What is the Therapeutic Potential of Stem Cells?

Stem cells possess an extraordinary ability to self-renew and generate specialized cells, such as embryonic stem cells (ESC), induced pluripotent stem cells(iPSC), and a host of differentiated cells. Their potential to differentiate into various tissue cells makes them highly promising for regenerative medicine applications. Despite numerous registered stem cell therapy-related clinical trials, only a limited number of these therapies have been successfully translated into clinical practice. Currently, the only FDA-approved stem cell therapy is the transplantation of hematopoietic progenitor cells for hematopoietic and immunological reconstitution in patients with hematopoietic disorders. However, there have been approvals for specific clinical uses of stem cell products derived from mesenchymal stem cells or tissue-specific stem cells, such as Prochymal for acute graft-versus-host disease and Holoclar for corneal injury repair. The challenges in translating stem cell therapies to clinical applications include issues like the size of stem cells causing lung accumulation and infusion toxicity, immune responses triggered by allogeneic stem cells, and the potential for oncological complications. Additionally, the therapeutic effects of stem cells may be attributed to their paracrine action rather than the engraftment of transplanted stem cells.

Stem cell therapies are currently being investigated for the treatment of various diseases, such as hematological, immune, neurodegenerative conditions, and tissue injuries. An alternative approach involves using stem cell-Exo(stem cell-Exo), which offers similar clinical benefits without the concerns associated with the transplantation of living cells.

Unlocking the Power of Exosomes: A Cell-Free Alternative to Stem Cells?

Exosomes, which are membrane-bound vesicles released by cells, including stem cells, have gained attention for their therapeutic potential. Exosomes are nanovesicles with sizes ranging from 30 to 150 nm and are formed through a specific biogenesis pathway. They carry various cargo, including nucleic acids, lipids, and proteins, which play essential roles in cell signaling and regulation. The composition of exosomes depends on the donor cell type and the physiological conditions. Exosomes are recognized by recipient cells through adhesion factors and can be absorbed through different pathways depending on the target cell's proximity. The contents of exosomes, particularly nucleic acids, contribute to their functional activities. Various proteins, lipids, and markers, such as tetraspanins and heat shock proteins, are associated with exosomes and serve as specific identifiers. The lipid bilayer membrane surrounding exosomes protects them.

The composition of exosomes varies depending on the donor cell type, but several conserved proteins serve as specific markers, including the tetraspanin family (CD9, CD63, and CD81), integrins, antigen presentation molecules (MHC), HSP70, HSP90, Alix, TSG101, and GTPases. Exosomes are protected by a lipid bilayer membrane, which contains cholesterol, sphingomyelin, ceramide, and lipid rafts that reflect the characteristics of the cell source. Exosomes are stable and can be stored at low temperatures without losing functionality.

Exosomes have been found to possess immunomodulatory potential and play a role in intercellular communication. They carry cytokines with antimicrobial properties and signaling molecules involved in the innate immune response to viral and bacterial infections. The composition of exosomes is influenced by the donor cell source, epigenetic changes, and physiological or pathological microenvironments. Due to their ability to maintain homeostasis and transfer molecules between cells, exosomes have gained significant attention in medical research, particularly in the last two decades, with a focus on their therapeutic applications.

Among different types of exosomes, Mesenchymal Stem Cell-derived Exosomes (MSC-Exo) have received considerable attention due to their immunomodulatory, regenerative, and anti-inflammatory functions. MSC-Exo plays roles in various cellular processes and has beneficial effects similar to those of their parent MSCs. They have been shown to stimulate angiogenesis, suppress fibrosis, increase neuronal survival and differentiation, induce extracellular matrix remodeling, inhibit local inflammation responses, and regulate immune cell activities. MSC-Exo has demonstrated therapeutic potential in animal models of cardiovascular, kidney, liver, lung, and neurodegenerative diseases. They also possess antimicrobial properties and have shown promise in the treatment of microbial infections.

