Another look

Exosomes: an innovation in aesthetic medicine

Sarah Berndt, PhD

Doctor of Biomedical and Pharmaceutical Sciences
Head of Cellular Therapy Research at Regen Lab SA (Switzerland)

Introduction

Exosomes are small round extracellular vesicles produced by almost all cell types and present in all biological fluids. They constitute a privileged means of communication between the different cells of the organism.

Each exosome can contain many constituents of a cell such as cytosolic and cell surface proteins, lipids, DNA or RNA and metabolites. At its destination, the exosome will fuse with the membrane of the target cell and its contents will be integrated.

Vesicles are very specific, since the nature of the cell that produces this exosome influences its physicochemical properties (Figure 1).

The physiological role of exosomes is thought to be the maintenance of cellular homeostasis by regulating excess or unnecessary protein constituents in cells [1].

Exosomes are now being evaluated as

biomarkers of different pathophysiological states
or as potential vehicles for drug delivery [2] such as
- short interfering RNAs,
- antisense oligonucleotides,
- chemotherapeutic agents
- and immune modulators
- with the main advantage of targeted delivery to the desired cell.

Figure 1: Schematic illustration of exosome biogenesis and composition.

Exosomes and microvesicles are invaginations of the plasma membrane that belong to the family of extracellular vesicles.
The composition of exosomes includes lipids, cytosolic proteins, DNA, RNA and surface membrane proteins.

Exosomes are produced by different cell types and are being studied for their diagnostic and therapeutic potential.

Among the main physiological functions of exosomes, research has highlighted roles in

cell survival,
proliferation,
migration,
differentiation,
senescence,
immunomodulation,
angiogenesis,
healing,
neoplasia, among others [1].

Given their difference in size, cargo cell of origin, and distinct combination, exosomes are known to be highly heterogeneous in structure and function [1] (Figure 2).

Figure 2: Exosomes are highly variable in size, content, function and source. Extracted from [1].

Currently, 9,769 proteins, 2,838 miRNAs, 3,408 mRNAs and 1,116 lipids have been described in their composition [3].

Exosomal proteins are different depending on the nature of the primitive cells or tissues [4].

The main exosomal proteins are proteins:

transport and membrane fusion,
chaperones,
adhesion molecules,
of the MHCs,
of the cytoskeleton proteins
and lipid-bound proteins [5].

Cells produce specific exosomes with unique characteristics and content.

Exosomes from cancer cells are likely to make the cancer worse,
while exosomes produced by stem cells are likely to promote anti-inflammatory and regenerative activity like their cell of origin [5].

Methods for exosome isolation

Extracellular vesicles, found in all biological fluids, are a family that includes:

exosomes (40 nm to 160 nm) from endosomes/multivesicular bodies
and microvesicles (150 nm to 1000 nm) from the plasma membrane [6].

Exosomes are now considered by many biotechnology companies as emerging vectors [7].

A wide range of methods for the isolation of exosomes from biological fluids has been developed [8].

Exosomes are isolated based on size, density and immunoaffinity by the following methods:

centrifugation,
chromatography,
filtration,
polymer-based precipitation
and immunological separation (see Table 1) [9]..

Ultracentrifugation is now considered the gold standard and many research groups use this technique [10].
However, ultracentrifugation has several drawbacks, including the need for a large volume of biological fluids, long processing times, and poor reproducibility [11].

Methods of exosome production, isolation, and purification have significant effects on yield, viability, and function (Table 1).

Exosomes from different specimens may have different protein/lipid and luminal contents and different sedimentation characteristics.

For example, exosomes from adipose tissue have a high lipid content and require adaptation of their isolation methods [12].
If exosomes are to be isolated from culture media, it is very important to use serum-free media or fetal bovine serum without exosomes.

Table 1. Methods of exosome isolation (after Chesmi et al. 2020 [13]).

Exosomes in diagnosis

Exosomes are key players in intracellular communications and therefore have the ability to determine disease progression. They therefore have great potential as diagnostic tools and biomarkers for many diseases [2].

The content of exosomes is modified during active disease, whether it is

of cancer,
cardiovascular disease,
of immunological diseases,
infections or CNS pathologies (Alzheimer and Parkinson).
The more an inflammatory process is underway, the more exosomes are exuded by the cells [14].

Monitoring of exosomes secreted by cancer cells can be done by liquid biopsy, as all body fluids contain exosomes released by cancer [15].

Exosomes can protect the rapid degradation of nucleic acids and remain very stable in plasma [16].
There is growing evidence that this technique can be used to predict the existence of a tumor as well as the state of invasion and metastasis.
Exosomal membrane markers can be used to subtype exosomes to track the cell of origin.

The exosome diagnostics market is in the early stages of development. Technical advances and a better understanding of exosome biology must be made before this technique can be used in routine diagnostic tests.

Exosomes as therapeutics

Exosomes are powerful communicators in the body. Some suggest that almost all stem cell activity is a result of the exosomes they produce [17]. Many ongoing studies aim to decipher the role of exosomes in tissue regeneration [5].

Recently, there has been increasing interest in using endogenous stem cells to repair tissue damage.

