Mesenchymal Stem Cell Exosomes

Where are the legendary exosomes? How do you view "the application of exosomes in clinical medicine"? Today, we invite Mr. Bubble to chat with you!

With the promotion of mesenchymal stem cells (MSCs) culture technology, exosomes, as a high-tech concept product, have rapidly become popular in the cell therapy and cosmeceutical industries. Of course, these applications you have heard are just the tip of the iceberg of exosome applications.

In fact, when it comes to exosomes, they are also well-known, and it can be said that they are also Nobel Prize-winning. The 2013 Nobel Prize in Physiology or Medicine was awarded to American scientists James Rothman, Randy Schekman and German scientist Thomas Zuderhoff for their discovery of the regulation mechanism of vesicle transport in cells. Exosomes are one type of vesicles .

In a word, exosomes are just a bunch of messy things (including miRNA, mRNA, lncRNA, cytokines, and maybe a little DNA), which are then secreted out of the cell. Then enter another cell, unload the car and finish.

Where are exosomes sacred?

Exosomes are a type of extracellular vesicles with a diameter of 30-150nm and a complete membrane structure, which are mainly responsible for the transport of substances and information transmission between cells.

In 1983, exosomes were first discovered in sheep reticulocytes, and in 1987, Johnstone named them "exosomes". Originated from endosomes formed during endocytosis, and finally released from the cell to the outside.

As a means of communication between cells, a variety of cells can secrete exosomes under normal and pathological conditions, such as cell culture fluid, blood, saliva, urine, cerebrospinal fluid and breast milk. It is mainly derived from the multivesicular body formed by the invagination of lysosomal particles in the cell, and is released into the extracellular matrix after the fusion of the outer membrane of the multivesicle and the cell membrane.

Electron microscopy of exosomes is a routine method to identify the shape and size of exosomes, and the electron microscope shows a typical cup-shaped shape. Extracellular vesicles (EVs) are a means of long-distance intercellular communication in the body. At present, it is mainly divided into three categories: exosomes, microvesicles and apoptotic bodies. The main focus of clinical research is on exosomes. Exosomes (30-150nm in diameter, rich in RNA and protein), the larger ones are microvesicles (100-350nm in diameter, rich in metalloproteinases and phosphatidylserine), and the larger ones are apoptotic bodies (500-350nm 1000nm, rich in histones), produced by apoptosis.

Biological effects and isolation methods of exosomes

What are the ways in which exosomes exert their biological effects? There are two main types:

(1) Direct action. Protein molecules or lipid ligands on the surface of exosomes directly activate receptors on the surface of target cells, generate signaling complexes and activate intracellular signaling pathways;

(2) Delivery function. Exosomes can fuse with the plasma membrane of target cells or endocytose directly into cells, and bring their own active molecules such as proteins, nucleic acids, and lipids into cells, thereby regulating cell functions and biological behaviors.

Ultracentrifugation is the standard method for isolating exosomes. Centrifuge at 300G, 2000G, and 10000G at 4°C to remove cells and cell debris in sequence, and then ultracentrifuge at 100000G to obtain exosomes. However, the amount of samples processed by centrifugation is small each time, and repeated high-speed centrifugation will cause exosomes to rupture and affect the quality. Continuous gradient sucrose density gradient centrifugation can be used according to the exosome density . Sucrose is non-toxic to cells, has low viscosity and neutral pH. The obtained exosomes are of high purity, but contain high-density chemicals that are difficult to remove, which is not suitable for clinical use application. Exosomes can be isolated by ultrafiltration using an ultrafiltration membrane with a pore size of less than 100 nm for a short time at low speed, but the pressure applied during ultrafiltration is relatively high, resulting in deformation and rupture of large vesicles.

We can also precisely separate antibodies against exosome-specific proteins such as CD63. In addition, methods based on the precipitation of polymers such as polyethylene glycol ( PEG6000 ) have also been used to isolate exosomes. There are also researchers who cleverly combine microfluidic technology and acoustics to form an acoustofluidic device. First, use lower frequency (19.6 MHz) sound waves to remove larger red blood cells, white blood cells, and platelets in blood; then use higher frequency (39.4 MHz) ) to separate exosomes from extracellular vesicle subcomponents (apoptotic bodies, larger extracellular vesicles, etc.), and collect exosomes at the designated outlet of the channel.

Exosomes for tissue repair and regeneration

Delivery mediated by exosomes has several advantages: 1. They are highly biocompatible and at the same time immunologically inert if they are derived from appropriate cells such as mesenchymal stem cells (MSCs) or immature DCs ; 2. It can cross the thick tissue barrier of the human body, such as the blood-brain barrier (BBB). Normally, exosomes can be directly used as potential therapeutic agents for immunomodulation and immunomodulation.

Although it is said that stem cell exosomes have become a research hotspot may benefit from the vigorous development of stem cell research, stem cell exosomes do have many advantages.

1. Low immunogenicity . Exosomes are less immunogenic than cells. Easier development of "shelf" products.
2. Easy to save . Exosomes can be stored at -20°C for 6 months and -80°C for long-term storage without losing their biochemical activity.
3. stable . Exosomes protect their contents from degradation, thereby preventing the rapid degradation of certain active ingredients.
4. Available for mass production . Exosomes can be enriched in large quantities in the culture medium .
5. controllable . Exosome function can be altered by altering the cellular environment.