There are currently over 150 clinical trials investigating exosome-based therapies for diseases like respiratory diseases, infectious diseases, and cancer. Stem cell-Exo, particularly those derived from mesenchymal stem cells, are being explored as an alternative to traditional stem cell therapy. Preclinical and clinical studies suggest that these exosomes may replicate some of the therapeutic effects of their donor cells and offer advantages over stem cell therapy, including their small size, stability, and ability to be administered through different routes. The engineering and modification of exosomes' membrane proteins further enhance their potential for therapeutic applications, such as using lung stem cell-Exo as an inhalable COVID-19 vaccine. The field of engineered exosome therapy is evolving and may open new avenues for therapeutic applications.

stem cell-Exo is particularly promising due to its immunomodulatory, regenerative, anti-inflammatory, and anti-microbial properties. Stem cell-Exo offers advantages over parent cells, such as their small size, stability, low immunogenicity, absence of tumorigenicity, and ease of storage. Recent research has explored the use of stem cell-Exo in treating microbial diseases caused by bacteria, viruses, fungi, and parasites. Although most studies are currently in the preclinical stage, there is ongoing progress toward clinical applications. However, challenges remain in translating stem cell-Exo therapy from the laboratory to clinical settings, including safety concerns and the need for standardized isolation methods and characterization of exosomes.

Stem cell-Exo, such as MSC-Exo, offers advantages over stem cell therapies, including their stability, small size, low immunogenicity, ease of storage, and simpler delivery procedures. They mimic the beneficial effects of parent stem cells without safety concerns associated with cell transplantation. Stem cell-Exo can be manipulated to load therapeutic compounds and transfer RNA molecules, enhancing their efficacy. They have been investigated as drug delivery vehicles, including for antibiotics. However, there are challenges to overcome, such as the lack of standardized isolation and purification methods for exosomes, incomplete understanding of their cargo, and the heterogeneity of released vesicles due to changes in the extracellular environment. Further research is needed to fully explore and harness the potential of stem cell-Exo for therapeutic applications.

What Are the Market Prospects and Challenges for the Clinical Translation of Stem Cell-Exosome Therapy?

The market prospects for both stem cell and exosome therapy are promising, with the global stem cell market projected to reach US$31.6 billion by 2030 and the global exosome market anticipated to reach 1.03 billion by 2030. Regulatory standards and ethical issues regarding stem cell research have been addressed through guidelines issued by organizations like the NIH and international associations. However, there is still a need for comprehensive standards and regulations specific to stem cell and exosome therapy. The establishment of good manufacturing practice (GMP) facilities for large-scale manufacturing of stem cells and exosomes is crucial for their clinical translation. While GMP facilities for cell therapy products like HPC transplantation and chimeric antigen receptor (CAR)-T cells exist, GMP facilities for exosomes are limited. Alternative approaches, such as bioreactor systems and hollow-fiber membranes, are being explored for large-scale exosome production. Quality control measures are also necessary to ensure the batch-to-batch consistency of exosomes. Advanced instruments like ZetaView, Amnis, ImageStream, and ONI Nanoimager are used for the accurate characterization of exosomes. Standardized operating procedures and a streamlined closed operation system should be established for large-scale manufacturing and quality control of exosomes. Additionally, the therapeutic potential of stem cell-Exo in microbial diseases is being investigated, offering a potential alternative to traditional treatments with antibiotics and chemical compounds.

How Can The Manufacturing Challenges for Large-Scale Production of Exosomes Be Overcome?

