The development of epilepsy, a process known as epileptogenesis, often occurs later in life following a prenatal or early postnatal insult such as cerebral ischemia, stroke, brain trauma, or infection. These insults share common pathophysiological pathways involving innate immune activation including neuroinflammation, which is proposed to play a critical role in epileptogenesis. This review provides a comprehensive overview of the latest preclinical evidence demonstrating that early life immune challenges influence neuronal hyperexcitability and predispose an individual to later life epilepsy. Here, we consider the range of brain insults that may promote the onset of chronic recurrent spontaneous seizures at adulthood, spanning intrauterine insults , perinatal injuries , and insults sustained during early postnatal life—such as fever-induced febrile seizures, traumatic brain injuries, infections, and environmental stressors. Importantly, all of these insults represent, to some extent, an immune challenge, triggering innate immune activation and implicating both central and systemic inflammation as drivers of epileptogenesis. Increasing evidence suggests that pro-inflammatory cytokines such as interleukin-1 and subsequent signaling pathways are important mediators of seizure onset and recurrence, as well as neuronal network plasticity changes in this context. Our current understanding of how early life immune challenges prime microglia and astrocytes will be explored, as well as how developmental age is a critical determinant of seizure susceptibility. Finally, we will consider the paradoxical phenomenon of preconditioning, whereby these same insults may conversely provide neuroprotection. Together, an improved appreciation of the neuroinflammatory mechanisms underlying the long-term epilepsy risk following early life insults may provide insight into opportunities to develop novel immunological anti-epileptogenic therapeutic strategies. Epilepsy may develop later in life following a prenatal or early postnatal insult such as cerebral ischemia, stroke, brain trauma, or infection. These so-called “acquired epilepsies” account for approximately one-third of all human epilepsies , and preThe developing brain undergoes significant dynamic changes during fetal and early postnatal life, rendering it particularly vulnerable to insults and stressors which can have either transient or permanent effects on neuronal function . Indeed,Several mechanisms have been proposed to explain how and why prenatal, perinatal, and postnatal insults, such as those described above, result in a vulnerability to develop acquired epilepsy later in life. For example, modulation of gene transcription and epigenetic programming, acquired channel and synaptopathies, and neuronal network connectivity all likely play an important role, as discussed elsewhere . Here, wNeuroinflammation, defined as an inflammatory response within the brain or spinal cord, is a common consequence of brain injuries, insults, and immune challenges . Charactvia neuroimmune modulation. Such an understanding is necessary to inform the development and appropriate application of novel therapeutic agents targeting the relevant biological mechanisms, with the goal of disrupting and preventing the epileptogenic process from occurring.In this review, we will consider the most common early life insults linked to the development of epilepsy later in life—including prenatal immune activation, perinatal injuries, and immune challenges sustained during early postnatal life  Figure 1in utero exposure to infections and/or inflammation considered to be an environmental risk factor for neurodevelopmental and psychiatric disorders including autism and schizophrenia —revealed increased seizure susceptibility compared to those exposed to saline control  is an experimental substrate frequently used to mimic viral infections. When administered to gestating animals in a model known as maternal immune activation (MIA), this toll-like receptor 3 (TLR3) agonist results in long-lasting physiological perturbations . Poly I:ocarpine .Infections in Postnatal Life) provokes an acute elevation of pro-inflammatory cytokines via gene transcription of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in neurons and glia of the mother and offspring  axis have been proposed to be one of the key mechanisms by which maternal stress mediates network reorganization and epileptogenesis in the developing offspring . Anotheral weeks . Neonataal weeks . This stal weeks .Importantly, alterations in the stress response—resulting in elevated stress hormone levels—have been shown to promote chronic priming and activation of microglia, which then generate increased cytokines IL-1β, IL-6, and TNFα in response to a secondary immune challenge . This meHypoxic–ischemic injury (HI) is a significant cause of brain damage in newborn infants, and is associated with a high incidence of neurodevelopmental disabilities. Neonatal hypoxic–ischemic encephalopathy, defined as a syndrome of disturbed neurological function during the first days of life, often occurs following moderate to severe HI, and is the most common cause of neonatal seizures . These aA receptor antagonist and pro-convulsive agent pentylenetetrazol (PTZ), an agent widely used to assess brain excitability  or younger (p5) rats . These aThe contribution of inflammation to development of epilepsy in this context was recently demonstrated, by use of a novel therapeutic drug Vitexin. This anti-inflammatory botanical flavonoid was found to reduce cytokine release, neutrophil infiltration, and blood–brain barrier (BBB) permeability alongside a reduction in epilepsy susceptibility after HI in neonatal rats . In the Febrile seizures (FS), typically provoked by fever, are common during infancy and early childhood, affecting approximately 3–5% of children between 6 months and 5 years of age . When reA subunit composition, and inflammation  and GABAA receptor subunit protein expression in the hippocampus  to p10–16 rodent pups . Dependipocampus .Similarly, LPS has been shown to potentiate fever-induced FS at p14 in both rats and mice, at least acutely . Adminisper se, but rather indirectly from the long-term effects of the acute inflammatory response on the immature brain, and its subsequent developmental trajectory , in a manner dependent on myeloid differentiation primary response 88 (MyD88)  . Constit (MyD88) .As noted earlier, a particular window of enhanced vulnerability to seizures has been identified in rodent models during the second week of postnatal life, coincidental with significant synaptogenesis and synaptic pruning . As micrSE is defined as a condition in which abnormally prolonged seizures occur, which may have long-term consequences including neuronal loss and altered neuronal networks . In expevia an NMDA receptor–dependent mechanism  microglia  has a reported incidence of up to 35% after severe TBI . While sAn established model of experimental TBI to the p21 mouse, utilizing the controlled cortical impact model of unilateral injury to the parietal lobe, results in progressive neuropathology and chronic neurobehavioral and neurocognitive dysfunction consistent with what is commonly observed in toddler-aged children after TBI . This movia IL-1  interact with genetics to promote epileptogenesis remains in its infancy. However, there is increasing evidence that genetic predisposition to epilepsy will increase an individuals' likelihood of developing late-onset seizures after an acquired insult. For example, a higher risk of post-stroke epilepsy was recently reported in individuals with a family history of epilepsy compared to those without a family history, even when adjusted for stroke injury severity . SimilarContrary to the above-described literature, there is also preclinical evidence that an early life insult inducing a modest inflammatory response can alternatively attenuate the response to a second-hit insult. This phenomenon, termed “preconditioning,” occurs when the brain develops resistance to injury after exposure to a low dose, typically subthreshold stimuli, such as brief ischemia, hypoxia, or low dose endotoxin. Preconditioning in the context of brain insults has been well documented in adult animals, but less so in the immature brain. Even fewer studies have considered the effect on neuronal excitability and seizure vulnerability.via preconditioning. Acting via TLR4, LPS is thought to reprogram the intracellular response to a subsequent insult, resulting in broad neuroprotection via activation of anti-inflammatory factors, alongside the downregulation of NF-κB   is one oof NF-κB . One stuof NF-κB . Anti-icof NF-κB . This efinvolved . Of noteA role for astrocytes in modulating the relative levels of pro- versus anti-inflammatory mediators has been reported as a biological mechanism associated with this phenomenon, as well as microglial priming . In addiIn this review, we have summarized and critically discussed the most common known causes of acquired epilepsy following injury or insult during early life, from maternal infection exposure through to TBI during young childhood. We have excluded from our discussion some other causes of acquired epilepsy, such as brain tumors  or malfoper se; indeed, genetic predisposition, environmental factors, and interactions between all of these variables are likely to determine an individual's risk status  at a later time Figure 1k status . Experimk status . HoweverFuture studies to identify and characterize the key factors mediating the chronic consequences of such insults may allow for the development of predictive tests to more readily identify those individuals at greatest risk. Further, novel immune-based therapies may provide therapeutic benefit by aborting the epileptogenesis process prior to the onset of spontaneous recurrent seizures, or even mitigating its severity after the onset of epilepsy (i.e. disease modifying).BS conceptualized and drafted the manuscript. LD and TO'B provided critical revision and intellectual input, and LD generated the figure. BS and LD incorporated reviewer feedback to revise the manuscript for resubmission. All authors approve the manuscript and are accountable for its content.The authors acknowledge funding support from the National Health and Medical Research Council of Australia  and Monash University, Australia. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer AV declared a past co-authorship with one of the authors, TO'B, to the handling editor.


