injured brain is replaced with differentiated MSCs because only a small number of transplanted MSCs actually survive in injured brain tissues and fewer differentiate into neural cells (Mahmood et al., 2001).

The therapeutic effects of MSCs may be attributed to their generation and release of extracellular vesicles (EVs) but other molecules secreted by MSCs are also associated with the MSC therapeutic effects (Eleuteri and Fierabracci, 2019; Ahangar et al., 2020; Pinho et al., 2020; Muhammad et al., 2022). Due to the lack of consensus about specific markers of EV subtypes, the use of physical characteristics of EVs such as the size is suggested (Gould and Raposo, 2013; Thery et al., 2018; Witwer and Thery, 2019). Thus, small EVs are referred to those EVs with a diameter < 200 nm. Given that microvesicles are 100 to 1000 nm in diameter, some of them could be also small EVs (Gould and Raposo, 2013). Therefore, using the term exosome will not be completely appropriate. In this review, we will use the term EVs. International Society for Extracellular Vesicles endorses EVs as the generic term for particles naturally released from the cells that are delimited by a lipid bilayer and cannot replicate, i.e., do not contain a functional nucleus (Thery et al., 2018; Witwer and Thery, 2019). EVs can be released by almost all cells and contain functional molecules such as proteins, lipids, cell surface receptors, enzymes, cytokines, metabolites, and nucleic acids including messenger RNAs (mRNAs), microRNAs (miRNAs) and DNA (Keerthikumar et al., 2016; Witwer and Thery, 2019). EVs play a pivotal role in intercellular communication via transfer of their cargos to recipient cells as a regenerative medicine.

Search Strategy

Studies cited in this review published from 2001 to 2023 were searched based on multiple databases (PubMed, Google Scholar, and ClinicalTrials.gov) using the following keywords: mesenchymal stem cells, extracellular vesicles, exosomes, biomarkers, traumatic brain injury, stroke, miRNAs, intracerebral hemorrhage, neuroinflammation, neuroprotection, neurorestoration, neuroplasticity. The literature search was performed between January and March 2023.

Neurorestorative Effects of Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles on Angiogenesis, Neurogenesis, and Neuroinflammation

Neurorestorative potential of MSC-derived EVs has been well established and it has emerged as a novel treatment for TBI. In a rat model of TBI, MSC-derived EVs have been shown to improve functional recovery with delayed intravenous administration in a wide range of effective doses (50–200 μg protein/rat) for treatment of TBI with an extended therapeutic window from 1 day to 7 days postinjury (Zhang et al., 2020). Furthermore, monkey bone marrow MSC-derived EVs administered 24 hours after injury are able to enhance recovery of fine motor function in a monkey cortical injury model (Moore et al., 2019). Beneficial effects of MSC-derived EVs are mediated by reducing inflammation, and promoting endogenous angiogenesis and neurogenesis in rats after TBI (Zhang et al., 2020). Endogenous neurovascular plasticity occurs after TBI, including neurogenesis, angiogenesis, axonal sprouting, and synaptogenesis, which may contribute to spontaneous functional recovery after brain injury (Xiong et al., 2010). Spontaneous recovery is limited after brain injury. There is a compelling need to develop novel therapeutics to improve functional recovery after TBI by enhancing neurovascular plasticity.

Increased endogenous neurogenic response occurs in the subventricular zone and subgranular zone in the dentate gyrus of the hippocampus in injured brain and is associated with cognitive functional recovery after TBI (Patel and Sun, 2016). Neural stem cells in the subventricular zone and subgranular zone continuously generate new neurons throughout adulthood in mammals and develop into mature neurons. It is well established that adult-born dentate gyrus granule cells can functionally integrate into the existing circuitry (Fares et al., 2019). Treatment with MSC-derived EVs (starting 24 hours after injury) significantly promotes neurogenesis and angiogenesis in the injured brain after TBI, which may in part contribute to functional recovery after TBI (Zhang et al., 2015). The adult brain vasculature is quiescent under normal conditions but activated after injury. Activated vasculature may secrete growth factors that facilitate neurorestorative processes including neurogenesis and synaptogenesis, which may lead to improved functional recovery after brain injury (Zhang et al., 2012).