Developing standardized protocols for isolating and purifying exosomes is paramount for large-scale production. Optimization and validation of isolation techniques specific to different cell types or body fluids are crucial to ensure reproducibility and consistency. By establishing standardized methods, researchers and manufacturers can achieve greater uniformity in exosome production. Efficient large-scale production of exosomes heavily relies on optimizing the culture conditions of donor cells. Culturing cells in large quantities and under optimal conditions is essential for achieving high exosome yields. This may involve fine-tuning cell culture media, growth factors, and oxygen levels to promote robust exosome production. By systematically optimizing cell culture parameters, researchers can maximize the output of exosomes. The implementation of bioreactor systems offers a promising solution for scaling up exosome production. Bioreactors provide a controlled environment for the expansion of cell cultures, allowing for better monitoring and adjustment of various culture parameters. Utilizing bioreactors enables researchers to enhance the scalability of exosome production and meet the demands of large-scale manufacturing. Robust quality control measures are vital to ensure the purity, integrity, and functionality of exosomes produced on a large scale. Implementing rigorous characterization techniques, such as nanoparticle tracking analysis, electron microscopy, protein profiling, and functional assays, allows researchers to assess the quality of exosome preparations. These measures help ensure that the produced exosomes meet the required standards for safety and efficacy. Long-term storage of exosomes without compromising their functionality is crucial for large-scale production. Developing optimized cryopreservation protocols enables exosomes to be stored for extended periods without loss of efficacy. Selecting suitable cryoprotectants, optimizing freezing and thawing processes, and validating the stability of exosomes during storage contribute to their long-term preservation and ease of distribution. The introduction of automation and process optimization plays a significant role in enhancing the efficiency and reproducibility of large-scale exosome production. Automating cell culture, isolation, purification, and characterization processes reduces human error and increases throughput. By implementing automated systems, researchers can streamline production workflows and achieve higher levels of scalability. To address the challenges of large-scale exosome production comprehensively, collaboration among researchers, industry experts, and regulatory bodies is essential. Sharing knowledge, experiences, and best practices accelerates progress in the field and fosters the development of standardized protocols and guidelines. Collaborative efforts promote consistency, reliability, and regulatory compliance in large-scale exosome production.

How Are Exosomes Characterized to Ensure Quality?

Physicochemical Characterization Techniques:

1. Atomic Force Microscopy (AFM): AFM is a powerful technique for studying exosomes at the nanoscale. It detects and records interactions between a probing tip and the sample surface, enabling the characterization of exosome abundance, morphology, biomechanics, and biomolecular makeup. AFM provides sub-nanometer accuracy for measuring out-of-plane dimensions of nano-objects. It allows quantification and simultaneous probing of the structure, biomechanics, and biomolecular content of individual exosomes. AFM has contributed significantly to our understanding of exosomes' structural and functional properties.
2. Dynamic Light Scattering (DLS): DLS, also known as photon correlation spectroscopy, measures the size of exosomes by analyzing time-dependent fluctuations in scattering intensity caused by their Brownian motion. While DLS is a simple and widely used technique, it cannot visualize individual particles. It is most suitable for analyzing monodispersed suspensions and may face challenges when larger vesicles are present in the sample. DLS provides information on the diameter range of analyzed vesicles but does not offer biochemical data or cellular origin information.
3. Flow Cytometry: Flow cytometry is a molecular approach used to characterize exosomal surface proteins and measure the size and structure of exosomes. Conventional flow cytometers have limitations in detecting smaller particles, but high-end flow cytometers with advanced detection capabilities enable the detection of exosomes smaller than 300 nm. Flow cytometry allows the analysis of the cellular origin, size, and antigen expression levels of individual exosomes
4. Nanoparticle Tracking Analysis (NTA): NTA is an optical particle tracking method that measures the concentration, size distribution, and movement of exosomes in liquid suspension. By tracking the Brownian motion of individual nanoparticles, NTA provides valuable information on exosome size, size distribution, concentration, and phenotype. NTA can detect a wide range of exosome sizes, including those as small as 30 nm, making it a versatile technique for exosome characterization. Its quick sample preparation, fast measurement time, and ability to recover samples in their native form make NTA attractive for routine analysis.
5. Resistive Pulse Sensing (TRPS): TRPS is a technique used to measure the size distribution and concentration of exosomes and colloidal particles. It enables non-subjective characterization on a particle-by-particle basis and has been successfully employed for various nanoparticle suspensions. However, TRPS measurements can be affected by system stability and sensitivity issues. Optimization of system parameters can enhance sensitivity and stability. TRPS has been extensively utilized to measure the size distributions of exosomes designed for therapeutic applications.
6. Transmission Electron Microscopy (TEM): TEM is widely used for visualizing the structure, morphology, and size of biological components, including exosomes. It involves passing a beam of electrons through a sample to create images. TEM requires extensive sample preparation, including fixation and dehydration, which may alter the morphology of exosomes. Cryo-electron microscopy (cryo-EM) offers an alternative, preserving samples in a near-native state. TEM and cryo-EM provide valuable insights into exosome morphology and size, but cryo-EM is preferred for avoiding dehydration artifacts.