MSCs exosomes have immunomodulatory, anti-inflammatory and anti-fibrosis effects, inhibit oxidative stress, and enhance angiogenesis. This mainly depends on the protein and nucleic acid components in it. For example, MSCs exosomes are rich in glycolysis-related enzymes, which can increase ATP production and reduce tissue cell death. MSCs exosomes also contain cytokines such as VEGF, TGF-β1, IL-6, IL-10, and HGF, which are beneficial to angiogenesis and immune regulation. In April 2018, the FDA approved Aegle's first new drug (IND) application for extracellular vesicles to begin clinical trials . The company treats severe skin diseases, including burns and epidermolysis bullosa, by isolating and purifying extracellular vesicles secreted by MSCs.

Exosomes can promote skin wound healing: promote the transformation of local macrophages in the wound from pro-inflammatory M1 to anti-inflammatory M2 to help tissue repair, inhibit myofibroblast differentiation and excessive fibrosis, thereby reducing scar formation. Studies have shown that exosomes derived from human adipose stem cells (ADSCs), which are rich in miRNA-125A and miRNA-31, can significantly promote angiogenesis. Exosomes derived from human amniotic epithelial cells regulate the deposition of extracellular matrix by stimulating the expression of matrix metalloproteinase 1 (MMP1), thereby promoting wound healing. MSCs exosomes can also be used to regulate the function of BMSCs in osteoporosis caused by systemic lupus erythematosus, increase bone density and promote bone regeneration.

miRNA in exosomes can regulate gene expression, and its ratio is higher than that in cells, such as miR-155, let-7f, miR-199a, miR-221, miR-125b-5p and miR-22, etc., involved in many Physiological and pathological processes. For example, miR-125b-5p released from exosomes helps to improve the function of myocardial infarction area.

Exosomes for tumor diagnosis and treatment

Exosomes of tumor cells contain a large number of nucleic acids and proteins associated with tumors, which have important research and application value in the occurrence and early detection of cancer. For example, by using 49 antibodies to detect exosomal proteins from 431 lung cancer patients and 150 healthy individuals, it was found that compared with healthy individuals, CD171 and CD151 were highly expressed in patients, and A marker for lung cancer.

Exosome content as a potential diagnostic marker for tumors (data source: Chinese Journal of Laboratory Medicine)

Parasites can also transfer biologically active substances into host cells by secreting exosomes, regulate host immunity, and mediate immune escape of parasites. For example, Plasmodium, Trypanosoma, Schistosoma and Toxoplasma can all affect host immunity through exosomes.

Dendritic cell exosomes can activate tumor-specific cytotoxic T lymphocyte responses and inhibit tumor growth in vivo. Various clinical trials on the application of dendritic cell exosomes have shown their great potential as a therapeutic method , but the overexpression of PD-L1 on the surface of dendritic cell exosomes limits its efficacy, and the combination therapy based on dendritic cell exosomes , such as the combination with PD-1/PD-L1 neutralizing antibodies, can provide Anti -tumor therapy provides a new way.

How to treat the druggability of exosomes

Due to the advantages of small size, easy crossing of cell membrane and low immunogenicity, exosomes can be used as good carriers of drugs. The structure of exosomes is relatively stable and can cope with adverse physical and chemical environments.

Currently, many phase I/II clinical trials based on exosomes are underway. Exosomes from different sources show different homing abilities in vivo, which may be related to their membrane surface receptors and extracellular matrix binding proteins. Intravenous injection is the most widely used route of administration for exosome-based drug delivery. Intraperitoneal injection, intratumoral injection, oral and nasal administration have also been tried in various disease models. After 24 h of intravenous injection of exosomes, the fluorescence signal was strongest in the liver, followed by spleen, gastrointestinal tract and lung.

Therefore, the distribution of exosomes after injection, as well as the long-term effects and safety, will still need to be further explored and elucidated.

Exosome drug loading can be divided into three types: exogenous drug loading, endogenous drug loading and transgene.

1. Exogenous drug loading method : first isolate the exosomes, and then introduce the drug into the exosomes through transfection or electroporation;
2. Endogenous drug loading method : the drug is first introduced into the cells, and when the drug enters the exosomes and is released from the donor cells, the drug-loaded exosomes are obtained by separation and purification;
3. Transgenic method : Through transgenic methods, the cells express themselves, and finally secrete them out of the cells with exosomes.

The key to the large-scale application and development of exosomes lies in two points: one is to rely on the improvement of cell culture conditions , and 3D culture is one direction. The second is to rely on the improvement of exosome separation and purification methods , and membrane separation is one direction. Mammalian cells release less exosomes, and the yield is not high. Exosomes-mimetic nanovesicles can be extruded through polycarbonate membranes of 10 μm, 5 μm and 1 μm in sequence. Through enrichment, the yield is higher than that of natural exosomes More than 100 times, the yield is high and easy to purify, which provides the possibility for large-scale clinical application of exosomes.

In recent years, the number of research articles on exosomes has developed rapidly, whether it is the extraction and preparation process or as a therapeutic agent and drug , or as a new diagnostic marker for diseases, great progress has been made. Exosomes are expected to be a bridge to people's health.