Staphylococcus aureus is a leading cause of bacterial infections globally in both healthcare and community settings. The success of this bacterium is the product of an expansive repertoire of virulence factors in combination with acquired antibiotic resistance and propensity for biofilm formation. S. aureus leverages these factors to adapt to and subvert the host immune response. With the burgeoning field of immunometabolism, it has become clear that the metabolic program of leukocytes dictates their inflammatory status and overall effectiveness in clearing an infection. The metabolic flexibility of S. aureus offers an inherent means by which the pathogen could manipulate the infection milieu to promote its survival. The exact metabolic pathways that S. aureus influences in leukocytes are not entirely understood, and more work is needed to understand how S. aureus co-opts leukocyte metabolism to gain an advantage. In this review, we discuss the current knowledge concerning how metabolic biases dictate the pro- vs. anti-inflammatory attributes of various innate immune populations, how S. aureus metabolism influences leukocyte activation, and compare this with other bacterial pathogens. A better understanding of the metabolic crosstalk between S. aureus and leukocytes may unveil novel therapeutic strategies to combat these devastating infections. Staphylococcus aureus (S. aureus) is an opportunistic pathogen that colonizes approximately one-third of the human population and can cause invasive disease at an array of different sites throughout the body, including endocarditis, skin and soft tissue infection, bacteremia, pneumonia, osteomyelitis, and medical implant-associated infection  that is mediated, in part, by IL-10 production , significantly decreased the chronicity of biofilm infection by eliciting a heightened pro-inflammatory response . Host-adapted P. aeruginosa strains preferentially utilized the tricarboxylic acid (TCA) cycle to limit stress from reactive oxygen species (ROS) produced by leukocytes in the airway. This TCA metabolic bias in P. aeruginosa enhanced biofilm formation, which served to increase the chronicity of infection  of the electron transport chain (ETC), phagosomal acidification, and bacterial killing  and as the concentration of itaconate increases, SDH is progressively inhibited. This leads to succinate accumulation and decreased oxygen consumption via the inhibition of SDH, which is also complex II of the mitochondrial ETC  activity and nitric oxide (NO) production  expression that allows S. aureus to detoxify its environment by converting NO into nitrate, making it an iNOS-dependent virulence determinant (S. aureus is L-lactate dehydrogenase (ldh1), which is a NO-inducible gene. While hmp serves to detoxify the environment, Ldh allows S. aureus to maintain redox homeostasis by promoting the conversion of pyruvate to lactate  in anti-inflammatory polarized macrophages to produce ornithine. Ornithine is further metabolized into polyamines and proline for wound repair and cell growth processes . A similar reduction in glucose availability occurs during S. aureus biofilm formation, as reflected by a shift towards fermentative metabolism and lactate production  production that promoted their immunosuppressive effects , eliciting a cascade that culminates in the phosphorylation of mixed lineage kinase domain-like protein (MLKL) that damages the plasma membrane leading to cell death. Pyroptosis occurs in response to inflammasome activation that cleaves gasdermin D, which oligomerizes to form pores in the cell membrane. While both forms of cell death induce inflammation, the heightened production of inflammatory mediators associated with inflammasome activation makes pyroptosis more inflammatory in nature than necroptosis  racing to consume the same resources, which becomes more complicated in the context of polymicrobial infections. Glucose and oxygen are among the first resources that become restricted in these settings and their deprivation can be enough to bias the actions of responding immune cells or pathogens alike. These events can also dictate the nature of immune cell death, which has been reported to occur roptosis . The rolS. aureus metabolic adaptations under a variety of in vitro and in vivo conditions and the reader is directed to several excellent and comprehensive reviews on the topic (S. aureus utilizes a number of two-component regulatory systems to sense changes in its environment (S. aureus uses two catabolite control proteins (CcpA & CcpE) to modulate glucose utilization through central carbon metabolism. In glucose replete conditions, oxidative metabolism is repressed by CcpA, which inhibits the expression of critical TCA cycle enzymes   . LDNs ars (LDNs) . Increass (LDNs) , 133. ByicaADBC that encode polysaccharide intercellular adhesins that promote bacterial aggregation and adherence to host surfaces  mutants, our group demonstrated that biofilm-derived lactate was imported into MDSCs and macrophages where it inhibited histone deacetylase 11   that were able to deplete local fumarate stores via enhanced fumarate hydratase (fumC) expression  –162. A rpression , 164. Trroptosis Figure 4roptosis . This isroptosis . These eS. aureus biofilm infection to below the limit of detection .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Atherosclerosis retains the leading position among the causes of global morbidity and mortality worldwide, especially in the industrialized countries. Despite the continuing efforts to investigate disease pathogenesis and find the potential points of effective therapeutic intervention, our understanding of atherosclerosis mechanisms remains limited. This is partly due to the multifactorial nature of the disease pathogenesis, when several factors so different as altered lipid metabolism, increased oxidative stress, and chronic inflammation act together leading to the formation and progression of atherosclerotic plaques. Adequate animal models are currently indispensable for studying these processes and searching for novel therapies. Animal models based on rodents, such as mice and rats, and rabbits represent important tools for studying atherosclerosis. Currently, genetically modified animals allow for previously unknown possibilities in modelling the disease and its most relevant aspects. In this review, we describe the recent progress made in creating such models and discuss the most important findings obtained with them to date. Atherosclerosis lies at the basis of severe human diseases that account for a large part of global morbidity and mortality, such as ischemic heart disease, myocardial infarction, and stroke. This disease can affect any artery in the human body, but is especially dangerous in large vital vessels, such as the carotid and coronary arteries. Certain parts of blood vessels that have bends or bifurcations are more susceptible to