Compared to stem cell therapy, exosome therapy offers several advantages. Firstly, exosomes have a small size, ranging from 40 to 160 nm, which facilitates their delivery to target tissues and cells. This small size also minimizes the risk of immune responses and tumor formation, addressing concerns associated with stem cell transplantation. Additionally, exosomes are stable and can be stored long-term, either through freezing or lyophilization, allowing for convenient transportation and storage. This stability further enables sterilization by filtration, ensuring safety during administration.

Furthermore, exosomes offer versatility in terms of administration routes. For example, lung stem cell-Exo can be nebulized or lyophilized for inhalation, providing a potential treatment option for lung diseases. Exosomes can also be engineered and modified to display specific molecules or carry drug cargo, expanding their therapeutic potential. Recent advancements include the engineering of exosomes as inhalable COVID-19 vaccines, demonstrating the flexibility and adaptability of exosome-based therapies. Clinical trials, such as the exoSTING therapy for solid tumors, indicate the ongoing exploration of engineering exosomes for therapeutic applications.

Clinical Trials and Therapeutic Applications: ClinicalTrials.gov lists over 150 registered clinical trials investigating exosome-based therapies for various diseases, including respiratory diseases, infectious diseases, and cancer. Of these trials, 31 utilize exosomes derived from stem cells, predominantly MSCs from different tissues. Stem cell-Exo is being explored as an alternative to stem cell therapy, aiming to recapitulate the therapeutic effects of donor cells without the associated drawbacks.

The clinical investigations and preclinical studies indicate the potential of stem cell-Exo in various therapeutic applications. By harnessing the therapeutic cargo of exosomes, these studies aim to address the limitations of traditional stem cell therapies, such as oncological complications, immunogenicity, and ethical concerns. Stem cell-Exo offers a promising avenue for cell-free therapy, providing a safer and more accessible treatment option.

While exosome therapy shows great promise, several challenges need to be addressed for its widespread adoption. Manufacturing and purification processes for exosomes are difficult to upscale, leading to batch-to-batch variation. Further research and development efforts are required to optimize manufacturing processes and establish standardized protocols.

ESCRT Machinery: Orchestrating ILV Formation and Cargo Sorting Central to exosome biogenesis is the endosomal sorting complex required for transport (ESCRT) machinery. Comprising ESCRT-0, -I, -II, -III, and Vps4 complexes, ESCRT regulates membrane remodeling, cargo sorting, and ILV formation within MVBs. Ubiquitinated cargoes are recognized and sorted by ESCRT-0, which interacts with phosphatidylinositol-3-phosphate (PI3P)-enriched endosomal compartments. ESCRT-0 recruits ESCRT-I, initiating the inward budding of endosomal membranes. ESCRT-II further promotes ILV budding around clusters of ubiquitinated proteins. The ESCRT-III subunit, CHMP6, binds to ESCRT-II and recruits CHMP4, resulting in ILV pouch formation. Finally, Vps4 catalyzes ATP-dependent ESCRT-III disassembly, leading to the release of ILVs.