Mesenchymal stem cells (MSCs) have been extensively studied because of their differentiation potential, immunosuppressive effects and relative ease of culture.
These effects are known as the paracrine activity of MSCs, which helps to mediate them [18].
Many in vivo studies have demonstrated the therapeutic effects of MSC-derived exosomes [19].
Three main tissues are considered when harvesting stem cells with MSC potential: bone marrow, adipose tissue and placenta.

MSC-derived exosomes can act as a follow-up in skin wound healing (Figure 3) :

Reduction of inflammation (due to a reduction in pro-inflammatory T cells and an increase in the number of anti-inflammatory cells (T cells and macrophages) [20] ;
A regenerative action highlighted by the proliferation of fibroblasts and keratinocytes in the wound [21] ;
A reduction in cell death and anti-apoptotic activity[22] ;
Regulation of angiogenesis by direct activation of endothelial cells [23].

A systematic review evaluated more than 200 studies with MSC-derived exosomes, demonstrating benefit in 72% of the studies [24].

Exosomes have various advantages over cell therapy:

- exosomes are more stable and storable [5] ;

- exosomes can be loaded with therapeutic agents with targeted action on the site of interest[25] ;

- Stem cell-derived exosomes are less immunogenic than cell-based therapies[26] ;

- Stem cell therapy requires a delicate processing technology; exosomes are not cells and can be frozen or freeze-dried without loss of biochemical activity[27] ;

- stem cells can undergo neoplastic transformation whereas exosomes do not have this ability [28].

Figure 3. Biological activities of stem cell-derived exosomes on skin wound healing. From Lv et al [29].

Exosomes can limit inflammation by acting on inflammatory cells and macrophages.
Exosomes can trigger re-epithelialization by increasing the activities of fibroblasts and keratinocytes.
Exosomes can accelerate angiogenesis through direct activation of endothelial cells.
Exosomes can modulate tissue remodeling via collagen production and myofibroblast differentiation.

Technical and regulatory requirements for clinical use of exosomes

Theoretically, as an immunocompatible cell-free therapy, exosome technology should offer regulatory advantages over stem cell therapies in terms of process and manufacturing. However, to date, no exosome-based product is currently approved.

The FDA and other regulatory authorities around the world require extensive controls for quality, purity, potency and reproducibility.
The protocol for optimizing the purification method is not trivial while exosomes contain an unknown amount of exosomes, and are also contaminated with proteins, cell free DNA, vesicles, potentially viruses and other components of the same size range.
Exosomes can accumulate in various organs and cross the blood-brain barrier [30].
In addition, exosomes can lose or gain function during the isolation process, which can be potentially dangerous for the patient.

Mesenchymal stem cell (MSC) therapy has attracted scientific interest after the discovery of its therapeutic potential. However, their clinical use has been hampered due to their immunogenicity and tumorigenicity.

Relatively recently, it has been revealed that the mechanism by which MSCs promote healing is the secretion of exosomes.
This has sparked interest in the development of cell-free therapy, thus avoiding the obstacles that have prevented the application of MSC therapy in clinical practice.

Figure 4: Biological and technical limitations for the use of exosomes in clinic. From [26].

Origin of exosomes affects its biological features (immunity, toxicity, and therapeutic properties) as they are the same as the parental cells.

Isolation procedure (i.e. centrifugation) may damage the exosome membrane that may be damagable to patients and are difficult to predict.
Environmental culture conditions is affecting the quality and quantity of exosomes production. Injection technique is directly affecting the exosome biodistribution and pharmacokinetics.
Intravenous injection bears the risk of exosome phagocytosis but led to a better organ accumulation than after intramuscular or subcutaneous injection.

Status of fundamental research

Up to now, efficacy and safety are not already established in human trials. Exosomes in clinical trials need to comply with good manufacturing practice (GMP).

Three important issues are prevalent in GMP for exosomes, i.e., upstream of cell cultivation process, downstream of the purification process, and exosome quality control [8].

Kwon and colleagues were the first to propose exosomes as a clinical tool with presenting human clinical data in atrophic acne scars [31].
MSC-derived exosomes were topically applied after carbon dioxide laser treatment. No adverse effects were associated with exosome application. The treated patients displayed sooner and better improvement in atrophic acne scarring. MSC-derived exosomes used were hypo immunogenic and negative for human leukocyte antigen.
Early preclinical studies in aesthetics have demonstrated promising effects of exosomes on wound healing for skin rejuvenation and hair growth in in vitro and murine models (Table 2 and Table 3).
Despite this, only 1 clinical study has been published to date, and there are no FDA-approved products on the market [32].

Conclusion

Numerous clinical trials have investigated stem cells and their conditioned media (CM) for aesthetic purposes [31,37-42].

The conditioned medium of stem cells is of interest for preclinical studies because it contains paracrine mediators, including exosomes, which exert regenerative activities similar to those of the stem cells themselves.

This is an interesting free alternative to stem cell therapy.

In the field of aesthetics, the exosomes used are most often derived from adipose MSCs [43].
Purified exosomes have been used successfully in preclinical studies, but clinical trials in aesthetics are lacking [32].
Further animal and human studies on the safety and efficacy of exosomes are needed before exosome therapy becomes a reality.

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Lm3. Another look. Exosomes: an innovation in aesthetic medicine. Dr. Sarah Berndt Doctor in Biomedical and Pharmaceutical Sciences