atherosclerotic plaque formation. Growing atherosclerotic plaque can by itself reduce the vessel volume leading to pathological consequences for the alimented organ or tissue. However, thrombotic events that occur at the surface of so-called unstable plaques are more dangerous. Thrombosis associated with rupture or erosion of atherosclerotic plaques is the cause of many cases of sudden cardiac death .According to current understanding, atherosclerosis is a multifactorial disease that involves altered lipid metabolism, increased oxidative stress, impaired mitochondrial function, and chronic inflammation ,3. The iNon-resolving inflammation plays a crucial part in the formation of the most dangerous unstable plaques that are prone to thrombogenesis . It was Matrix metalloproteinases (MMPs) are secreted by macrophages and other inflammatory cells in the plaque. MMPs are responsible for matrix degradation that leads to collagen depletion in the fibrous cap of the plaque, and is typically associated with plaque rupture . CollageThe exact mechanisms of plaque erosion remain unclear. Among the pathways responsible for this process, different authors have named local platelet-mediated neutrophil activation, release of myeloperoxidase, TLR-2 signaling in the endothelium, neutrophil-mediated injury, and apoptosis of endothelial cells. Neutrophils appear to play a special role in this process. Activated neutrophils can release their contents, including DNA and proteins, into the extracellular space forming the so-called neutrophil extracellular traps (NETs) during the process called NETosis, a special type of cell death. Formation of NETs was shown to contribute to atherosclerosis ,13. In sCommon requirements for animal models of human diseases include compatibility with human anatomy and physiology, translational potential, relative ease of maintenance, and affordable cost. When modelling atherosclerosis, it is important that animal models share the topography of the lesions with that observed in humans. Reproduction of atherosclerosis features in animal models is based on accelerated plaque formation that can be achieved by different approaches. The most frequent methods include cholesterol-rich diets and modifications of genes involved in lipoprotein metabolism. Mice and rabbits remain the most common choice for atherosclerosis model creation, followed by pigs and non-human primates. This review aims to summarize the information on the most commonly used animal models of atherosclerosis and their specific featuresEach of the models has both advantages and limitations. Murine models are characterized by a short life cycle, high reproduction rate, and simplicity of manipulation that makes their use convenient for modelling atherosclerosis . RabbitsGenetically modified animals revolutionized the approaches to animal model creation in many disease areas, including atherosclerosis. Methods of introducing modifications to DNA through molecular manipulations are being constantly improved. Currently, it is possible not only to insert or inactivate genes of interest, but to create conditional knock-outs silencing certain genes in particular organs and tissues or in response to an external signal.Currently, genetically modified mice are commonly produced using one of the two basic technical approaches. The first method is used to manipulate a single gene, for example for knocking out or single nucleotide changing. In this method, embryonic stem cells are modified with a DNA construct containing DNA sequences homologous to the target gene, and then injected into blastocysts . The othRabbit models of atherosclerosis became less frequently used since 2000, when apolipoprotein E (apoE) and low-density lipoprotein (LDL) receptor knock-out mice were developed .-/-) animals  rabbit line was established based on a mutation that causes a defect in the LDL receptor. Such animals are characterized by spontaneously developing hypercholesterolemia and atherosclerosis . HomozygWatanabe heritable hyperlipidemic rabbit model for spontaneous myocardial infarction (WHHLMI) was created in the attempt to further refine the WHHL model by selective breeding of myocardial infarction-prone animals for several years . The res-/- rabbits were designed as a model for investigating the relationship between atherosclerosis and human hyperlipidemia  endonucleases, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs) methods. Even when kept on a normal diet, ApoE-/- rabbits develop mild hyperlipidemia. Total cholesterol level in such animals remains about 200 mg/dL, and can be increased up to 1000 mg/dL upon feeding with a cholesterol-rich diet (0.3% cholesterol and 3% soybean oil) for two weeks, while wild-type rabbits fed with a cholesterol-rich diet only demonstrate a cholesterol level increase up to 170 mg/dL. Moreover, ApoE-/- rabbits develop more pronounced aortic atherosclerosis than wild-type rabbits when fed a cholesterol-rich diet for 10 weeks. Due to important roles of both ApoE and LDL receptor in the regulation of cholesterol metabolism, using ApoE-/- rabbits together with LDL receptor-deficient WHHL rabbits appears to be promising for modelling human hyperlipidemia (ApoEipidemia . These aipidemia .The transgenic rabbit model expressing human apolipoprotein(a) was developed using white Japanese rabbits. Unlike rodents, rabbit apoB is capable of binding to recombinant human apo(a) forming lipoprotein(a) (Lp(a))-like particles in the plasma . TransgeapoE-/- mice strains were created independently by 2 research groups in the early 1990’s (apoE-/- mice is the ability to develop atherosclerosis even while being fed standard rodent chow diets. Another commonly used murine model of atherosclerosis is LDL receptor-deficient (ldlr−/−) mice that replicate human familial hypercholesterolemia  and the LDLR-related protein 1 (LRP-1) . The hom protein .apoE-/- mice, atherosclerosis development is initiated spontaneously, even when animals are kept on a regular rodent diet, but can be accelerated by applying cholesterol- and fat-enriched Western type diet. Atherosclerotic lesions typically develop in the aortic root, the aortic arch, innominate artery, carotid arteries, and other arteries, depending on the diet and the duration of cholesterol-rich feeding  were created using the gene targeting approach in 1994  is a plasma membrane protein that mediates the hepatic clearance of plasma lipoproteins containing apolipoproteins apoB100 or apoE . Mutatio in 1994 . These mlar diet . Without00 mg/dL ,48. In lpurposes –51. In purposes .A novel murine model of atherosclerosis, PCSK9 adeno-associated virus mice, was developed without using germline genetic engineering . Designih/h): SR-BI KO/ApoeR61h/h mice. The most significant feature of the SR-BI KO/ApoeR61h/h mouse is the development of atherosclerosis and coronary heart disease in response to an atherogenic diet rich in fat, cholesterol, and