Complex Lipids: Shaping Membrane Curvature and Cargo Sorting Complex lipids, such as ceramide and sphingomyelin, contribute to the initial curvature of membranes during ILV formation. Sphingomyelinase, an enzyme involved in ceramide production, plays a crucial role in exosome secretion. Inhibition or loss of sphingomyelinase impairs exosome release and cargo loading. Ceramide's self-association into raft-like structures influences membrane curvature, highlighting the importance of lipid-mediated pathways in exosome biogenesis.

Tetraspanins: Scaffold Proteins Guiding Cargo Sorting and Membrane Organization Tetraspanins, highly conserved membrane integral proteins, are abundant in exosomes and play multifaceted roles in exosome biogenesis. CD9, CD63, and CD81, among others, act as scaffolds for protein anchoring and contribute to cargo sorting. Tetraspanin interactions with proteins such as syndecan and metalloproteinase influence ILV formation and cargo loading. Additionally, tetraspanins regulate the incorporation and secretion of other tetraspanins into exosomal membranes, further shaping exosome composition and function.

Rab GTPases: Orchestrators of Intracellular Vesicle Trafficking Rab GTPases, a family of proteins involved in intracellular vesicle transport, have emerged as key regulators of exosome secretion. Different Rab GTPases, including Rab27a, Rab27b, Rab11, Rab35, Rab7, and Rab2b, exert diverse effects on exosome secretion and cargo sorting. Their interactions with SNARE proteins facilitate exosome secretion and regulate MVB trafficking to lysosomes or plasma membranes. Rab GTPases contribute to the dynamic nature of exosome biogenesis and play cell-type-specific roles.

Autophagy-Related Proteins: Linking Exosome Biogenesis and Cellular Waste Management Certain autophagy-related proteins, including Atg5 and Atg16L1, have been implicated in exosome biogenesis. They participate in autophagy processes and contribute to the regulation of exosome secretion in specific cellular contexts. The interplay between autophagy and exosome biogenesis highlights the interconnectedness of cellular pathways and their impact on exosome formation.

Biodistribution and Routes of Administration: Exosomes exhibit diverse biodistribution patterns depending on the route of administration. Studies using both heterologous exosomes and those derived from biological fluids have shown distribution to multiple organs, including the liver, lung, kidney, pancreas, spleen, ovaries, colon, and brain. Intravenous administration predominantly results in sequestration in the liver, spleen, lungs, and gastrointestinal tract, while intratumoral injection allows for longer exosome detection in tumors. Intranasal administration favors delivery to the brain. Macrophages are commonly involved in exosome uptake in most tissues, while endothelial cells preferentially mediate uptake in the lungs. Additionally, exosome size influences transport and biodistribution, with larger extracellular vesicles preferentially accumulating in bones, lymph nodes, and the liver.

Specific Targeting Mechanisms: While non-specific uptake occurs across all cell types, specific targeting is crucial for delivering exosome content and exerting their function. The surface composition of exosomes plays a vital role in mediating targeting. For instance, incorporating cell-specific proteins like the rabies viral glycoprotein (RVG), which interacts with acetylcholine receptors, enables exosome delivery to the brain. The conservation of tropism between donor and recipient cells further contributes to exosome targeting. Cellular signatures present in secreted exosomes act as recognition motifs for uptake by the same recipient cell types. Cancer cells employ specific glycoproteins enriched with mannose and sialic acid on exosome surfaces to target recipient cells, such as ovarian cancer cells. Integrins, such as α6β4 and α6β1, are involved in lung and liver metastasis targeting, respectively. Different exosomes released by neuroblastoma cells target distinct neuronal compartments. The presence of certain receptors, such as CD47, facilitates evasion from the host immune system during circulation. Complex lipids, such as phosphatidylethanolamine and sphingomyelin, influence exosome targeting in cancer cells and other cell types like dendritic cells. Additionally, exosome composition, cell origin, and route of administration are all influential factors in determining exosome biodistribution.