The potential effects of MSCs-derived EVs on neuroinflammation and neurogenesis in TBI and, especially, on functional recovery have been well reviewed (Yang et al., 2017; Liu et al., 2023). A recent study demonstrates that human bone marrow MSC (BMSC)-EVs promote functional neurological recovery and stimulate post-stroke neurogenesis adjacent to the subventricular zone in aged rats post-stroke (Dumbrava et al., 2022). MSC-derived EVs from human BMSCs (100 μg protein, IV) effectively improve functional recovery in rats after intracerebral hemorrhage, possibly by promoting endogenous angiogenesis and neurogenesis (Han et al., 2018). Thus, cell-free, MSC-derived EVs may be a novel therapy for intracerebral hemorrhage. Intravenous administration of cell-free MSC-generated EVs post stroke improves functional recovery and enhances neurite remodeling, neurogenesis, and angiogenesis and represents a novel treatment for stroke (Xin et al., 2013; Zhang and Chopp, 2016; Venkat et al., 2018; Zhang et al., 2019). EVs could improve cognition function by protecting blood-brain barrier, inhibiting apoptosis, suppressing inflammation, and regulating autophagy in brain injuries via different molecules and pathways including microRNA (miRNA) (Zhang et al., 2022b). EVs obtained from adipose-derived MSCs promote neural differentiation of neural progenitor cells in vitro (Park et al., 2022). EVs from adipose-derived mesenchymal stem cells reduce autophagy in stroke mice by EV transfer of miR-25 (Kuang et al., 2020). EVs from human BMSCs improve neuroregeneration and prevent postischemic immunosuppression in a mouse model of stroke (Doeppner et al., 2015). Human BMSC-derived EVs attenuate neuroinflammation evoked by focal brain injury in rats (Dabrowska et al., 2019). MSC-derived EV-enclosed microRNA-93 prevents ischemic brain damage in rats (Shi et al., 2022). Intranasally administered EVs from umbilical cord stem cells provide neuroprotective effects and improve functional recovery after perinatal brain injury in rats (Thomi et al., 2019). Collectively, these studies support that treatment with EVs improves functional recovery through EVs-effects on neurorestoration, neurogenesis, angiogenesis, immune system, and neuroprotection.

Neuroinflammation is a hallmark for acute and chronic TBI. One of the emerging mechanisms for cell-cell communication involved in the immune response regulation is represented by EVs (Mot et al., 2022). Green fluorescent protein-tagged (GFP⁺) MSC-EVs can be taken up by neurons, microglia, and astrocytes in the TBI rat brain as early as 30 minutes after intravenous delivery of EVs, with more EVs detected in the injured hemisphere while GFP⁺ EVs are also co-localized with CD68⁺ macrophages in the spleen, liver, and thymus (Xiong et al., 2017). These data suggest that intravenous administration of EVs may have non-central effects to modulate peripheral immune responses in addition to central roles in enhancing neurovascular remodeling and regulating neuroinflammation. Non-central nervous system (CNS) effects if any, would be complementary, and do not in any way negate therapeutic effects of EVs on functional recovery and neurovascular remodeling. Further investigation of the effect of MSC-derived EVs on the CNS effects for TBI as well as non-CNS systemic effects on peripheral immune response is warranted.

Neuroprotective Effects of Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles

Early (15 minutes post injury) administration of MSC-EVs significantly reduces the lesion size and improves functional performance in mice after TBI through modulating the polarization of microglia/macrophages, increasing anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) expression but inhibiting expression of pro-apoptotic protein Bcl-2-associated X protein and pro-inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha (Ni et al., 2019). In translational research, the neuroprotective efficacy of MSC-derived EVs has been investigated in large animal models. In a combined swine model of TBI and hemorrhage shock, early (1 hour post injury) single-dose administration of MSC-derived EVs provides neuroprotection by reducing brain swelling, lesion size, and blood-brain barrier breach (Williams et al., 2020a). These data indicate that cell-free EVs have neuroprotective and neurorestorative effects for improving TBI functional recovery, supporting continued investigation of EVs as a novel treatment for TBI. Use of human bone marrow MSCs as the source of EVs may ensure the TBI study in large animals as translational as possible. Several references related to use of human BMSC-derived EVs in large animal (swine and monkey) models of TBI are described in Additional Table 1 (Moore et al., 2019; Williams et al., 2019, 2020a, b, c; Medalla et al., 2020; Muhammad et al., 2022).