cholate. It allows investigators to control the time of disease onset, and also the severity of symptoms (This model was generated by breeding two mice strains: SR-BI-deficient (SR-BI KO) mice and hypomorphic apoE mice , severe coronary heart disease, and even premature death seen in humans ,60.The recently developed apoE*3‐Leiden.CETP (E3L.CETP) mouse model of atherosclerosis appears to be the one that most closely replicates the features of human disease. Among the similarities are the ability to form atherosclerotic lesions of all stages (type I to V) in a diet-induced manner and the response of diseased animals to the treatment with such drugs as statins, fibrates, and ezetimibe. The model was created by combining the apoE*3‐Leiden transgene that provides reduced clearance of triglyceride‐rich lipoprotein, and the cholesteryl ester transfer protein (CETP) transgene that makes the cholesterol profile more humanized .apoE-/-) mice, and can therefore be used to study the features of human unstable plaques. ApoE-/-Fbn1C1039G+/- develop atherosclerosis in response to a high-fat diet, and this process is accelerated compared to regular apoE-/- mice (These mice are characterized by impaired production of fibrillin-1, which is responsible for the fragmentation of elastic fibers observed in aortic stiffening. This feature is known to be a potential cause of plaque rupture. The model also shares the common features of atherosclerotic (-/- mice .Animal models proved to be indispensable for studying human diseases, including atherosclerosis and searching for novel therapeutic approaches. Currently, several reliable rabbit and mice models of atherosclerosis have been developed and validated. Most of them are based on genetic modifications of key genes involved in atherosclerosis development, such as apolipoprotein E or LDL receptor genes. The models vary in terms of blood lipid profile, the ability to develop atherosclerotic lesions spontaneously or induced by a special diet, and the presence of complicated and unstable plaques. While induction of atherosclerosis lesions in model animals can be achieved reliably, modelling of complicated plaques, with such features as calcification, neovascularization, intraplaque hemorrhage, and thrombosis, is more challenging. Future studies should concentrate on creating such models that would allow testing new medications aimed at plaque stabilization.


Ancient schwannoma is an extremely rare benign, peripheral nerve sheath tumor. Despite its benign nature, its characteristic heterogeneous appearance and degenerative changes commonly lead to misdiagnosis of malignancy. Although schwannomas are extremely uncommon in the pediatric population, these neoplasms have been associated with underlying conditions such as neurofibromatosis type two, and appropriate recognition is important to ensure close monitoring of potential future symptoms secondary to other tumors. We report the imaging and laboratory findings of an ancient schwannoma of the vagus nerve in a 10-year-old female, the first documented case of such a tumor in a pediatric patient, and discuss its characteristic findings and diagnostic considerations. Awareness of this rare tumor can help promote correct diagnosis and avoidance of costly, high-risk diagnostic methods. Schwannomas are benign, neurogenic tumors originating from Schwann cells surrounding peripheral nerves. These rare lesions can arise from peripheral nerves surrounded by Schwann cells in any area of the body. They are most often found in the head, neck, and extremities -3. The vSchwannomas are divided into different subtypes based on their histologic characteristics. Ancient schwannoma is an uncommon subtype of these rare neoplasms, histologically characterized by degenerative changes and diffuse hypocellular patterning, which often leads to misdiagnosed malignancy despite the tumor’s benign nature ,5. ThereA 10-year-old female presented to her primary care provider with a three-day history of sore throat and cervical lymphadenopathy. The sore throat improved after four days of antibiotic therapy, but persistent right cervical lymphadenopathy warranted follow-up at her primary care clinic. Aside from periodic low-grade contralateral posterior neck pain and intermittent difficulty in swallowing, the patient was asymptomatic. White blood cell count, mononucleosis spot test, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) were unremarkable, and Epstein-Barr virus (EBV) studies were consistent with prior infection. A right neck ultrasound (US) revealed a solid hypervascular mass deep to the right sternocleidomastoid muscle (SCM) and posterior to the carotid artery and internal jugular vein, as seen in Figure Outside imaging was reviewed and, after unremarkable CRP, complete blood count, and urine and blood metanephrine studies performed at our institution, magnetic resonance imaging/angiography (MRI/MRA) was obtained. This identified a well-defined 3.0 x 2.3 x 4.1 cm ovoid-appearing, heterogeneous mass seen within the right carotid space. On postcontrast sequences, there was avid enhancement with internal areas demonstrating less prominent enhancement and no restricted diffusion. The mass displaced the right internal jugular vein anterolaterally and the internal carotid artery anteromedially with associated prominent and mildly enlarged cervical lymph nodes felt to be reactive in nature  performed for this patient are illustrative of the imaging characteristics of ancient schwannomas and makes this case unique in comparison to its counterparts which classically include only one or two imaging modalities. US in Figure Although imaging can assist in the diagnosis of ancient schwannoma, histopathology is the only method of confirmation. In this case, diagnosis of schwannoma was made via ultrasound guided FNA and ancient subtype was confirmed post-excision. FNA was chosen to aid in diagnosis, triage urgency of surgical excision, and could be performed with local anesthetic only. The use of FNA in schwannoma diagnosis remains controversial due to challenges in representative specimen acquisition and difficulties in making a confident diagnosis based on a limited sample ,8. DespiNeurogenic tumors make up only 2% of benign, pediatric neoplasms but they are commonly associated with underlying conditions such as neurofibromatosis types one and two  ,6. SchwaDespite the rarity of schwannomas in the pediatric population, clinicians should be aware of their presence due to the association with underlying genetic conditions and symptoms related to those conditions. FNA should be strongly considered to aid in diagnosis and accurate surgical planning and can be performed safely with minimal to no sedation. Understanding the histologic and radiologic characteristics of the ancient schwannoma subtype can help avoid misdiagnosis of malignancy and improve efficiency and accuracy of care.