When exosomes reach the target cell, they can initiate signaling through two main mechanisms: direct interaction with extracellular receptors or internalization by the recipient cell. In the direct interaction mode, transmembrane ligands present on the exosome surface bind directly to surface receptors on the recipient cell. This binding triggers downstream signaling cascades, leading to the activation of the target cell. This direct interaction route is commonly used to mediate immunomodulatory and apoptotic functions. For example, exosomes released from dendritic cells can activate T lymphocytes by binding to MHC-peptide complexes. Additionally, exosomes can bind Toll-like receptor ligands on bacterial surfaces to activate bystander dendritic cells and enhance immune responses. Moreover, exosomes expressing tumor antigens stimulate T cell proliferation and antitumor activity by interacting with specific receptors on T cells. Furthermore, ligands such as tumor necrosis factor (TNF), Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL) expressed on exosome surfaces can bind to TNF receptors on tumor cells, triggering caspase activation and apoptosis.

Exosomes can also fuse with the plasma membrane of the target cell and release their contents directly into the cytosol. This fusion process involves the formation of hemi-fusion stalks between the exosome's hydrophobic lipid bilayers and the plasma membrane, followed by expansion to form a continuous structure. The fusion process is mediated by families of proteins called SNAREs and Rab proteins. Additionally, lipid raft-like domains, integrins, and adhesion molecules present on the exosome surface facilitate interaction, attachment, and fusion with the target cell. By incorporating lipophilic dyes, such as octadecyl rhodamine B, into the exosome bilayer, researchers can distinguish fusion from endocytosis. This fusion process has been observed in dendritic and tumor cells. Although the evidence supporting this mechanism is limited, some authors speculate that the low pH of the tumor microenvironment and changes in lipid composition may facilitate exosome fusion with tumor cells.

The most common mode of exosome internalization is through endocytosis, a process by which cells engulf extracellular particles. Clathrin-mediated endocytosis is a well-characterized mechanism involving the assembly of transmembrane receptors and ligands into clathrin-coated vesicles. After internalization, the vesicles undergo uncoating and fuse with endosomes. This mode of exosome entry occurs in various cell types and is dependent on factors essential for clathrin-mediated endocytosis. Another endocytic mechanism involves lipid raft-associated membrane invagination, which influences exosome uptake by shifting cargo into early endosomes. Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol (GPI)-anchored proteins. Disrupting lipid rafts or inhibiting sphingolipid synthesis can impair exosome uptake. Caveolin-dependent endocytosis, mediated by integral membrane proteins called caveolins, is another potential mechanism for exosome uptake. However, there are conflicting reports on the role of caveolin-dependent endocytosis in exosome uptake, with evidence suggesting both positive and negative regulation depending on the cell type and exosome composition.

Phagocytosis, a process involving the engulfment of large particles, can also internalize small particles like exosomes. This pathway relies on cell membrane deformations that encircle extracellular particles, forming phagosomes. Phosphatidylinositol-3-kinase (PI3K) and phospholipase C (PLC) enzymes are involved in phagosome closure. Immune cells, such as macrophages and dendritic cells, predominantly use this route, depending on PI3K and actin cytoskeleton activity.

Macropinocytosis, on the other hand, utilizes actin-driven membrane invagination to form intracellular compartments called macropinosomes. These compartments enable the non-specific uptake of soluble molecules, nutrients, and antigens. Macropinocytosis relies on cholesterol-mediated Rac1 GTPase recruitment, Na+/H+ exchanger function, and, in some cases, dynamin. Exosome uptake can occur through macropinocytosis in various cell types, including HeLa cells, microglial cells, and epithelial cells.

