Emerging potential of exosomes for treatment of traumatic brain injury

Ye Xiong, Asim Mahmood Michael Chopp

1 Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA

2 Department of Neurology, Henry Ford Hospital, Detroit, MI, USA

3 Department of Physics, Oakland University, Rochester, MI, USA

Emerging potential of exosomes for treatment of traumatic brain injury

Ye Xiong1,＊, Asim Mahmood1, Michael Chopp2,3

1 Department of Neurosurgery, Henry Ford Hospital, Detroit, MI, USA

2 Department of Neurology, Henry Ford Hospital, Detroit, MI, USA

3 Department of Physics, Oakland University, Rochester, MI, USA

How to cite this article:Xiong Y, Mahmood A, Chopp M (2017) Emerging potential of exosomes for treatment of traumatic brain injury. Neural Regen Res 12(1):19-22.

Open access statement:is is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Traumatic brain injury (TBI) is one of the major causes of death and disability worldwide. No e ff ective treatment has been identi fi ed from clinical trials. Compelling evidence exists that treatment with mesen‐chymal stem cells (MSCs) exerts a substantial therapeutic e ff ect aer experimental brain injury. In addition to their soluble factors, therapeutic e ff ects of MSCs may be attributed to their generation and release of exosomes. Exosomes are endosomal origin small‐membrane nano‐sized vesicles generated by almost all cell types. Exosomes play a pivotal role in intercellular communication. Intravenous delivery of MSC‐de‐rived exosomes improves functional recovery and promotes neuroplasticity in rats aer TBI.erapeutic e ff ects of exosomes derive from the exosome content, especially microRNAs (miRNAs). miRNAs are small non‐coding regulatory RNAs and play an important role in posttranscriptional regulation of genes. Com‐pared with their parent cells, exosomes are more stable and can cross the blood‐brain barrier.ey have reduced the safety risks inherent in administering viable cells such as the risk of occlusion in microvascula‐ture or unregulated growth of transplanted cells. Developing a cell‐free exosome‐based therapy may open up a novel approach to enhancing multifaceted aspects of neuroplasticity and to amplifying neurological recovery, potentially for a variety of neural injuries and neurodegenerative diseases.is review discusses the most recent knowledge of exosome therapies for TBI, their associated challenges and opportunities.

traumatic brain injury; exosomes; microRNAs; mesenchymal stem cells; treatment; neuroplasticity; cell therapy

Accepted: 2016-12-30

Unmet Need forreatment forraumatic Brain Injury (BI)

TBI is one of the major causes of death and disability world‐wide. An estimated 1.7 million people sustain TBI each year in the United States, and more than 5 million people are coping with disabilities from TBI at an annual cost of more than $76 billion. Despite improved supportive and reha‐bilitative care of TBI patients, no e ff ective pharmacological treatments are available for reducing TBI mortality and im‐proving functional recovery because all phase II/III TBI clin‐ical trials have failed. Emerging preclinical data indicate that restorative therapies targeting multiple parenchymal cells including cerebral endothelial cells, neural stem/progenitor cells and oligodendrocyte progenitor cells enhance TBI‐in‐duced angiogenesis, neurogenesis, axonal sprouting, and oligodendrogenesis, respectively (Xiong et al., 2009).ese interacting neuroplastic events in concert improve neurolog‐ical function aer TBI.ere is a compelling need to develop novel therapeutics speci fi cally designed to stimulate neuro‐plasticity which subsequently promote neurological recovery aer TBI.

Mechanisms Underlying Cellerapy forBI

Cell therapies including bone marrow‐derived mesenchymal stem cells (MSCs) have shown promise in the fi eld of regen‐erative medicine for treating various diseases including TBI. Exogenously administered MSCs selectively target injured tissue (homing), interact with brain parenchymal cells, re‐duce expression of axonal inhibitory molecules, stimulate the production of growth and plasticity positive factors, which increase neurite outgrowth, promote neurorestoration and recovery of neurological function aer brain injuries (Chopp and Li, 2002). Administration of MSCs through different routes (intraarterial, intravenous, and intracerebral) exhibit robust therapeutic effects in experimental TBI. However, there are some disadvantages for each route. For example, relatively few MSCs can be injected intracranially; intraarte‐rial injection of MSCs can cause brain ischemia; and intra‐venous injection results in body‐wide distribution of MSCs. The efficacy of MSC transplantation in treating TBI has been observed to be independent of di ff erentiation of MSCs. The possibility that their therapeutic benefit is derived by replacement of injured tissue with differentiated MSCs ishighly unlikely because only a small proportion of trans‐planted MSCs actually survive and fewer differentiate into neural cells in injured brain tissues. MSCs secrete or express factors that reach neighboring parenchymal cellsviaeither a paracrine e ff ect or a direct cell‐to‐cell interaction, or MSCs may induce host cells to secrete bioactive factors, which promote survival and proliferation of the parenchymal cells (brain remodeling) and thereby improve functional recovery. It is well documented that the predominant mechanisms by which MSCs promote brain remodeling and functional recov‐ery aer brain injury are related to bioactive factors secreted from MSCs or from parenchymal cells stimulated by MSCs (Chen et al., 2002; Mahmood et al., 2004). Much of research on MSC secretion has centered on individual small mol‐ecules such as growth factors, chemokines and cytokines. Paradigm‐shiing fi ndings that therapeutic e ff ects of MSCs are mediated by secreted factors as opposed to the previous notion of di ff erentiation into injured tissues o ff er numerous possibilities for ongoing therapeutic development of MSC secreted products.

