Exosomes

Understanding Exosomes

Exosomes are lipid-bilayer-enclosed biological nanoparticles ranging in size from 30 to 150 nm, or about 1/1000th the size of the average cell. They are secreted by nearly every cell type and are roving packets of information, each filled with a cargo of potent signaling messenger molecules. They are the major means by which intercellular communication takes place and can be found in nearly every body fluid including plasma, urine, semen, saliva, bronchial fluid, cerebral spinal fluid (CSF), breast milk, serum, amniotic fluid, synovial fluid, tears, lymph, bile, and gastric acid.
Exosomes can fuse with, enter, and affect the behavior of nearby recipient cells, or travel through the bloodstream to influence biologic responses of cells in distant organs. Stem cells are especially prolific producers of exosome.

Focus of Exosome Research

Paracrine signaling occurs when a cell secretes molecular signals that affect nearby cells. Studies have demonstrated that the in-vivo regenerative effects of stem cells are due to paracrine signaling via cytokines and growth factors that promotes tissue repair in the local environment. Exosomes have been shown to play a similar role in this process through the intercellular transmission of protein and nucleic acid components which in turn initiate downstream effects in targeted neighboring cells. Exosomes appear to be a major means by which intercellular communication takes place.

The highly regulated cellular expulsion of exosomes, including the specific composition of their cargo and cell-targeting specificity, are of immense biological interest. They have high potential as non-invasive diagnostic biomarkers as well as therapeutic nanocarriers for targeted drug delivery. As biomarkers, they appear useful in evaluating normal and pathological biologic processes and monitoring the response to therapeutic intervention. Exosomes can thus provide insights on diagnosis, prognosis, regression, or response to disease treatments. They possess the potential dual function to both diagnose and treat human disease.

Mesenchymal stem cells (MSCs) have been the topic of intensive research for their regenerative paracrine effects on wound healing. MSCs derived from human umbilical cord (UC-MSCs) have been shown to facilitate wound repair and upregulate angiogenesis which is important in re-establishing local vasculature following tissue wounding. Exosomes secreted from UC-MSCs have also been shown to facilitate pro-angiogenic paracrine signaling. Thus, regenerative treatment in ischemic tissue environments in various disease models can be therapeutic targets of these types of exosomes. In addition to tissue repair, MSCs from bone marrow have demonstrated potent anti-inflammatory and immunomodulatory effects making them attractive candidates for therapies that reduce tissue inflammation and its negative sequelae. Both umbilical cord and bone marrow mesenchymal stem cells are prolific producers of exosomes. XOStem has extensive experience with both types of MSCs, as well as others.

Exosomes in Health & Disease

Containing cell-of-origin cytoplasmic contents including proteins, mRNAs, miRNAs, lipids and other macromolecules, the exosome cargo has the potential to affect targeted cellular functions in either healthy or pathological ways. Hence, exosomes are intrinsic to normal cellular communication and function, as well as being incriminated in the genesis and metastatic behavior of malignancies.

As essential messenger emissaries functioning throughout the body, they are attractive candidates as possible therapeutic envoys.  For instance, because the blood brain barrier (BBB) prevents penetration of 98% of small molecule drugs, and exosomes have the ability to cross the BBB under inflammatory conditions, it may prove feasible to use exosomes in the treatment of neurological diseases and traumatic conditions. This could have profound implications in treatments for Parkinson’s and Alzheimer’s diseases, and other neurologic maladies including stroke and traumatic injury. Recently published research by members of our XOStem Scientific Team demonstrates the value of mesenchymal stem cell-derived exosomes in treating a mouse model of multiple sclerosis. (see URLs below)

Exosome Research is Rapidly Accelerating

Exosomes were originally observed 50 years ago when they were assumed to be the means by which cells disposed of waste products such as unneeded proteins and excess nucleic acids.  The recognition of the true nature of what we now call exosomes came in 1983, from studies on the loss of transferrin during the maturation of reticulocytes into erythrocytes. In the past decade, interest in exosomes has exploded. There was a  tenfold increase in publications from 2006 to 2015.  The PubMed search term “exosome” returns nearly 10,000 articles for the year 2018. The pace and magnitude of exosome research continues to accelerate rapidly. Nonetheless, despite 20 years of research, the very basics of exosome biology are in their infancy, and we know little of the part they play in normal cellular physiology, or their potential as therapeutic modalities.

Exosome Engineering & Cargo Modification

Using both proprietary and published methods, the authors have demonstrated an ability to modify or engineer the molecular cargo of exosomes derived from UC-MSCs. Furthermore, a diverse spectrum of scientific publications has shown that such engineered and modified exosomes confer augmented paracrine effects,  i.e., more powerful, and more potent downstream impact on targeted cellular behavior. This has important, and potentially revolutionary, impact on the diagnostic, prognostic and therapeutic uses of exosomes discussed above.  Umbilical cord mesenchymal stem cells are obtained from umbilical cords donated by patients at an Orange County, California medical facility. Full pertinent medical safety information is provided on both mother and infant. Bone marrow mesenchymal stem cells are obtained from paid young adult volunteers medically tested and monitored to be free of contagious diseases.

Years of experience in the laboratory culture of bone marrow mesenchymal stem cells (for high science skincare, hair restoration, and vaginal health products) provides team members with valuable knowledge into methods useful in the modification of exosome content. Alterations in oxygen, nitrogen and carbon dioxide concentration, pathway stimulants and inhibitors, external energy delivery using light, electromagnetic waves, ultrasound, etc., have potential use. Nearly a decade of conditioned media bio-signal assays provides a historical database of observed effects, also useful in exosome manipulations.