Different modes of exosome entry can coexist within a cell. For example, ovarian tumor and melanoma cells primarily utilize cholesterol-associated lipid rafts for exosome uptake but also employ clathrin-mediated endocytosis, phagocytosis, and macropinocytosis simultaneously. Macrophage-derived exosomes use both macropinocytosis and clathrin-mediated endocytosis to penetrate hepatocytes and transfer interferon-induced resistance to the Hepatitis A virus. The uptake of exosomes by bone marrow-derived MSCs involves both clathrin-mediated endocytosis and macropinocytosis. In some cases, the roles of these pathways can be reversed, as observed in glioblastoma cells that stimulate exosome uptake through lipid rafts but inhibit it through caveolin-mediated endocytosis.

A recently discovered mode of entry is the filopodial pathway observed in fibroblasts. Filopodia are thin, actin-rich protrusions that allow cells to interact with their environment. Exosomes can surf on filopodia or encounter them through grabbing or pulling motions. This actin-dependent process facilitates the internalization and adhesion of exosomes, potentially mediated by transmembrane molecules like integrins.

Intracellular Signaling of Exosomes: Upon fusion with the plasma membrane or interaction with surface receptors of recipient cells, exosomes release their contents into the cytosol, initiating downstream signaling cascades. The fate of internalized exosomes follows the endosomal pathway, sorting into early endosomes, late endosomes, and multivesicular bodies (MVBs) that ultimately fuse with lysosomes for degradation. However, studies indicate that exosome cargoes can bypass degradation, leading to functional changes in recipient cells.

Gradual acidification in endosomal compartments can activate exosome cargoes, such as latent transforming growth factor beta (TGF-β1) and induce phenotypic changes in recipient cells. The fusion of late endosomes with lysosomes allows cargo uncoating and potential release into the cytosol. Some exosome contents may also passively diffuse across the cytoplasm, leading to potential leakage.

The endoplasmic reticulum (ER) can serve as a route for lysosomal escape and cargo release. Exosomes can be directed to ER-associated invaginations linked with late endosomes, allowing delivery of exosome components into the nucleoplasm. Other pathways for exosomal escape from lysosomal degradation include retrograde trafficking to the trans-Golgi network, the release of partially degraded materials from ruptured endosomal or lysosomal compartments, membrane fusion, or redirection back to the plasma membrane via recycling endosomes.

Why is defined media required for exosome production?

Defined media is required for the production of exosomes to ensure consistency, reproducibility, and control over the manufacturing process. It provides a precisely formulated composition of nutrients, growth factors, and supplements that meet the specific requirements for exosome production. Using defined media offers several advantages. Firstly, it allows for the elimination of undefined components, such as serum, which can introduce variability and impurities into the exosome preparation. This helps in achieving a more standardized and controlled exosome production process. Secondly, defined media enable better optimization of culture conditions for exosome production. Researchers can tailor the media composition to enhance exosome yield, purity, and functionality. This optimization can lead to improved scalability and efficiency in exosome manufacturing. Additionally, defined media reduce the risk of potential contaminants or unwanted biological effects associated with undefined components. By precisely defining the media composition, unwanted variations and impurities can be minimized, ensuring the production of high-quality exosomes.

 Conclusion and Future Prospects

Controversies in Exosome Research: Understanding Biogenesis and Heterogeneity

Exosome research has gained significant attention in recent years due to its role in intercellular communication and potential therapeutic applications. However, several controversies surround key aspects of exosome biology, including their biogenesis at the plasma membrane and their heterogeneity and characterization.