Figure 1 Exosomes derived from mesenchymal stem cells (MSCs) for treatment of traumatic brain injury (BI).

MSC-derived Exosome as a Novelerapy forBI

Recent studies indicate that therapeutic effects of MSCs are likely attributed to their robust generation and release of exosomes (Lai et al., 2010; Xin et al., 2013; Zhang et al., 2015). Exosomes are endosome‐derived small membrane vesicles, approximately 30 to 100 nm in diameter, and are released into extracellular fl uids by cells in all living systems. Administration of cell‐free exosomes derived from MSCs is su fficient to exert therapeutic e ff ects of intact MSCs aer brain injury (Xin et al., 2013; Zhang et al., 2015, 2016). A recent report demonstrates that extracellular vesicles (EVs) from MSCs are not inferior to MSCs in a rodent stroke model by comparing therapeutic e fficacy of MSC‐EVs with that of MSCs (Doeppner et al., 2015).e exosomes transfer RNAs and proteins to other cells which then act epigeneti‐cally to alter the function of the recipient cells.e develop‐ment of cell‐free exosomes derived from MSCs for treatment of TBI is just in its infancy (Zhang et al., 2015, 2016; Kim et al., 2016). In a proof‐of‐principle study, an intravenous deliv‐ery of MSC‐derived exosomes improves functional recovery and promotes neuroplasticity in young adult male rats sub‐jected to TBI induced by controlled cortical impact (Zhang et al., 2015), as shown in Figure 1. A recent study also demonstrated that isolated extracellular vesicles from MSCs reduce cognitive impairments in a mouse model of TBI (Kim et al., 2016). Administration of cell‐free nanosized exosomesmay avoid potential concerns associated with administration of living cells, which can replicate. Compared to their parent cells, exosomes may have a superior safety pro fi le, they do not replicate or induce microvascular embolism, and can be safely stored without losing function. Exosomes could substitute for the whole cell therapy in the treatment of TBI.is may open new clinical applications for “off‐the‐shelf” interven‐tions with MSC‐derived exosomes for TBI. MSCs are most typically grown in traditional 2 dimensional (2D) adherent cell culture. Three dimensional (3D) conditions such as spheroid culture have been shown to stimulate higher levels of trophic factor secretion compared to monolayer culture. MSCs seeded in the 3D collagen sca ff olds generated signi fi‐cantly more exosomes compared to the MSCs cultured in the 2D conventional condition (Zhang et al., 2016). Exosomes derived from MSCs cultured in 3D sca ff olds provided better outcome in spatial learning than exosomes from MSCs cul‐tured in the 2D condition, although an equal amount of exo‐somes isolated from MSCs in 2D or 3D conditions was ad‐ministered intraperitoneally into rats aer TBI (Zhang et al., 2016).ese data suggest that the content of the exosomes is responsible for the di ff erential therapeutic e ff ects, and the 3D conditioned exosomes likely contain a di ff erent pro fi le of proteins and genetic materials compared to 2D conditioned exosomes.e e fficacy of exosomes may critically depend on the exosome contents including proteins, RNAs, lipids and DNAs (mitochondrial origin).e mechanisms under‐lying regenerative activities of MSC‐derived exosomes are attractive subjects of ongoing investigation. A better under‐standing of the e ff ects of exosomes derived from MSCs on functional recovery and brain remodeling and the under‐lying mechanisms of their actions are prerequisite for the development of MSC exosomes as an e fficacious and novel therapy for TBI.