Table 1.

Treatment with MSC-derived EVs in animal models of TBI

TBI model	TBI animal species/sex	MSC source for EVs	Dosage	Injection time/ route	Main findings	References
CCI	Mouse/male	Rat bone mesenchymal stem cells (BMSCs)	30 μg protein/mouse	15 min PI/ retro- orbital	Reducing lesion size, improving neurobehavioral performance, inhibiting neuroinflammation	Ni et al., 2019
CCI	Mice/male	Human BMSCs	6.4 or 12.8 or 25.6 × 10^9 EVs/mouse	90 min PI/IN	Dose-dependently preventing long-term cognitive and mood impairments	Kodali et al., 2023
CCI	Mice/male	Human BMSCs	3.8, 7.5, 15, 30 μg EVs per mouse	1 hour PI/IV	Rescuing cognitive impairments after TBI, suppressing neuroinflammation in a dose-response manner	Kim et al., 2016
CCI	Rat/male	Human BMSCs	50, 100, 200 μg protein/rat	1 day PI/IV	Exhibiting a wide range of effective doses for treatment of TBI with a therapeutic window of at least 7 days post injury	Zhang et al., 2020
CCI	Rat/male	Human BMSCs	100 μg protein/rat	1 day PI/IV	Improving functional recovery, promoting endogenous angiogenesis and neurogenesis, reducing neuroinflammation	Zhang et al., 2015
CCI	Rat/male	Human BMSCs	100 μg protein/rat	1 day PI/IV	EVs derived from hMSCs cultured in 3D scaffolds providing better outcomes in spatial learning than EVs from hMSCs cultured in the 2D condition.	Zhang et al., 2017b
CCI	Rat/male	Human BMSCs	100 μg protein/rat	1 day PI/IV	miR-17-92 cluster enriched EVs have better therapeutic effects on improvement in functional recovery, by reducing neuroinflammation and cell loss, enhancing angiogenesis and neurogenesis versus naïve exosome treatment	Zhang et al., 2021
CCI	Rat/male	Human BMSCs	100 μg protein/rat	1 day PI/IV	Attenuating miRNAs in MSC-derived EVs abolishes EV treatment-induced beneficial effects in TBI recovery	Zhang et al., 2023
CCI	Rat/not described	Rat BMSCs	100 μg protein/rat	1 hour PI/IV	Reducing neurological damage by alleviating glutamate-mediated excitotoxicity	Zhuang et al., 2022
CCI	Rat/male	Rat BMSCs	100 μg protein/rat	1 day PI/IV	BDNF-induced MSCs-EVs with higher level of miR- 216a-5p have better effects on improving cell migration and inhibiting apoptosis than MSCs-EVs in rats after TBI	Xu et al., 2020
Feeney’s weight-drop method	Rat/male	Human adipose MSCs	20 μg protein/rat	1 day PI/ICV	hADSC-EVs specifically enter microglia and macrophages and suppress their activation during brain injury, thereby inhibiting inflammation and facilitating functional recovery	Chen et al., 2020
CCI combined with hemorrhage shock	Swine/female	Human BMSCs	1 × 1012 particles/swine	1 hour PI/IV	Reducing brain swelling and lesion size, levels of blood-based cerebral biomarkers, and improving BBB integrity	Williams et al., 2020a
CCI combined with hemorrhage shock	Swine/female	Human BMSCs	1 × 1013 particles/swine	9 hours, 1, 5, 9, and 13 days PI/IV	Attenuating the severity of neurologic injury with faster neurologic recovery	Williams et al., 2019
CCI combined with hemorrhage shock	Swine/female	Human BMSCs	1 × 1013 particles/swine	1 hour PI/IV	Attenuating cerebral inflammatory networks and promoting neurogenesis and neuroplasticity	Bambakidis et al., 2022
Cortical injury in the mapped hand representation.	Monkey/female	Monkey BMSCs	4 × 1011 particles/kg	1 day and 14 days PI/IV	Enhancing recovery of motor function	Moore et al., 2019
Cortical injury in the mapped hand representation.	Monkey/male and female	Monkey BMSCs	4 × 1011 particles/kg	1 day and 14 days PI/IV	Dampening injury-related hyperexcitability and restoring excitatory/inhibitory balance in the ventral premotor cortex, thereby normalizing activity within cortical networks for motor function.	Medalla et al., 2020