Anti-müllerian hormone (AMH) is produced by granulosa cells of small, growing follicles in the ovary. Serum AMH levels strongly correlate with the number of growing follicles, and therefore AMH has received increasing attention as a marker for ovarian reserve. This review summarizes recent findings and limitations in the application of serum AMH in ovarian reserve assessment.A PubMed search was conducted to find recent literature on the measurements and use of serum AMH as a marker for ovarian reserve.Serum AMH levels are measured to assess the “functional ovarian reserve,” a term that is preferred over “ovarian reserve,” since AMH levels reflect the pool of growing follicles that potentially can ovulate. Serum AMH levels are used in individualized follicle-stimulating hormone dosing protocols and may predict the risk of poor response or ovarian hyperstimulation syndrome but has limited value in predicting ongoing pregnancy. Serum AMH levels are studied to predict natural or disease-related age of menopause. Studies show that the age-dependent decline rates of AMH vary among women. The generalized implementation of serum AMH measurement has also led to an increase in diagnostic assays, including automated assays. However, direct comparison of results remains problematic.Serum AMH remains the preferred ovarian reserve marker. However, the lack of an international standard for AMH limits comparison between AMH assays. Furthermore, little is known about endogenous and exogenous factors that influence serum AMH levels, which limits proper interpretation of AMH values in a clinical setting. Anti-müllerian hormone (AMH) is a member of the transforming growth factor beta family that has derived its name from its role during male sex differentiation by inducing the regression of the müllerian ducts. To date, AMH is best known as a serum marker for ovarian function, with assessment of AMH levels at both ends of the spectrum, that is, ovarian reserve and polycystic ovarian syndrome. In the ovary, AMH is expressed by granulosa cells of growing follicles from the primary up to the small antral stage. After follicle-stimulating hormone (FSH)-dependent selection, AMH expression disappears, although some expression remains in cumulus cells of preovulatory follicles. Also, in atretic follicles and corpora lutea, AMH expression is lost. This window of expression is largely preserved among species and in the adult human ovary. Increasing expression levels of AMH are detected in follicles up to 8 mm, and expression is absent in follicles >8 mm. This expression pattern is positively matched by AMH concentrations in follicular fluid, showing highest levels in follicles up to 8 mm and a sharp drop thereafter  1)..1).Since AMH is expressed by growing follicles prior to FSH-dependent selection and has been shown to be detectable in circulation, serum AMH has taken momentum as a marker for ovarian function, in particular in the assessment of the quantitative aspect of the ovarian reserve, which is the focus of this review. By definition, the ovarian reserve is constituted by the quality and quantity of the primordial follicles, which both decline with increasing age . The numSince serum AMH is only an indirect marker, this has led to confusion or even misinterpretation of the term ovarian reserve. To make a clear distinction between the pool of resting primordial follicles and the pool of growing follicles, the term functional ovarian reserve (FOR) has been suggested . FOR conSerum AMH levels are negatively correlated with age in adult women. However, studies aimed to develop normative data for AMH also showed that this correlation depends on the age category analyzed. From birth onward, AMH levels increased to plateau at approximately age 25 years , 12. Up Serum AMH levels are generally measured during the early follicular phase, similar to other hormonal markers of ovarian function, such as FSH, estradiol, and inhibin B. However, it has been questioned whether the variations in serum AMH levels could be explained by differences during the menstrual cycle. While initial studies suggested that AMH levels are relatively stable during the menstrual cycle , more reN) dimer and a 25kDa C-terminal mature region (AMHC) dimer, which together form a stable noncovalently bound complex  , a mid-region (AMHM) and a mid-mature region  (AMH is produced as a 140-kDa disulfide-linked homodimer (proAMH), consisting of covalently bound monomers each of 560 amino acids (aa) . The AMH . In vitr . An addi .The clinical importance of AMH measurement has led to the development of several AMH assays. Current frequently used manual assays include the modified Gen II assay (Beckman Coulter) and the ultra-sensitive AMH enzyme-linked immunosorbent assay (ELISA) and picoAMH assay (both Ansh Labs), which use different antibodies than the Gen II AMH assay . The picSeveral studies have compared the manual and automated assays and present regression equations to allow comparison of serum AMH levels measured by different assays . HoweverThese differences between assays depending on AMH levels remain puzzling and contribute to conflicting results in clinical studies assessing AMH. The lack of an international AMH standard, even 20 years after the development of the first AMH ELISA assay, is one of the main problems hampering AMH assay comparison. Absence of uniformly calibrated assays limit the development of standardized AMH cutoff values needed to enhance patient safety and to prevent misinterpretation by clinicians unaware of this interassay variability.The differences might be partly explained by a wide variation in population characteristics. Some studies have included women undergoing in vitro fertilization (IVF) treatment, whereas others included women undergoing laparoscopic sterilization or samples with an unclear clinical status related to fertility. Moreover, the sample size was relatively small (ranging from 23 women to 142 women), and also the age range was quite variable (23-56 years). Therefore, more research is needed to allow proper comparison of the different assays, using larger, clearly defined cohorts stratified by age.N,C. However, little is known about the presence and concentration of other AMH isoforms in the circulation. Although the AMHC fragment is the biologically active form of AMH, Pankhurst et al suggest that it is not detectable in circulation . This result was obtained using Ansh Labs ELISAs that either use a different antibody pair or sample treatment to distinguish the different isoforms , yet reported effects of HC use on serum AMH levels are conflicting. A systematic review by Amer et al , reassesSeveral studies have identified body mass index (BMI) to negatively influence AMH levels. In a study by Moslehi et al  reanalyzVitamin D (VitD) has increasingly been recognized to influence AMH levels. VitD levels exhibit seasonal variation with higher levels in summer compared with winter. Dennis et al  demonstrThese studies suggest that when counseling women on their FOR based on AMH levels, insight into factors that influence AMH expression but also follicle dynamics is important. It remains to be determined whether changes in AMH expression also lead to changes in number of primordial follicles, that is, the ovarian reserve, and whether such changes are in the same direction. This emphasizes the use of FOR over ovarian reserve in relation to AMH assessment.In the Western world, the age at which a woman decides to have her first child has increased, and thereby also the risk of age-related involuntary infertility . Given tIn the above-discussed meta-analysis by Depmann et al , compareMost prediction models are based on a single AMH measurement and assume a comparable decline pattern in each woman. Recent studies analyzing longitudinal AMH measurements suggest that AMH may not follow a uniform decline trajectory. Analysis of the population-based Doetinchem cohort study with data available from 5 visits over a 20-year follow-up period, showed an age-dependent decline in AMH levels, which varied significantly for individual women. Furthermore, it was shown that the decline rate changed with age, accelerating after the age of 40 years . Hence, Although the study of Ramezani Tehrani et al  did not Based on current studies, the predictive value of serum AMH for age of menopause remains controversial. The majority of these studies have analyzed different age ranges, duration of follow-up, and AMH assays, making direct comparison of studies difficult. It also remains unclear whether current results obtained in regularly cycling women can be