Exosomes are a fascinating area of research in healthcare. Discovered 30 years ago and initially thought to be the means by which cells “eject” waste products and debris, they are now recognized as playing an essential role in intercellular communication and bio-signal transport in nearly all tissues. With the realization of their importance in normal physiology, and the potential therapeutic value they appear to have in disease, the number or scientific articles published about exosomes has grown exponentially since their discovery.
For decades, exosomes were considered extracellular byproducts, but these nano-sized vesicles are now becoming increasingly recognized for their critical role in intercellular communication for both healthy and diseased cell types.
Exosomes have an extraordinary ability to transfer proteins, DNA, mRNA, and non-coding RNAs i.e. miRNA (microRNA).
A rapidly growing body of research suggests that understanding the role of exosomes in cancer will be critical to the future of human health, because exosomes have the potential to be used in cancer prevention, diagnosis, and therapeutic intervention.
WHAT ARE EXOSOMES?
Exosomes are small vesicles excreted from the membranes of many kinds of cells. They are miniscule double-layered lipid “envelopes” that carry cytokines, chemokines, DNA, RNA, miRNA and other proteins from the cells that excrete them to neighboring or distant cells, thereby altering the function of the target cells. They exert close-range paracrine function when affecting nearby cells and have endocrine-like effect on distant cells. We have written about the importance of bio-signals (cytokines and growth factors) in human health and disease for years here on BFT. It’s now time to inform our readers about exosomes – the next “new” thing.
Ranging from 30 to 150 nanometers in size, exosomes are naturally found in nearly all bodily fluids and are crucial for facilitating a range of important cellular functions. In the same way that a cells secretome differs depending on the cell type, the specific contents of exosomes reflect the biological repertoire of the cell from which they are secreted.
DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are well- recognized scientific terms, miRNA not so much. MicroRNA (miRNA) molecules are short sequences of non-coding genetic material that are only 20 or so base pairs (average is 22) in length. This is in comparison to an individual human gene which may consist of 27,000 up to 2 million base pairs. The rules of base pairing (or nucleotide pairing) are fixed: A with T: the purine adenine (A) always pairs with the pyrimidine thymine (T) C with G: the pyrimidine cytosine (C) always pairs with the purine guanine (G).
Families of miRNAs are abundant in many mammalian cell types and appear to target about 60% of the genes of humans and other mammals. Many miRNAs are evolutionarily conserved, meaning they are present in humans and more primitive life forms, implying they have important biological functions. For example, 90 families of miRNAs have been conserved since at least the common ancestor of mammals and fish, and most of these conserved miRNAs have important functions, as shown by studies in which genes for one or more members of a family have been genetically “knocked out” in mice, resulting in significant negative health impacts.
The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Rather than code for the production of a specific protein, which genes generally do, miRNA influences the turning “on” or “off” of a gene, sort of like a control switch. Thus, they are profoundly powerful, even though extremely tiny in size. Because of their innate ability to impact cellular behavior, and additional potential to be “loaded” with drugs or genetic “snippets” that can be smuggled into diseased or malignant cells, they hold great promise as a potent treatment modality.
HOW SMALL ARE EXOSOMES?
Exosome are very small, ranging in diameter from 30 nm on the low end to 150 nm on the high end. That is only 1/1000 the size of an average human cell. A nanometer (nm) is extremely tiny, measuring only one billionth of a meter. For comparison, a human hair, depending on from where it is extracted, varies in size from 40,000 to 60,000 nm. Because exosomes are so small and difficult to visualize, separation of exosomes from similar sized cells and other kinds of vesicles is challenging. Very specialized techniques are required to isolate them.
Isolation of pure populations of exosomes may involve physical techniques based on size and density, as well as techniques that utilize biochemical parameters. More about this below.
The changes induced by exosomes upon interaction with recipient cells can vary widely depending on the type and physiological state of the secreting cell, and can either help ward off disease or, in some cases, exacerbate it.
WHERE ARE EXOSOMES FOUND?
Exosomes are abundant and present in nearly all body fluids, including:
- Blood
- Urine
- Saliva
- Synovial fluid
- Amniotic fluid
- Semen
- Vaginal fluid
- Breast milk
- Serum and plasma from cancer patients
- Cultured medium of cell cultures
- And more
CANCER EXOSOMES AND WHY THEY ARE IMPORTANT RESEARCH SUBJECTS
Exosomes are released in high quantities from rapidly growing cells, including all forms of cancer. It is now documented that cancer exosomes contribute to metastasis (spread to other parts of the body) through intercellular communication. Differences in cancer cell exosomes are being investigated to determine if they may have value in predicting the propensity of a given cancer to metastasize. Exosomes may hold clues as to which cancers must be aggressively treated and which ones may pose less risk, making diligent surveillance a treatment option.