BMSCs: Bone marrow MSCs; BDNF: brain-derived neurotrophic factor; CCI: controlled cortical injury (an open head injury, focal contusion); EVs: extracellular vesicles; hADSC: human adipose mesenchymal stem cells; IN: intranasal administration; IV: intravenous administration; MSC: mesenchymal stem/stromal cells; PI: post injury; TBI: traumatic brain injury.

Although we focused on therapeutic effects of MSC-derived EVs in TBI, EVs from many other cells including neural stem cells, astrocytes, and microglia have therapeutic effects on improving functional recovery in TBI (Hering and Shetty, 2023). Adipose tissue is usually treated as waste material and discarded, which makes it a valuable source of cells (Bunnell et al., 2008; Strioga et al., 2012). Adipose tissue has proven to serve as an abundant, accessible, and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications (Bunnell et al., 2008). A previous study showed that bone marrow as an MSC source had significant disadvantages compared to the use of adipose tissue, including less stability in culture conditions and a smaller number of cells. Despite the minor differences between these MSC populations, adipose tissue stem cells seem to be as effective as BMSCs in clinical application, and, in some cases, may be better suited than BMSCs (Strioga et al., 2012). Recent studies reveal that BMSC-derived EVs showed superior regeneration ability, and adipose tissue MSC-derived EVs played a significant role in immune regulation, whereas umbilical cord MSC-derived EVs were more prominent in tissue damage repair (Wang et al., 2020). Proteomics analyses also reveal functional differences between EVs from umbilical cord MSCs and EVs derived from the adipose tissue and the different functions of adipose tissue- and umbilical cord-MSC EVs may be related to the differences in their immunomodulatory activities (Liu et al., 2021). The choice of MSC type as cell source of EVs may depend on the specific treatment and the desired outcomes of the therapy. Of note, MSC-derived EVs have been entered in clinical trials (https://clinicaltrials.gov) for many diseases mainly ARDS and COVID-19, acute ischemic stroke, degenerative meniscal injury, knee osteoarthritis, and type I diabetes mellitus. MSC-derived EVs have proven to be a promising strategy in treating TBI. It is important to understand their mechanisms of action to maximize their treatment potential for TBI.

Role of miRNAs from Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles in Traumatic Brain Injury Treatment

The mechanisms underlying the MSC-derived EV treatment-induced beneficial effects in TBI remain elusive. EVs contain miRNAs, small non-coding regulatory RNAs (usually 18 to 25 nucleotides), which regulate gene expression at the post-transcriptional level for a wide array of cellular processes via binding to complementary sequences on target mRNA transcripts and causing mRNA degradation or translational repression and gene silencing (Chopp and Zhang, 2015). Argonaute 2 (Ago2) one of the primary miRNA machinery proteins is required for packaging miRNAs into EVs and performing biological functions in the recipient cells. Knockdown of endogenous Ago2 in MSCs reduces the level of Ago2 proteins and miRNAs in EVs and diminishes the effect of EVs on promotion of axonal growth (Zhang et al., 2017a). Although treatment with various miRNAs or mimics has shown beneficial effects post-TBI, only recently have essential roles of miRNAs been investigated in TBI by determining the effects of miRNA-depleted EVs harvested from human bone marrow MSCs with Ago2 knockdown on neurovascular remodeling, neuroinflammation, and neurological recovery in a clinically relevant rat model of TBI (Zhang et al., 2023). This study demonstrates that MSCs with Ago2 knockdown have reduced global levels of miRNAs in MSC-derived EVs and these miRNA-reduced EVs have significantly fewer effects on reducing neuronal cell loss, inhibiting neuroinflammation, and augmenting angiogenesis and neurogenesis, as well as improving functional recovery in TBI as compared to naïve MSC-derived EVs, suggesting the essential roles of miRNAs from EVs in therapeutic effects on TBI.