translated to infertile women in whom the ovarian reserve may be compromised. The potential impact of ethnicity on AMH decline rates also has not been analyzed in much detail. Thus, validation studies that incorporate additional variables are required to determine specific AMH thresholds in the prediction of age at menopause.Previous studies have also shown that AMH levels may aid in the prediction of ovarian response to controlled ovarian hyperstimulation (COH) protocols. Low AMH levels are correlated with a low response, defined as retrieval of less than 5 oocytes or cycle cancellation. Currently, 2 different ovarian stimulation approaches are widely used: a gonadotropin-releasing hormone agonist or a gonadotropin-releasing hormone antagonist in combination with recombinant or urinary FSH . To imprAccurate and reliable calculation of the individual dosage is important since both under- and overstimulation could lead to cycle cancellation. In addition, an excessive response could result in the development of ovarian hyperstimulation syndrome, a potentially life-threatening condition. It is, however, unclear whether the use of these clinical characteristics significantly improves the prediction of ovarian response and clinical outcomes. Broer et al performed 2 meta-analyses to investigate the added value of ORTs to the patients’ characteristics of age, BMI, and duration of subfertility , 64. Of The question remains whether individualizing treatment based on these parameters also improves clinical outcomes. Based on the studies from Broer et al, both AMH and AFC showed a very low predictive value for pregnancy rate after IVF, with an AUC of only 0.50 and 0.55, respectively , 64. A mFriss Petersen et al  investigDrawing conclusions based on these findings remains complicated as the majority of these studies have used different cutoff values for ORTs and used different AMH assays. Therefore, the clinical application of AMH levels in ORT-based dose adaptation still needs to be demonstrated.Women with autoimmune diseases are at risk for early menopause and therefore at increased risk for infertility and menopause-associated diseases such as osteoporosis and cardiovascular diseases. Hence, several studies have analyzed serum AMH levels to evaluate FOR in women with autoimmune diseases.A recent meta-analysis of 19 studies analyzing patients with systemic lupus erythematosus concluded that serum AMH levels were significantly lower in patients with systemic lupus erythematosus compared with healthy control participants . In addition, AMH levels were further lowered by the immunosuppressant cyclophosphamide  .Women with recent-onset rheumatoid arthritis (RA) had comparable AMH levels with healthy control participants, and methotrexate use did not affect AMH levels . HoweverIn women with autoimmune thyroid disease (ATD), conflicting results have been reported. Adolescent girls newly diagnosed with ATD had either normal or higher AMH levels compared with age- and BMI-matched control participants . In adulWomen with type 1 diabetes (T1D) are suggested to have an earlier age at menopause , althougOverall, these recent studies suggest that the relationship between autoimmune diseases and diminished FOR, as assessed by AMH, remains inconsistent. Additional well-controlled studies are needed to analyze the impact of disease onset, duration, and therapy on AMH levels.Chemotherapy is known to have adverse effects on ovarian function and increases the risk of primary ovarian insufficiency (POI) posttreatment. In recent years, a number of studies have analyzed pre- and posttreatment AMH levels in patients with cancer, particularly in breast cancer, being the most common cancer in women worldwide. Studies agree that upon chemotherapy, AMH levels rapidly decline to (nearly) undetectable levels , 89. ThiIn women treated for differentiated thyroid cancers, radioactive iodine (RAI) treatment has been suggested to induce early menopause. Although the impact of postsurgical RAI treatment on AMH levels seems less severe than chemotherapy, AMH levels did declined by at least 50% and only showed a partial recovery . FurtherThe improved sensitivity and automation of AMH assays has strengthened the role of serum AMH levels as a marker for the FOR. Assessment of serum AMH levels in the prediction of ovarian response to COH and age of menopause, being either natural or iatrogenic, may be useful. However, limitations have also surfaced. For proper interpretation of AMH levels, more knowledge is needed on endogenous and exogenous factors that regulate AMH expression. Studies further suggest that heterogeneity exists in AMH trajectories, which may hamper application in personalized patient counseling. Over the last years, the number of assays for AMH measurement has increased. However, differences exist between assays. An international standard for AMH is therefore urgently needed to establish assay-independent cutoff values.


This editorial aims to summarize the six scientific papers that contributed to this Special Issue. Metabolism is broadly defined as the sum of biochemical processes in living organisms that either produce or consume energy. It is widely acknowledged that metabolic perturbations—often genetically programmed—accompany common human diseases. Among these, cancer is a prime example of a disease with genetically-defined, pathological metabolic perturbations. Indeed, metabolic reprogramming, such as enhanced aerobic glycolysis, mutations in the tricarboxylic acid (TCA) cycle metabolic enzymes, and dependence on lipid and glutamine metabolism are key characteristics of cancer cells. Therefore, addressing such metabolic perturbations is a very promising strategy for anti-cancer therapies that, in combination with conventional chemotherapeutic agents, may enhance therapeutic efficacy and clinical outcomes.The understanding that metabolic changes occurring in cancer cells may have an impact on immune cell functionality, and contribute to tumor immune evasion, has fueled a growing interest in developing techniques that target metabolism for immunotherapy . As elegAlterations in mitochondrial function have long been considered a hallmark of cancer . MultiplMany nuclear oncogenes or tumor suppressors play a direct role in regulating transcription, and nearly all such proteins are modified by O-GlcNAcylation, namely the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to the protein . A singlIt is widely known that mitochondrial dysfunction and oxidative stress are associated with the development of neurodegenerative diseases. Among them, amyotrophic lateral sclerosis (ALS) is an incurable progressive neurodegenerative disease that affects nerve cells in the brain and spinal cord, causing loss of muscle control. ALS reduces muscle strength, modulates muscle metabolism through lowering citrate synthase (CS) and increasing cytochrome c oxidase and malate dehydrogenase activities, and elevates oxidative stress markers in skeletal muscle . TherefoS-dependent cyclic adenosine-monophosphate (cAMP) signaling, as well as other signaling pathways [The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin–melanocortin pathway . Mutatiopathways . To studpathways . OverallThe crucial role played by thyroid hormone (TH) on energy expenditure and body weight in humans is well recognized. Hyperthyroidism is characterized by increased resting energy expenditure, weight loss, and heat intolerance. On the other hand, reduced resting energy expenditure, weight gain, and cold intolerance are hallmarks of hypothyroidism. The action of metabolically active TH metabolites involves a wide variety of stimuli and conditions that have still not been completely unraveled. Rutigliano et al. elegantly review the relevance of 3-iodothyronamine (T1AM) in the regulation of energy homeostasis and metabolism, along with its potential therapeutic applications in metabolic disturbances . As highIn summary, all articles published in this Special Issue thoroughly describe the most interesting current directions in metabolic reprogramming studies. Cellular metabolic reprogramming is an important mechanism by which cells rewire their metabolism to promote proliferation and cell growth. This process has been mostly studied in the context of pathological processes, with a special focus on cancer, while little is known about its relevance for non-pathological conditions and how it affects animal physiology as a whole. It is a pleasure for the Guest Editor to thank all the authors for their important contributions to this Special Issue and the advancement of knowledge in future studies.