Cancer exosomes are also unique because they are surrounded by protein “ribbons” known as chaperones. While these ribbons can vary in size and shape on the surface of exosomes from different types of rapidly growing cells, they are always present.
Because exosomes exhibit biomarkers specific to the cancerous cells that have released them, exosomes also have potential to be used within theranostic applications. (Theranostics is a field of medicine that provides patient-specific therapy based on findings from targeted diagnostic tests.)
Finally, cancer exosomes have great potential to be used within diagnostic tools and liquid biopsies for non-invasive detection of cancer.
There are three ways in which cancer cell exosomes may prove highly valuable:
- Exosomes facilitate cancer activity, including tumor formation (tumorigenesis), spread (metastasis), blood vessel growth (angiogenesis), and immune evasion
- Cancer exosomes can be used as biomarkers within diagnostic tools
- Cancer exosomes can be used to select targeted treatments for cancer patients based on their unique disease progression
EXOSOMES AS THERAPEUTICS AND CARRIERS OF DRUGS
Increasingly, exosomes are being recognized as potential therapeutics because of their demonstrated ability to elicit potent cellular responses in both cell cultures and animal models. They elicit regenerative outcomes in injury and disease that replicate the observed bioactivity of stem cell populations. Exosomes from bone marrow mesenchymal stem cells were found to activate several signaling pathways important in wound healing and bone fracture repair. They induce the expression of a number of growth factors (hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), nerve growth factor (NGF), and stromal-derived growth factor-1 (SDF1)).
Exosomes have distinct advantages that also position them as highly effective drug carriers. Composed of cellular membranes with multiple adhesive proteins on their surface, exosomes are known to specialize in cell–cell communications and provide an exclusive approach for the delivery of various therapeutic agents to target cells. For example, researchers used exosomes as a vehicle for the delivery of cancer drug paclitaxel. They placed the drug inside exosomes derived from white blood cells, which were then injected into mice with drug-resistant lung cancer. Importantly, incorporation of paclitaxel into exosomes increased cytotoxicity more than 50 times as a result of nearly complete co-localization of airway-delivered exosomes with lung cancer cells. This has huge implications in the development of future cancer treatments!
The first extracellular vesicle (EV) product has been cleared by the FDA to enter clinical trials in humans. In May 2018, the FDA approved the first Investigational New Drug (IND) application for a product clinical trial in severe second degree burn patients. This product utilizes allogeneic bone marrow mesenchymal stem cell (BM-MSC) derived extracellular vesicles.
Epidermolysis bullosa is a genetically inherited malady and one of the first targeted diseases for exosome treatment study.
Another product is intended to address management of epidermolysis bullosa, a group of genetic conditions that cause the skin to be very fragile and to blister easily. Blisters and skin erosions form in response to minor injury or friction, such as rubbing or scratching – a topically applied “cure” would be life-changing!
Because it appears that all cells make exosomes, and all cells can take them up, exosomes may be amenable for use in every therapeutic area.
The graphic illustrates the potential for exosome-based tests and treatments.
Of particular interest to your BFT hosts is the utilization of exosomes derived from our “favorite” stem cell, the bone marrow mesenchymal stem cell. This is already a focus of our laboratory research.
The table below shows the large number of studies studying the effects of microvesicles and exosomes derived from mesenchymal stem cells.
CHALLENGES IN EXOSOME RESEARCH
Despite the growing amount of data on the changes induced by exosomes on target cells, all such studies were performed on vesicles concentrated in vitro i.e. in laboratory equipment, not derived directly from living tissue. In most cases, cultures were held in conditions that minimized cell death to ensure that the analyzed vesicles were not contaminated with random cellular debris spilled out by broken, dying cells. Given this technical challenge, reliable purification of exosomes from whole organs is still impossible. Mechanical or chemical tissue disruption will necessarily break open some cells and make it impossible to separate exosomes naturally present in the extracellular space from vesicles artificially released from broken cells.
Furthermore, distinguishing exosomes from other secreted microvesicles shed by the cellular membrane remains a formidable challenge. Like exosomes, membrane microvesicles also contain various active molecules, such as cytokines, growth factor receptors, and RNAs. Exosomes can be separated from vesicles of different sizes using ultracentrifugation at different speeds, with the larger vesicles concentrating at lower speed than the smaller ones, but similar-size vesicles of different intracellular origins (e.g., exosomes and certain plasma membrane-derived vesicles) are not separated by this method, making it difficult to identify the effects unique to exosomes.