The miR-17-92 cluster as a master regulator of neurogenesis controls the proliferation and neuronal differentiation of neural stem/progenitor cells in both developmental and adult brains (Xia et al., 2022). Engineering MSCs with miR-17-92 cluster may potentiate the MSC-derived EV therapeutic effects for treatment of TBI. MSC-derived EVs with overexpression of the miR-17-92 cluster present a significant therapeutic effect on improvement in functional recovery than do natural MSC-derived EVs, likely by reducing neuroinflammation and neuronal cell loss, enhancing angiogenesis and neurogenesis after TBI (Zhang et al., 2021). Beneficial effects of miR-17-92 cluster-enriched MSC-EVs enhanced neurofunctional recovery may be mediated in part via the activation of the phosphatidylinositol 3-kinase 3/protein kinase B/mechanistic target of rapamycin/glycogen synthase kinase 3 beta signaling pathway induced by the downregulation of phosphatase and tensin homolog (PTEN, a validated miR-17-92 cluster targets) as demonstrated in a rat stroke model (Xin et al., 2021). In addition, EVs isolated from BMSCs cultured in the presence of brain-derived neurotrophic factor have better effects on promoting neurogenesis and inhibiting apoptosis than naïve MSCs-derived EVs in rats after TBI, which may be mediated by the higher expression of miR-216a-5p in brain-derived neurotrophic factor-treated MSCs-derived EVs (Xu et al., 2020). These findings indicate that MSC-derived EVs with increased specific miRNAs will be a novel cell-free therapy for TBI.

Role of Proteins from Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles in Traumatic Brain Injury Treatment

Although miRNAs play an important role in the EVs-mediated plasticity and functional recovery after TBI, MSC-derived EVs may work through other cargos including proteins.

Significant differences in proteomic profiles exist among bone marrow (BM), adipose tissue (AT), and umbilical cord-MSC-derived EV (Wang et al., 2020). BMSC-derived EVs show superior regeneration ability, and AT-MSC-derived EVs play a significant role in immune regulation, whereas umbilical cord-MSC-derived EVs have more prominent effects on tissue repair revealed by bioinformatics analysis (Wang et al., 2020). Comprehensive analyses of MSC-derived EV cargos may help select optimal source cells in future EV-related studies for potential applications in different diseases. Bioinformatic analysis of miRNAs and proteins revealed that the majority of their content was shared by both AT-MSC and BMSC-EVs, but relevant differences in the molecules selectively expressed in these EVs can explain their different biological activity. Specifically, AT-MSC-EVs are more effective than BMSC-EVs in accelerating wound healing (Pomatto et al., 2021). Most of 591 proteins detected in human AT-MSC-EVs are involved in signal transduction in regulating inflammation, apoptosis, angiogenesis, and cell proliferation while 604 miRNAs negatively regulate gene expression (Alonso-Alonso et al., 2022).

MSC-derived EVs have great potential to replace conventional MSC-based cell therapy as a modern approach in regenerative medicine. Although numerous cargos including proteins and miRNAs have been discovered in MSC-derived EVs from different origins, the study of MSC-derived EV cargos and their functions is still in the infant stage. It is unknown whether functional miRNAs and proteins work independently or synergistically in the recipient cells. MSC-derived EVs are heterogeneous in different bioactive molecules from different donors. It is a challenge to comprehensively understand the complete components of MSC-derived EVs because their cargos dynamically change in response to surrounding microenvironment (2D vs. 3D culture, preconditioning/metabolic programming such as hypoxia and serum deprivation, culture medium/nutrition, pH), isolation methods, engineering approaches, and post-isolation loading with therapeutic molecules. MicroRNAs, siRNAs, proteins, and small molecule compounds have been successfully loaded into isolated EVs by various methods including electroporation, sonication, freeze and thaw cycles, extrusion, and co-incubation of modified cargos with membrane permeabilizers (Luan et al., 2017).

