Regenerative medicine is not a subject traditionally found in the curriculum of U.S. medical schools. This gap in physician training has resulted in much confusion within the field despite the rapid growth of technological advances and an explosion in patient demand. Having spent over 20 years in academia and biotech studying regenerative medicine, I have noticed how the adoption of regenerative medicine has outpaced education and sound knowledge.
In part one of this three-part editorial, I described how the term “stem cell” has been assumed as a genericization for anything relating to regenerative medicine, much like the adoption of Kleenex to describe tissue paper or Xerox when referencing photocopiers.
In part two, I aim to bring transparency to the subject of exosomes, extracellular vesicles, nanoparticles and signaling modalities currently being utilized in regenerative medicine as an alternative to cellular therapy. Finally, in part three, I will discuss the emergence of personalized, precision medicine as a strategy to deploy safe, effective and reliable regenerative medicine in modern healthcare.
In cell biology, eukaryotic cells communicate through direct contact (juxtacrine signaling) or by the secretion of soluble factors such as growth factors, hormones and cytokines. These soluble factors can act on the cell itself (autocrine signaling), neighboring cells (paracrine signaling) or distant cells (endocrine signaling). In the past few decades, extracellular vesicles (EVs) have been recognized as the vehicles which facilitate intercellular communication, both in eukaryotes and prokaryotes. EVs are membrane-bound nanoparticles released in an evolutionarily conserved manner by cells for the protection and delivery of multiple different messengers, collectively referred to as “cargo.”
In 1999, I graduated from the Liverpool School of Tropical Medicine in England with a master’s degree in Parasitology. I was interested in the mechanisms by which human prokaryotic parasites communicate with the host in order to hijack the immune system to avoid destruction and, in many cases, to establish a home. During the parasite-host interaction, those organisms release EVs with the potential to mimic the characteristics of host EVs to modulate the immune system and promote their survival. For example, leishmania can produce EVs taken up by phagocytic cells, enabling the delivery of immunomodulatory proteins contributing to the creation of a permissive environment for infection. These parasites inhibit pro-inflammatory cytokine production (e.g., TNF-α) while promoting immunosuppressive cytokine production (e.g., IL-10) in human monocytes. To me, these details suggested a vast unexplored potential for the management of human autoimmune diseases and other immune-mediated medical conditions, such as wound healing.
The first indications of the relevance of EVs in medicine came in 1946 by the observations made by Erwin Chargaff and Randolph West, who described their procoagulant activity in normal plasma. These particles would later become known as “platelet dust” thanks to Peter Wolf in 1967. Decades later, Raposo and colleagues demonstrated that these nanoparticles, now termed exosomes, isolated from Epstein–Barr virus-transformed B lymphocytes, could induce T cell responses. By 2006, with the discovery that EVs contain RNA cargo, including microRNA, EVs acquired substantially renewed interest as mediators of cell-to-cell communication. EVs have now been isolated from most cell types and biological fluids such as saliva, urine, nasal and bronchial lavage fluid, amniotic fluid, breast milk, plasma, serum and seminal fluid.
Exosomes are a sub-population of EV identified by the expression of specific surface markers (pan-EV markers) called tetraspanins (CD9, CD63, CD81). However, these proteins have recently been observed in apoptotic bodies and microvesicles, which are non-regenerative. As such, it has been challenging to identify the populations of exosomes that specifically contribute to regenerative medicine. The contents, size and membrane composition of EVs are highly heterogeneous and dynamic and depend on the cellular source and environmental conditions, contributing to significant confusion within the industry. Additional problems facing the field include a lack of standardization of both isolation procedures and methods for EV characterization. Proteomic profiles obtained from various EV populations highly depend on how EVs were isolated. Such methods may include isolation by filtration, differential ultracentrifugation, density gradient centrifugation (sucrose or iodixanol gradients) and size-exclusion chromatography. There is currently a major push by scientists to come to a consensus on isolation procedures and characterization.
Extracellular Vesicle Biology
EVs function by triggering intracellular signaling pathways through simple interaction with the surface receptors or ligands of target cells or by undergoing internalization. EVs can induce changes in the cell phenotype by transfer to the target cell of biologically active proteins or RNAs, collectively referred to as “cargo.” The presence of functional RNA within EVs was first described in 2006 from mouse stem cell-derived EVs. Cellular mRNA varies in size from 400 to 12,000 nucleotides, whereas RNA in EVs has a predominant size of <700 nucleotides. EVs can contain intact mRNA, mRNA fragments, long non-coding RNA, microRNAs (miRNA), PIWI-interacting RNA, fragments of tRNA and ribosomal RNA (rRNA). While most studies report the absence of ribosomal 18S and 28S in EVs (a typical cellular RNA), some recent studies report a substantial proportion of rRNA (up to 80%) in some EV sub-groups, including certain MSC-derived EVs. Its function, though, in EVs is currently unknown.