DIFFERENTIAL CENTRIFUGATION SEPARATION
Multiple steps are required to separate and concentrate exosomes, with the easier separated components being removed stepwise leaving the exosomes as the final component at the end of the process.
Because the specific gravity differences are so small among the constituent components of cells, including their secreted exosomes, centrifugation treatments needed for separation of exosomes are long (up to an hour) and huge (100,000 times the force of gravity!)
To aid with separation, the centrifugation process often employs use of viscosity gradients. These are added substances that have specific gravities that match that of the various constituents in the mixture to be separated. The components then “stratify” in layers or zones matched by specific gravity.
ALL CONDITIONED MEDIA CONTAINS EXOSOMES
By now, it should be apparent that the liquid broth (conditioned media) in which cells are cultured in the laboratory must contain exosomes. And it does, albeit not in any concentrated form. The concentration step requires lots of effort and lots of very precise science, exactly what was discussed above. This means that all skincare products that have conditioned media from cell cultures (regardless of type of cell being cultures) as one of their ingredients must therefore contain exosomes, or more accurately, what is left of them after the mixing and heating and agitation and emulsification steps required to make a homogeneous product.
The truth is, no exosome could possibly survive this process intact. Exosome are extremely fragile because miniscule lipid bilayers are not robust structures. In fact, they are exceedingly delicate. We fully understand this and freely admit that the exosomes in our conditioned media are long gone in the final products we currently produce. However, all their powerful contents continue to be present, just not in exosome form.
BUYERS BEWARE!
Marketers are a cunning lot who routinely purloin scientific terms to sell products. Marketing claims are already being made about skincare products that contain exosomes. To be blunt, this is bull crap, except, of course, if they explain that the exosomes were all blown to smithereens during product manufacturing, so maybe they are telling a half-truth.
Typical suspicious language:
“Ours is the only cream in the world infused with exosomes—more than 150 million in every bottle.”
“Each product is uniquely formulated [….] utilizing Exosome delivery.
EXOSOMES CAN BE PROTECTED BUT IT’S NOT EASY
If one wants to produce skincare or any other kind of product containing exosomes (and your BFT hosts freely admit this is a focus of our ongoing research and development), one must treat exosomes with great care. This is possible but it is not easy, and such products will certainly not be one size fits all complex formulations with emulsifiers and lots of other ingredients that are bound to destroy any exosomes within a country mile.
There are ways to properly do this, ways that will protect these tiny yet mighty cell-produced structures. Such products hold great promise to perform near miracles.
We know what these ways are. We’ll discuss them when the time is right.
SELECTED REFERENCES
Concise Review: MSC-Derived Exosomes for Cell-Free Therapy. Stem Cells. 2017 Apr;35(4):851-858.
A Comprehensive Review on Exosomes and Microvesicles as Epigenetic Factors. Curr Stem Cell Res Ther. 2017;12(1):31-36
Exosomes: New players in cancer (Review). Oncol Rep. 2017 Aug;38(2):665-675.
Progress in Exosome Isolation Techniques. Theranostics. 2017 Jan 26;7(3):789-804.
Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics Proteomics Bioinformatics. 2015 Feb;13(1):17-24.
The role of exosomes in cancer metastasis. Semin Cancer Biol. 2017 Jun;44:170-181
Exosomes: Therapy delivery tools and biomarkers of diseases. Pharmacol Ther. 2017 Jun;174:63-78
Exosome biogenesis, bioactivities and functions as new delivery systems of natural compounds. Biotechnol Adv. 2018 Jan – Feb;36(1):328-334.
Role of stem cell derived exosomes in tumor biology.Int J Cancer. 2018 Mar 15;142(6):1086-1092.
Exosomes and their roles in immune regulation and cancer. Semin Cell Dev Biol. 2015 Apr;40:72-81
Exosomes: new molecular targets of diseases. Acta Pharmacol Sin. 2018 Apr;39(4):501-513.
Functions and application of exosomes. Acta Pol Pharm. 2014 Jul-Aug;71(4):537-43.
Exosomes in cancer theranostic: Diamonds in the rough. Cell Adh Migr. 2017 Mar 4;11(2):151-163
Exosomes: biology, therapeutic potential, and emerging role in musculoskeletal repair and regeneration. Ann N Y Acad Sci. 2017 Dec;1410(1):57-67.