Exosome Isolation Techniques

Exosomes are tiny, delicate and comprise a very small volume of the conditioned media obtained from laboratory cell culture of MSCs. Sophisticated methods are required to isolate and concentrate them. The most common isolation technique involves the use of ultracentrifugation in which conditioned media is exposed to long duration centrifugal forces in excess of 100,000 times gravity. During centrifugation, pure exosome nanoparticles are separated from the larger extracellular vesicles (>100 nm) found in the media. A major obstacle to developing a commercially feasible and scalable method of exosome isolation using ultracentrifugation is the relatively small starting volumes of conditioned media available for processing under most laboratory circumstances.  The authors are actively exploring a novel isolation technique based on tangential flow filtration (TFF), whereby flowing culture conditioned media passes through a series of filter membranes, resulting in a concentrated exosome isolate. This technique is amenable to smaller scale isolation, making commercialization of exosome technology feasible, regardless of whether its intended purpose is diagnostic or therapeutic. Exosome quality and quantity are preserved.

Application of Exosomes in XOStem Type I Diabetes Research

Exosome research at XOStem is expected to contribute to refinement, standardization, commercialization, and market deployment of a proprietary technology created over the past two-decades by members of our Scientific Advisory Board: an implantable “artificial pancreas” to treat and potentially “cure” Type 1 diabetes for months to years. This device will first be clinically tested in animals, and after adequate proof of safety and efficacy, in humans.

The device, branded STEM-DM®, is made of biocompatible material with chambers into which insulin producing cells are placed. These cells must be continuously supplied with adequate nutrients and oxygen to remain viable. For this reason, the device is surgically implanted subcutaneously for a  period of time prior to being “loaded” with insulin-producing cells. During this period, new tissue, including vascular structures, grow into the device, thereby delivering the nutrients and oxygen required to sustain the transplanted cells, potentially for months or years. Work by XOStem scientists demonstrated exosomes can be of significant benefit in enhancing this process, as well playing important roles in combating inflammation and immunologic attack of transplanted insulin-producing cells.

The insulin producing cells will be human or xenograft islets from swine, immunologically isolated from attack with a proprietary hydrogel protective barrier. Other possible candidate insulin-producing cells include mesenchymal stem cells induced to produce insulin, and even to clump into what is termed “organoids”, morphologically similar to how cells exist as clusters within islets in the pancreas.

Application of Exosomes in XOStem Neuroinflammation Research

Inflammation, nature’s protective and defensive response to trauma and microbial invasion, is ironically a cause and contributor to tissue damage and disease throughout the body. This is especially true in the central nervous system. Many published studies establish the etiologic link between neuroinflammation and CNS maladies including dementias, nerve and motor abnormalities, and chronic traumatic encephalopathy (CTE). XOStem scientists are exploring ways to modify exosomes (“engineered exosomes”) to enhance and harness their intrinsic anti-inflammatory nature to address neuroinflammation. Part of the research effort includes determining the optimal means of administration to provide maximum benefit, including exploring intranasal administration which has demonstrated efficacy as a mean of transmitting molecules into the brain.

Selected References

Moghadasi S, Elveny M, Rahman HS, Suksatan W, Jalil AT, Abdelbasset WK, Yumashev AV, Shariatzadeh S, Motavalli R, Behzad F, Marofi F, Hassanzadeh A, Pathak Y, Jarahian M. A paradigm shift in cell-free approach: the emerging role of MSCs-derived exosomes in regenerative medicine. J Transl Med. 2021 Jul 12;19(1):302. doi: 10.1186/s12967-021-02980-6. PMID: 34253242; PMCID: PMC8273572. https://pubmed.ncbi.nlm.nih.gov/34253242/

Riazifar M, Mohammadi MR, Pone EJ, Yeri A, Lässer C, Segaliny AI, McIntyre LL, Shelke GV, Hutchins E, Hamamoto A, Calle EN, Crescitelli R, Liao W, Pham V, Yin Y, Jayaraman J, Lakey JRT, Walsh CM, Van Keuren-Jensen K, Lotvall J, Zhao W. Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders. ACS Nano. 2019 Jun 25;13(6):6670-6688. doi: 10.1021/acsnano.9b01004. Epub 2019 May 29. PMID: 31117376; PMCID: PMC6880946. https://pubmed.ncbi.nlm.nih.gov/31117376/

Chu DT, Phuong TNT, Tien NLB, et al. An Update on the Progress of Isolation, Culture, Storage, and Clinical Application of Human Bone Marrow Mesenchymal Stem/Stromal Cells. Int J Mol Sci. 2020;21(3):708. Published 2020 Jan 21. doi:10.3390/ijms21030708 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037097/

Mendt M, Rezvani K, Shpall E. Mesenchymal stem cell-derived exosomes for clinical use. Bone Marrow Transplant. 2019 Aug;54(Suppl 2):789-792. doi: 10.1038/s41409-019-0616-z. PMID: 31431712.  https://pubmed.ncbi.nlm.nih.gov/31431712/

Yaghoubi Y, Movassaghpour A, Zamani M, Talebi M, Mehdizadeh A, Yousefi M. Human umbilical cord mesenchymal stem cells derived-exosomes in diseases treatment. Life Sci. 2019 Sep 15;233:116733. doi: 10.1016/j.lfs.2019.116733. Epub 2019 Aug 5. PMID: 31394127. https://pubmed.ncbi.nlm.nih.gov/31394127/

Alatyyat SM, Alasmari HM, Aleid OA, Abdel-Maksoud MS, Elsherbiny N. Umbilical cord stem cells: Background, processing and applications. Tissue Cell. 2020 Aug;65:101351. doi: 10.1016/j.tice.2020.101351. Epub 2020 Mar 19. PMID: 32746993. https://pubmed.ncbi.nlm.nih.gov/32746993/