Use of Brain-Derived Extracellular Vesicles as Biofluid Biomarkers of Traumatic Brain Injury

MSC-derived EVs have been currently investigated as a cell-free cell therapy for treatment of many diseases including TBI. Studies have also shown that brain-derived EVs can cross the blood-brain barrier and enter the bloodstream, allowing for non-invasive detection of TBI (Saint-Pol et al., 2020). EVs secreted by various cell types within the brain, including neurons, astrocytes, and microglia, can contain a variety of proteins, nucleic acids, and lipids that reflect the pathological changes associated with TBI (Devoto et al., 2020; Guedes et al., 2020). Brain-derived EVs and their cargos can be identified from blood (Kenney et al., 2018; Ko et al., 2020), saliva (Cheng et al., 2019), cerebrospinal fluid (Manek et al., 2018) as potential biofluid biomarkers for TBI (Guedes et al., 2020; Khan et al., 2022). For example, EVs derived from neurons have been found to contain proteins such as tau and amyloid beta, which are associated with TBI-induced neurodegeneration and cognitive impairment (Goetzl et al., 2019; Karnati et al., 2019). Similarly, EVs derived from astrocytes and microglia have been found to contain various pro-inflammatory cytokines and chemokines, reflecting TBI-induced neuroinflammation (Beard et al., 2021). However, the composition of brain-derived EVs can be highly variable and dependent on the specific injury and individual differences, making their use as biomarkers challenging (Guedes et al., 2020). Although brain-derived EVs represent a promising biomarker for TBI, further research is needed to fully characterize their potential clinical utility.

Current Limitations of Extracellular Vesicle-Based Therapies in Traumatic Brain Injury

Biological/functional differences of EVs and the therapeutic potential of EVs from different tissues are not clearly understood

EVs secreted by various cell types are involved in intercellular communication and have potential therapeutic applications. EVs from different tissues have been shown to differ in their biological and functional properties, including their protein and RNA content, size, and surface markers (Kalluri and LeBleu, 2020; Almeria et al., 2022). These differences may contribute to variations in their therapeutic potential for different diseases. For example, EVs derived from mesenchymal stem cells have been shown to have immunomodulatory and regenerative properties, while EVs derived from cancer cells may promote tumor growth and metastasis (Kalluri and LeBleu, 2020). Despite ongoing research efforts, the precise mechanisms underlying these differences are not yet fully understood. Further research is needed to elucidate the functional and therapeutic implications of tissue-specific EVs.

Number (threshold) of EVs required to elicit a therapeutic benefit is unknown in the neurological disease/injury and/or various neurological conditions

The optimal number of EVs required to elicit a therapeutic benefit in TBI is not yet well established, as there is limited research available on the topic. However, preclinical studies have shown promising results using EVs in TBI models. For example, in a study using a rat model of TBI, intranasal administration of EVs derived from MSCs improved cognitive and mood function and reduced brain inflammation in a dose-dependent manner at a concentration range of 6.4 or 12.8 or 25.6 × 10⁹ EV particles/mouse per dose (Kodali et al., 2023). Other studies using a swine model of TBI combined with hemorrhage shock showed that intravenous injection of EVs derived from human BMSCs at a concentration of 1.0 × 10¹² to 1.0 × 10¹³ EV particles per dose reduced brain inflammation, brain damage, and improved neurological function (Williams et al., 2019, 2020a, b, c). Another study using a monkey cortical injury model showed that intravenous injection of EVs derived from monkey BMSCs at a concentration of 4.0 × 10¹¹ EV particles per kg of body weight enhances recovery of motor function (Moore et al., 2019). Currently, it is not well established whether EV numbers should be varied with the severity of the neurological disease or injury, or with various neurological conditions. The optimal concentration and dosage of EVs for therapeutic use in different neurological diseases or injuries are still largely unknown, and research is ongoing to determine these parameters. The number of EVs required to achieve a therapeutic benefit in TBI may depend on various factors such as the severity of the injury, injury type, the type of EVs, and the route of administration.