Very interesting! Maybe this is jumping the gun, but do you have any vague potential estimates as to when you might release a product using this?
Our laboratory is busy at work perfecting culture, separation and concentration methods that will allow us to develop products based on exosome technology. Because they are delicate, although packed with powerful bio-signaling molecules, products will not resemble our current offerings. Concentrations of exosomes will require novel preservation methods to retain potency. We have some tricks up our sleeves we have confidence will do the job. Stay tuned.
Hi doctors; love reading your blog. Here’s a question:
Could you write a blog commenting the article Stem cell competition orchestrates skin homeostasis and ageing Published: 03 April 2019 on Nature?
https://pubmed.ncbi.nlm.nih.gov/30944469/
True exploration of this topic would require a serious deep dive into the science, HELOX scuba tank and all. Because of our diverse audience, with very few having PhD level knowledge of a given topic, we limit our dives typically to snorkel and mask depths only. For those readers who want to go to bathysphere depths, we suggest you get the article, read it and at least peruse its bibliography if even deeper knowledge is sought.
Stem cell competition orchestrates skin homeostasis and ageing
Here’s the abstract from the Nature April 3, 2019, article:
“Stem cells underlie tissue homeostasis, but their dynamics during ageing—and the relevance of these dynamics to organ ageing—remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention.”
COL17A1 is the official name for collagen type XVII alpha 1 chain. The gene that encodes for this specific collagen protein is critical to production of a structural transmembrane protein that is a structural component of hemidesmonsomes, the multiprotein complexes that mediate adhesion of keratinocytes to the underlying dermal-epidermal basement membrane. Mutations that cause aberrations in this gene are associated with epidermolysis bullosa, a skin condition where minor friction and trauma can cause the epidermis to disrupt from the dermis. The article in Nature makes the case that competition exists among stem cells within the skin, foundationally based on the ability of cells to express higher levels of COL17A1. Cells better at producing it are favorably selected for survival but eventually lose the ability to maintain those level of output. The result in hemidesmosome fragility and stem cell delamination which promotes skin aging.
Hope that helps. Heading back to the surface to get more air.
I wish you guys would develop a product that increases subcutaneous fat (aka filler without the needles). I read about a product in development, TAT4 Gel, that claims to do this but is not yet approved for sale and use. Because of my limited understanding, I Googled “upregulate adipogenesis” and found there may be other substances that increase fat. Exosomes sound awesome! Your very informative article makes me wonder if you might have an exosome product on the way!
We are active and busy in the exosome field and anticipate having exosome-based products eventually. Because the field is young and the science complex and challenging, applications for exosome use are still being explored. Product development will follow identification of appropriate indications, which to be frank, may include therapies above and beyond topical skin application in the “cosmetic” realm. Which indications we target, and the means by which these delicate but powerful bio-signal containing “packets” are best delivered, will determine the regulatory pathway we need to pursue. Stay tuned but also be prepared to be patient.
Keracell produces products that claim to contain exosomes, specifically stating: “Our passage of cells contains naturally occurring tiny nano-lipids (1000 times smaller than cells) called exosomes for efficacious delivery.”
I don’t know what ‘passage of cells’ means. I think they’re referring to the stem cell media? They have a lot of products with stem cell conditioned media as an ingredient, even cleansers. One called Re-Cu-perateTM Serum appears to have ingredients that include exosomes. Do you think exosomes could survive in such a product?
Good question. “Passage of cells” simply refers to the laboratory culture of cells in sequential “generations”. This is necessary because as the cells grow in number, they tend to slow their growth and division as the become “crowded.” Diluting them so they can be re-cultured in multiple flasks where they have more room to grow is called a passage. After several passages, the cells tend to lose their “vigor” and secretory potency, or differentiate from a stem cell into another type of tissue cell. They should be discarded before this occurs.
We’ve written elsewhere on BFT that exosomes are relatively delicate structures, certainly not robust enough to withstand agitation, mixing, and combination with the types of ingredients in cosmetic serums that very likely would destroy their membrane envelope. Exosomes can be preserved in the frozen state, and also if they are freeze-dried, or lypholized. We have done sophisticated analysis of other similar types of serum products, looking for intact exosomes. Nary a one was found. Within a serum, we seriously doubt they could exist. Their interior contents, however, i.e., mRNA, lipids, proteins, growth factor, cytokines, etc. would be dispersed throughout the final product, but not the exosomes per se.