The prevalence of kidney diseases is emerging as a public health problem. Stem cells (SCs), currently considered as a promising tool for therapeutic application, have aroused considerable interest and expectations. With self-renewal capabilities and great potential for proliferation and differentiation, stem cell therapy opens new avenues for the development of renal function and structural repair in kidney diseases. Mounting evidence suggests that stem cells exert a therapeutic effect mainly by replacing damaged tissues and paracrine pathways. The benefits of various types of SCs in acute kidney disease and chronic kidney disease have been demonstrated in preclinical studies, and preliminary results of clinical trials present its safety and tolerability. This review will focus on the stem cell-based therapy approaches for the treatment of kidney diseases, including various cell sources used, possible mechanisms involved, and outcomes that are generated so far, along with prospects and challenges in clinical application. Kidney diseases have become a global public health problem due to their rapidly growing incidence. These diseases affect over 10% of the global population, because of a global rise in the aging population, as well as increase in the frequency of their main etiologies, such as diabetes, cardiovascular diseases, and hypertension . As a roStem cells (SCs) are described by their self-renewal abilities and the capability to develop into various functional cells under certain conditions. Based on the advantages of plasticity, infinite amplification, and ease of genetic manipulation, stem cell therapy opens new avenues for almost all human diseases. The application of SC therapy in treating a variety of diseases such as immunological, vascular, cardiac, and renal diseases has been extensively explored , 7. The Before we go into further details regarding the current SC-based approaches, a brief summary of the background information regarding potential sources of stem cells has to be put forward. SCs are self-renewable cells that are capable of differentiating into specialized cell types under appropriate conditions, and could be divided into four categories according to their differentiation potential Fig. .Fig. 1CWith the greatest differentiation potential, totipotent stem cells (TSCs) possess the omnipotentiality to differentiate into any type of cells, including the extraembryonic lineages and the germline , 13. ThePluripotency refers to a stem cell with potential to differentiate into any of the three germ lineages: endoderm , mesoderm , or ectoderm  . PluripoAdult SCs that exist in the postnatal organisms are either multipotent or unipotent and are restricted to a limited range of cell types if iPSCs were not taken into consideration. Multipotent SCs, including hematopoietic stem cells (HSCs) , mesenchProgenitor cells exhibit differentiation potential between SCs and mature cells and were found in many organs, including the bone marrow , gastroiESCs that are characterized by features of self-renewability and multilineage differentiation hold great promise in cell therapy and kidney regeneration. Numerous studies have shown that mouse embryonic stem cells (mESCs) can integrate into kidney compartments, suggesting a possible efficacy for kidney repair. In addition, the exposure of ESCs to factors required for renal specification, such as retinoic acid, activin A, and bone morphogenic proteins (BMPs), induces differentiation of these cells into renal lineage cells in vitro –35.Geng et al. have reported that the ESC-loaded gelatin microcryogels on 5/6 nephrectomized rats slowed down the progression of chronic kidney disease and reduced glomerular injury . SimilarPatients require renal replacement therapy when their kidney disease progresses to ESRD. Although dialysis offers a temporary solution for patients, it has several limitations. Dialysis does not address the loss of hemostatic and endocrine function of the kidney and its associated complications . Kidney Morizane et al. have successfully induced differentiation of ESCs into nephron progenitor cells (NPCs), which were then transformed into self-organized nephron-like structures containing glomeruli, proximal tubules, loop of Henle, and distal tubules in a contiguous, ordered arrangement that are analogous to that of nephrons . TakasatAlthough scientists thought they have found a strategy to isolate hESCs from single blastomeres without destroying the embryos, legal and ethical controversies still limit the research and applications of ESCs . AnotherInduced pluripotent stem cells sharing many of the regenerative properties of ESCs are considered effective alternative cell sources for ESCs. Indeed, iPSCs could retain both genetic background and peculiar epigenetic memory of the cells of origin, providing indisputable advantages in cell therapy, kidney regeneration, and other biomedical applications.According to the earliest report on iPSCs put forward in 2006, scientists obtained PSCs by introducing four transcription factors  into mouse adult fibroblasts, and this technique has earned Shinya Yamanaka and John Gurdon the Nobel Prize in Physiology or Medicine 2012 . To date7 iPSCs affected the smaller arteries of the kidneys with cellular emboli, leading to the reduction of blood reperfusion after ischemia. When carrying out cell therapy for treatment of AKI using iPSCs, the cell dose for transplantation and the monitoring of renal blood perfusion should be taken care of [Induced pluripotent stem cells are considered as an effective means to alleviate renal tissue damaged in AKI and CKD, and paracrine pathways may be the major mechanism. The administration of iPSC-derived conditioned medium attenuated AKI by downregulating the oxidative stress response in ischemia-reperfusion rats . The rec care of . For the care of .The pluripotency of PSCs raises concerns on high risk of maldifferentiation of the cells and even tumor formation, when these cells are administered without pre-differentiation. An attractive alternative consists in the derivation of renal progenitors from iPSCs, which could be achieved by the controlled activation of the correct network of nephric transcription factors. The injected iPSC-derived renal progenitor cells robustly engrafted into damaged tubuli and restored renal function and structure in cisplatin mice with AKI . SimilarInduced pluripotent stem cells are regarded as the best source for producing new kidney tissues for transplantation, which are potentially derived from patients and used as a renal replacement therapy without immunosuppression. Although it is difficult to regenerate the entire kidney in vitro, recent advances in the field of SCs have enabled in vitro generation of organoids –73.The kidney organoid formation involves stimulation of a stepwise differentiation process, in which movement occurs from monolayered iPSCs to primitive streak then intermediate mesoderm to kidney lineage cells –71, 74. Bioengineering represents one possible approach for generating the complex branching structures of the kidney. Scaffolds are temporary physical support obtained from a variety of biomaterials and help to accommodate cells and support their three-dimensional (3D) growth during tissue developmental stage . ScaffolAlthough not yet clinically available for transplantation, hiPSC-derived kidney organoids provide dominant models for studying the pathophysiology of kidney diseases, through which we can better understand gene mutations and disease phenotypes of the disease, mimicking disease progression. Preserving the naturally occurring genetic mutations as well as the genetic background of their parental somatic cells, hiPSCs can be applicable to disease/patient-specific modeling. Genetically characterized hiPSCs have been efficiently derived from patients with autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive PKD (ARPKD), Alport syndrome, systemic lupus erythematosus, and Wilms’ tumor and patients undergoing hemodialysis , 