Scalability of the EV treatment needs further investigation

EVs have shown promising therapeutic potential for the treatment of TBI. However, there are several challenges associated with scaling up the use of EVs for clinical use, as well as some perspectives for the future (Zhang et al., 2022a). One of the primary challenges is the scalability of EV production. EVs are typically isolated from cell culture media or bodily fluids, and the yield of EVs can vary depending on the source and method of isolation. Current methods of large-scale production of EVs are limited, and there is a need for more efficient and scalable methods to produce EVs in sufficient quantities for clinical use. Another challenge is the lack of standardized methods for EV isolation, characterization, and quality control. There is currently no consensus on the optimal methods for EV isolation, and the heterogeneity of EVs makes it difficult to develop standardized protocols. This can impact the reproducibility and consistency of EV-based therapies. In addition, the optimal route of administration and dosing regimen for EV therapy in TBI is still unclear. Preclinical studies have used different routes of administration and dosing regimens, and it is not clear which approach is most effective or feasible for clinical use. Despite these challenges, there are several perspectives for the future of EV-based therapies for TBI. One potential avenue is the development of engineered EVs with specific therapeutic cargos, such as drugs or RNA molecules, to enhance their therapeutic potential. Another perspective is the development of biomimetic EVs that mimic the natural properties of EVs in the body, which could enhance their therapeutic efficacy. While there are several challenges associated with the use of EVs for the treatment of TBI, the potential benefits of this approach make it an area of active research and development. Further research is needed to address the challenges and optimize the use of EVs for clinical use in TBI and other neurological conditions.

Heterogeneity of the CNS may alter the response to the EV-based therapeutics

The heterogeneity of CNS may alter the response to EV-based therapeutics. TBI is a complex and multifactorial condition, and the severity and location of the injury can vary greatly between patients. This variability can affect the response to any therapeutic intervention, including EV therapy. The response to EV-based therapeutics may also depend on the type and source of the EV used. EVs derived from different cell types or different donors may have varying therapeutic effects in different types of TBI. In addition, the immune response to EVs may vary between individuals, which could impact their therapeutic efficacy. Furthermore, the timing of EV therapy may also play a role in the response to treatment. The optimal timing of EV therapy after TBI is not well established, and the response to EVs may vary depending on the stage of injury and the timing of treatment. Therefore, the heterogeneity of TBI is likely to have an impact on the response to EV-based therapeutics.

Understanding of the degree of specificity of EVs to a particular neural target is still limited

There is still limited understanding of the degree of specificity of EVs to a particular neural target for the treatment of TBI. EVs can be derived from a variety of cell types, and they can contain a diverse range of molecules, including proteins, lipids, and nucleic acids. These molecules can have various functions and interactions with different cell types in the brain, making it difficult to predict the specific effects of EVs on neural targets. While some studies have shown that EVs can selectively target specific cell types in the brain, such as neurons or glial cells (Xiong et al., 2017; Chen et al., 2020), the mechanisms of this specificity are not yet well understood. The composition of EVs, as well as the presence of specific proteins or receptors on the surface of EVs, may play a role in their targeting abilities. Similarly, the heterogeneity of TBI can also impact the specificity of EV targeting. The location and severity of the injury, as well as the type and stage of injury, can all influence the expression of specific proteins or receptors on neural cells, which in turn may affect the targeting of EVs to these cells. Further research is needed to better understand the mechanisms of EV targeting and to optimize their therapeutic potential in TBI and other neurological conditions.