microRNAs in MSC-derived Exosomes as Possible Mediators of Neuroplasticity

microRNAs (miRNAs), small non‐coding regulatory RNAs (usually 18 to 25 nucleotides), regulate gene expression at the post‐transcriptional level (Chopp and Zhang, 2015),viabinding to complementary sequences on target message RNA (mRNA) transcripts, and cause mRNA degradation or translational repression and gene silencing. In eukaryotic cells, miRNAs constitute a major regulatory gene family. Di ff erent cell types and tissues express di ff erent sets of miR‐NAs. By a ff ecting gene regulation, miRNAs are likely to be involved in most biological processes such as developmental timing and host‐pathogen interactions as well as cell di ff er‐entiation, proliferation, apoptosis and tumorigenesis in vari‐ous organisms. We propose that exosomes transfer miRNAs to the brain, which subsequently promote neuroplasticity and functional recovery after brain injury. For example, functional miRNAs transferred from MSCs to neural cellsviaexosomes promote neurite remodeling and functional recovery of stroke rats (Xin et al., 2012). As a control of exosomes, treatment with liposome mimic consisting of the lipid components of the exosome (no proteins and genetic materials) provides no therapeutic benefit compared with na?ve exosome treatment after TBI (Zhang et al., 2016), indicating that the therapeutic effects of exosomes derive from the exosome content, including proteins and genetic materials, such as miRNAs. Additional research is warranted to determine the role of active miRNAs (master regulators of gene translation) of exosomes in promoting functional recovery and neurovascular remodeling, and regulating neu‐roin fl ammation and peripheral immune response as well as brain growth factors.

Conclusions and Future Perspectives

MSC‐derived exosomes have shown promise in the fi eld of regenerative medicine including treatment of TBI, and 3D MSC culture further enhances generation of exosomes and therapeutic e ff ects. Exosomes play an important role in in‐tercellular communication.e re fi nement of MSC therapy from a cell‐based therapy to cell‐free exosome‐based ther‐apy o ff ers 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 exosomes, which allows a longer shelf‐life and half‐life in patients. As such, exosomes are more amenable to develop‐ment as an “o ff‐the‐shelf” therapeutic agent that can be de‐livered to patients in a timely manner.ey also reduce the safety risks inherent in administering viable cells such as the risk of occlusion in microvasculature or unregulated growth of transplanted cells. Exosome‐based therapy for stroke and TBI does not compromise efficacy associated with using complex therapeutic agents such as MSCs (Xin et al., 2013; Zhang et al., 2015; Kim et al., 2016; Zhang and Chopp, 2016). In contrast to transplantation of exogenous neural stem/pro‐genitor cells, MSC‐derived exosomes that stimulate endog‐enous neural stem/progenitor cells to repair injured brain may have several main advantages including: (1) no ethical issue of embryonic and fetal cells, (2) less invasiveness, (3) low or no immunogenicity, and (4) low or no tumorigenici‐ty. Exosomes are promising therapeutic agents because their complex cargo of proteins and genetic materials has diverse biochemical potential to participate in multiple biochemical and cellular processes, an important attribute in the treat‐ment of complex diseases with multiple secondary injury mechanisms involved, such as TBI. A clinical trial using exosomes from cord blood β‐cell mass for treatment of Type I diabetes mellitus is ongoing (NCT02138331). Devel‐oping a cell‐free exosome‐based therapy for TBI may open up a variety of means to deliver targeted regulatory genes (miRNAs) to enhance multifaceted aspects of neuroplasticity and to amplify neurological recovery, potentially for a variety of neural injuries and neurodegenerative diseases. Further investigation is warranted to take full advantage of regener‐ative potential of cell free MSC‐derived exosomes, including the choice of MSC sources and their culture conditions, as these have been shown to impact the functional properties of the exosomes. In addition to the role of miRNAs, furtherinvestigation of exosome‐associated proteins is warranted to fully appreciate the mechanisms of trophic activities un‐derlying exosome‐induced therapeutic e ff ects in TBI.

The ongoing and next steps for exosome research in the translational regenerative medicine would be to determine the mechanisms (central and peripheral e ff ects) of the exo‐somes underlying improved functional recovery after TBI, maximize generation of exosomes by the MSCs, identify the optimal sources of cells used for generating exosomes and determine potential e ff ects of age and sex of donor cells on exosome generation and contents, re fi ne the isolation proce‐dure for exosomes, de fi ne the optimal dose and therapeutic time window and potential routes of administration, identify the contents of exosomes, modify the content contained in or on exosomes for targeted treatment, develop exosomes as a drug delivery system that can cross the blood‐brain barrier and facilitate drug penetration into the brain, scale up cell manufacturing and exosome preparation by developing and re fi ning 3D culture methods such as sca ff olds, or tissue‐en‐gineered models, cell spheroids, and micro‐carrier cultures, monitor potential adverse e ff ects, and move towards trans‐lating these studies into therapies for TBI and other diseases. Although exosomes provide promising bene fi cial e ff ects in the rodent TBI model, the field of using exosomes as a re‐generative medicine needs to address additional questions, including exactly how exosomes pass the blood‐brain barrier into the brain, how their contents including mRNAs, miR‐NAs, lipids and proteins are transferred from exosomes to parenchymal cells, and which factor(s) play a major role in the treatment of TBI.

Author contributions:

Con fl icts of interest:None declared.

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Ye Xiong, M.D., Ph.D., yxiong1@hfffffbs.org.

10.4103/1673-5374.198966

＊< class="emphasis_italic">Correspondence to: Ye Xiong, M.D., Ph.D., yxiong1@hs.org.

orcid: 0000-0001-9770-6031 (Ye Xiong)