EV miRNAs are ~21 nucleotide regulatory molecules packaged into EVs to allow those miRNA messages to circulate protected within the body and avoid degradation from blood RNAse activity. EV-mediated transfer of miRNAs has been described to have substantial immunological effects. For example, the transfer of some miRNAs (such as miR-335) from T-cells to antigen-presenting cells modulates gene expression in the recipient cells. In addition, EVs isolated from saliva have been shown to contain platelet tissue factor and the T cell activator, CD26. Platelet tissue factors may initiate blood coagulation, and it has been suggested that saliva EVs could be important in the process in which humans and animals lick a bleeding wound to promote coagulation, antimicrobial activity and subsequent wound healing.
Extracellular Vesicles and Medical Applications
In addition to proteins and RNAs, bioactive lipids, such as eicosanoids, prostaglandins, fatty acids and cholesterol, can be transferred between cells by EVs. Vesicle-bound lysophosphatidylcholine has been proposed to play a role in the maturation of dendritic cells and can trigger lymphocyte chemotaxis. Interestingly, for my current research, placenta-specific miRNAs are also packaged into EVs and mediate crosstalk between the fetus, placenta and the mother during pregnancy. In 2007, EVs were detected in the amniotic fluid (AF) of laboratory mice and four samples from women undergoing routine amniocentesis. It is believed that the origin of AF-derived EVs is from both the mother and the fetus. The fetal kidney releases EVs containing proteins, such as TIMP1, TIMP2, CD24 and annexin-I, which are of significant interest to medicine. Successful pregnancy relies upon EVs, which are constantly present in the maternal blood throughout pregnancy and influence the maternal immune system controlling both maternal adaptive and innate immunity during pregnancy. EVs from the amniotic fluid are thought to modulate the mother’s immune response to maximize fetal survival. In this case, EVs from the amniotic fluid were shown to be captured by human monocytes and stimulate cytokine release in a TLR-dependent manner. Our research has seen EVs from AF modulate “cytokine storm” related cytokines in response to microbial challenges. This is very interesting, considering the current COVID-19 pandemic.
EVs have also been defined as novel players in neural cell communication with pleiotropic physiological effects, both in the developing fetal nervous system and throughout the adult body. Cerebrospinal fluid has been described to have many functions as an intermediary between blood and the brain for the transport of nutrients and growth factors. CSF is also involved in the elimination of toxins and other metabolic by-products. Because of the potential importance of EVs in the context of the CNS and neurological diseases, the presence of EVs in human CSF is of medical interest. EVs have been proposed to neutralize the synaptic-plasticity disrupting activities of amyloid β-protein, primarily by sequestering Aβ oligomers by EV surface proteins, such as PrPC. These indicate a protective role of EVs against Aβ accumulation. In cardiac studies, using a myocardial ischemia-reperfusion injury mouse model, researchers found that EVs were responsible for observed cardioprotection, including neoangiogenesis. EVs from human liver stem cells have been demonstrated to accelerate morphological and functional recovery in a rat model of 70% hepatectomy, while EVs derived from human adult MSCs have been found to protect against ischemia-reperfusion kidney injury and lead to enhanced survival in a model of lethal acute kidney injury.
As we celebrate the 75th Anniversary of the first experiments that yielded identifiable EVs in the lab, we are still learning about the medical potential packaged into these nano-sized, lipid membrane-bound transport vehicles. Their protective shell allows RNAs to be transported around the hostile environment of the body (e.g., the current COVID-19 RNA vaccines), and their receptor profile facilitates tissue targeting to sites of inflammation and injury. These robust packages of information and raw materials are immune evasive (to be distinguished from immune-privileged) and small enough to travel through the narrowest of capillaries and even pass the blood-brain barrier. We know from pre-clinical data and clinical trials that EVs derived from various cellular sources can dramatically affect inflammation and disease by stimulating the patient’s innate regenerative cells. The road to universal acceptance of EVs as a modality for medical intervention has been long, but the future is bright.
In the third and final part of this three-part series, I will discuss how EVs and other regenerative tools are currently being employed in medicine as I discuss the emergence of personalized, precision medicine.
Dr. Ian White is an expert in regenerative medicine with over 20 years of experience in both academia and the industry. Dr. White is the founder and CSO of Neobiosis, a perinatal tissue manufacturing CDMO, the scientific advisor to Top Doctor Magazine, and Vice President of The American College of Regenerative Medicine. Dr. Alessandro Salerno is an expert in lipid metabolism, cell culture techniques and regenerative medicine. Dr. Salerno is the Director of Manufacturing at Neobiosis, where he oversees tissue production and quality control for clinical research and clinical trial grade perinatal tissue manufacturing.
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