Stability and storage of EVs remain poorly defined

The stability and storage of EVs remain poorly defined as a potential treatment of TBI. EVs are small, lipid-bound vesicles that can be easily degraded or damaged under certain storage conditions. Therefore, it is important to understand the optimal storage conditions for EVs to maintain their stability and therapeutic efficacy. One factor that can affect the stability of EVs is the storage temperature. EVs are typically stored at –80°C to maintain their stability. MSC-derived EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4–6 weeks at –20°C and –80°C and after lyophilization (Levy et al., 2022). However, the optimal storage temperature for EVs may depend on their source, isolation method, and composition, and further research is needed to determine the optimal conditions for different types of EVs. In addition to temperature, the storage buffer and conditions can also impact the stability of EVs. EVs are typically stored in buffer solutions containing stabilizing agents, such as sucrose or trehalose, to prevent aggregation and degradation (Almeria et al., 2022). However, the optimal composition and concentration of these stabilizing agents may vary depending on the specific EV preparation, and further research is needed to optimize the storage buffer conditions for different types of EVs. Furthermore, the stability of EVs during transportation and administration is also an important consideration. EVs may need to be transported long distances for clinical use, and the conditions during transportation can impact their stability and therapeutic efficacy. Additionally, the method of EV administration, such as intravenous injection or direct injection into the brain, can also impact their stability and therapeutic efficacy. The stability and storage of EVs remain poorly defined as a potential treatment of TBI, and further research is needed to optimize the storage and transportation conditions for different types of EVs. It is important for ensuring the therapeutic efficacy and safety of EV-based therapies for TBI and other neurological conditions.

Conclusions

The refinement of MSC therapy from a cell-based therapy to cell-free EV-based therapy offers several advantages, as it eases the arduous task of preserving cell viability and function, storage, and delivery to patient because their bi-lipid membranes can protect their biologically active cargo allowing for easier storage of EVs, which allows a longer shelf-life and half-life. MSC-derived EVs contain the bioactive molecules and paracrine factors that reflect those of the parent MSCs. EV-based therapy for TBI does not compromise efficacy associated with using complex therapeutic agents such as MSCs. They reduce the safety risks inherent in administering viable cells such as the risk of occlusion in microvasculature or unregulated growth of transplanted cells. MSC-derived EVs can be engineered to carry certain cargos with therapy-specific functions (for example, anti-apoptosis, anti-inflammation, immune-modulation, promotion of angiogenesis and neurogenesis). EVs from allogeneic MSCs collected from a healthy donor may result in better therapeutic outcomes than autologous MSCs because the health and quality of MSCs harvested from a patient as autologous cell sources of EVs may be questionable given disease may affect these aspects of MSCs and their EV cargos. Developing an EV-based therapy for TBI opens up a wide variety of means to deliver targeted regulatory genes to enhance multifaceted aspects of central nervous system plasticity and to amplify neurological recovery for neural injury and neurodegenerative diseases. The key points of MSC-EVs for treatment of TBI and brain-derived EVs as biomarkers are summarized in Figure 1.

Figure 1.

Extracellular vesicles (EVs) as a treatment and biomarker for traumatic brain injury (TBI).

In TBI, primary brain injury occurs at the time of impact, and results from displacement of the physical structures of the brain. Secondary brain injury occurs gradually over a period of time from hours to days, even months after the initial injury. Excitatory amino acid neurotoxicity, calcium overload, neuroinflammation, cellular energy deficits, oxidative stress, autophagy, and neural apoptosis participate in secondary injury which leads to further brain damage and functional deficits. EVs can be released from almost all tested cells. EVs contain many cargos including proteins, metabolites, lipids, nucleic acids including DNA, mitochondrial DNA (mtDNA), RNA, and microRNA (miRNA). The lipid bilayer of EVs protects their cargos from degradation and improves their biological stability. EVs play an important role in intercellular communication by transferring their cargos into recipient cells to regulate multiple biological activities in many physiological and pathological processes. Brain-derived EVs can cross the blood-brain barrier (BBB). EVs can be isolated from a variety of biological fluids, including blood, cerebrospinal fluid, and urine, making them a non-invasive and easily accessible biomarker for TBI. Additionally, administration of EVs derived from many cells including bone marrow mesenchymal stem/stromal cells can improve functional recovery after TBI by enhancing neuroprotection, neurorestoration (enhancing angiogenesis, neurogenesis, and axonal remodeling) and reducing neuroinflammation. In summary, EVs hold promise as both a therapeutic tool and a diagnostic biomarker for TBI. Created with PowerPoint and Photoshop.

Additional files:

Additional file 1: Open peer review report 1 ^{(82.1KB, pdf)} .

Additional Table 1: Treatment with MSC-derived EVs in animal models of TBI.