One area of debate is the site of exosome biogenesis. Traditionally, exosomes were believed to originate from the endosomal pathway, specifically from multivesicular bodies (MVBs). These MVBs would fuse with the plasma membrane, releasing intraluminal vesicles (ILVs) as exosomes into the extracellular space. However, some studies have proposed an alternative mechanism, suggesting that exosome formation can occur directly at the plasma membrane within distinct domains. For example, certain domains enriched in exosome proteins and lipids have been identified on the plasma membrane of Jurkat T cells, referred to as "endosome-like" domains. These domains may facilitate rapid and direct exosome biogenesis. Observations of outward vesicle budding rich in exosomal proteins from the plasma membrane within these domains have provided further evidence. Additionally, other studies have shown that exosome markers such as CD9 and CD81 can bud more efficiently from the plasma membrane than from endosomal compartments. However, the biogenesis of exosomes at the plasma membrane is still a topic of debate, and further research is required to provide conclusive evidence. The controversies may arise from the limited characterization of the studied vesicles and the lack of definitive markers to differentiate between different types of vesicles.

Another significant challenge in exosome research is their heterogeneity and characterization. Exosomes exhibit considerable variability in size, composition, function, and cellular origin, which adds complexity to their study. Researchers have identified distinct subpopulations of exosomes based on size and density. Advanced fractionation techniques have classified exosomes as large (90-120 nm) or small (60-80 nm) based on size and as high or low density based on density centrifugation. These differences in exosome subpopulations can result from variations in the limiting membrane of MVBs during ILV formation or differences in the molecular routes involved in exosome biogenesis. The heterogeneity of exosomes also extends to their content, with more than 4400 proteins, 200 lipids, 1600 mRNA, and 750 miRNA having been identified. Notably, not all exosome proteins are shared among all exosomes, and only a fraction of the cargo is cell-specific, reflecting the cell type and physiological condition. Factors such as cellular biology and the microenvironment influence exosome loading, leading to differential qualitative and quantitative content of cargoes. This heterogeneity contributes to diverse organ biodistribution and distinct biological functions of exosomes.

Accurately characterizing exosomes is crucial to understand their contents and functional roles and to facilitate their differentiation from other extracellular vesicles (EVs). Various isolation methods, including ultracentrifugation, size exclusion, and immunoaffinity isolation, coupled with analysis techniques such as nanoparticle tracking, electron microscopy, flow cytometry, and western blots, are currently employed for exosome generation and characterization. Global and targeted proteomics analyses further aid in the characterization process. However, the lack of standardization in these methods has resulted in substantial overlap in the protein profiles of isolated EVs. Differentiating between microvesicles (MVs) and exosomes, in particular, is challenging due to the absence of specific or universal markers. To address this issue, guidelines provided by the International Society for Extracellular Vesicles (ISEV) are continuously being reviewed to adapt to the evolving nature of EVs and exosome research. The Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines recommend categories of markers, such as size, density, biochemical composition (surface markers), and cellular origin, to be analyzed in all bulk EV preparations. Furthermore, the identification of markers exclusive to specific isolation methods or parental cells is also proposed as a means of standardization.

Several pitfalls hinder exosome research and contribute to controversies in the field. One challenge is the involvement of molecular players in exosome biogenesis that are also part of other cellular trafficking pathways. Loss and gain of function experiments targeting these molecules can have direct or indirect effects, altering their function in other cellular vesicular pathways such as Golgi, lysosomes, and autophagy, which, in turn, can influence exosome production or secretion. Moreover, the variation in parent cell types, culture conditions, and the lack of standardized exosome generation and characterization methods can impact experimental reproducibility and lead to overlapping properties between different EVs. Implementing multiple, complementary characterization methods and carefully tracking for any co-isolated non-EV/exosome components is essential for accurate classification. However, not all studies incorporate rigorous characterization, resulting in mixed populations of vesicles and hampering the investigation of intended exosomes. Additionally, contamination from mycoplasma and other microorganisms can introduce unintended effects by altering the physiology of donor cells and releasing their exosomes, which need to be considered and accounted for. Furthermore, the effects of pre-analytical variables from biofluids and conditioned media and the influence of tissue processing on exosome analysis should be explored. Factors like processing and storage can also impact exosome physiology and affect research outcomes. Identifying and addressing these experimental artifacts is crucial for reliable advancements in exosome research.