92–97. Recently, gene editing technologies such as CRISPR/Cas9 make it possible to treat inherited kidney diseases by correcting the pathogenic mutation in an iPSC line. For example, a family with adult-onset autosomal dominant focal segmental glomerulosclerosis (FSGS) was recently found to carry a new germline missense heterozygous mutation (p.G189R) in the octapeptide domain of the transcription factor PAX2 . TrionfiBesides the therapies based on HSCs, MSC-based therapies are currently the most advanced SC-based therapies in terms of clinical testing. Firstly, MSCs can be readily expanded in culture, generating a large number of therapeutic doses. Secondly, MSCs do not express blood group, DR antigens, and costimulatory CD40, CD80, and CD86 proteins to render them hypoimmunogenic, thus facilitating their safe use in allogeneic off-the-shelf protocols . AccumulThe priority of MSCs to traffic to the sites of ischemia, hypoxia, and inflammatory response following injury plays a crucial role in the success of these as cellular therapy for organ injuries. MSC homing is defined as the arrest of MSCs within the vasculature of a tissue followed by transmigration across the endothelium –108. TheTreatment with intravenous injection of MSCs has efficiently induced improvement of the repaired kidney both morphologically and functionally. Therapeutic properties of MSCs were originally derived from their engraftment in the injured kidney and subsequent transdifferentiation of MSCs into renal-specific cells to repopulate the kidney. The possible role of MSCs in response to kidney injury was first observed when the female kidney transplants were transferred into male recipients, in which the Y chromosome staining of tubular epithelial cells was found , 124. SeMSCs secrete a broad repertoire of cytokines, chemokines, and growth factors, including IL-6 and IL-11, vascular endothelial growth factor (VEGF), granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), basic fibroblast growth factor (bFGF), monocyte-chemoattractant protein-1 (MCP-1), insulin-like growth factor (IGF), leukemia inhibitory factor (LIF), and hepatocyte growth HGF , 130–133Rather than being a constant mixture of molecular factors, MSCs’ secretome is known to be dependent on the diverse stimuli present in the microenvironment that MSCs encounter. The crosstalk and interplay of MSCs and local environment reversely control and regulate the paracrine activity of MSCs. MSC growth conditions such as oxygen tension, growth factor composition, and mechanical properties may serve to directly influence paracrine activity , 149. DaMSCs comprise immunomodulatory and tolerogenic properties. Extensive in vitro and in vivo studies have demonstrated that MSCs are capable of suppressing T cell proliferation, influencing dendritic cell maturation and function, suppressing B cell proliferation and terminal differentiation, and modulating other immune cells such as natural killer (NK) cells and macrophages. Besides, MSCs also induce regulatory T cells (Tregs) and maintain the capability of Tregs to suppress self-reactive T-effector responses . The immClinicalTrials.gov).Despite promising preclinical results obtained in animal models, which indicated effectiveness of MSCs in reducing acute and chronic kidney injuries, clinical trials still remained in the early phases and largely aimed to investigate the safety and efficacy of MSC infusion. To date, more than 40 clinical trials were conducted worldwide, completed or ongoing, involving the use of MSCs in the treatment of kidney diseases as reported by the US National Institute of Health database  or severe adverse events (SAEs). Moreover, MSC infusion prevented postoperative renal failure (0% versus 20% AKI incidence when compared to case controls) and the length of hospital stay and readmission rates were reduced by 40% , 160. A Although clinical translation of MSC-based therapeutics for human CKD is still in an early phase, it has demonstrated the potential to modify the progression of CKD. A single-center, randomized, placebo-controlled, phase II/III clinical pilot study investigated the safety and therapeutic efficacy of MSC-derived EVs in CKD patient stage III and IV after following up for a period of 12 months. Although kidney biopsy specimens that are obtained 3 months after the therapy showed no significant histologic changes, EV treatment exhibited a significant improvement in renal function  . A multiThe safety and tolerability of MSCs in patients with LN have also been described. Numerous clinical studies have shown that allogeneic MSC transplantation in patients with SLE resulted in the amelioration of disease activity, improvement in serological markers, and stabilization of renal function –171. A sGiven the immune-regulatory properties, MSCs have been administered to kidney transplant recipients, with the aim of controlling the host immune response towards the graft and minimizing immunosuppression, possibly leading to transplant tolerance. A previous study conducted by Perico et al. have reported that 2 recipients of kidney transplant from a living-related donor treated with autologous bone marrow-derived MSC autologous (BM-MSCs) have had transient renal insufficiency (engraftment syndrome) 7–10 days after single infusion . Kidney It is widely accepted that embryonic renal SCs with nephrogenic potential would deplete soon after (mice) or several weeks before (humans) birth. Interestingly, at least 6000 cells derived from different nephron segments are lost every hour in the urine, suggesting that the progenitor cells are required for continuous replacement of loss of cells during physiological processes . AdditioTo our knowledge, there is no single bona fide marker specific for RSPCs at present. Various attempts have been made to identify and characterize RSPCs from differentiated cells in adult kidney, including label-retaining assays, flow cytometry using cell surface markers, and side population assays.In general, SCs have a slow turnover rate and display minimal physiologic differentiation. When labeled nucleic acids were incorporated, BrdU or 3H-thymidine, SCs retain the label for longer periods of time, while other cells lose . Label-r+ and embryonic renal marker PAX2 [+ renal stem cells always co-express CD24 in adult human kidney tissue [+CD133+ cells had self-renewability and pluripotent differentiation potential, forming a subset of parietal epithelial cells (PECs) in Bowman’s capsule by expressing SC-specific transcription factors Oct-4 and BmI-1 [+CD133+ cells from adult human kidney represent a multipotent adult resident stem cell population [+CD133+cells could be isolated from human urine, which exhibit the same phenotypic and functional features of RSPCs obtained from human kidney tissue and show the potential to differentiate into tubular cells as well as podocytes [By using SC marker expression analyses, the RSPCs were identified in the proximal tubules, in the medullary papilla region, and in the Bowman capsule of human kidneys, which are commonly characterized by the expression of CD133ker PAX2 , 190. Fuy tissue . The CD2nd BmI-1 . Numeroupulation , 191–193odocytes . Moreoveodocytes , 196.high cells expressed CD24 and CD133 and displayed typical SC properties such as sphere formation and anchorage-independent growth [Other selection strategies based on functional assays were utilized to identify and select RSPCs from human renal tissue. With the ability to effectively efflux the Hoechst dye, RSPCs can be detected via flow cytometry and are termed as the side population (SP) cells, which were found in murine kidney interstitial space in several groups –200. Aldt growth . Althoug+ cells are capable of differentiating into epithelial or endothelial cells in in vitro and in vivo [+ cells into some proximal and distal tubules of SCID mice with glycerol-induced acute tubulonecrosis were observed [+CD24+PDX− cells when injected into mice with adriamycin-induced nephropathy glomerular, which are engrafted into the glomerular structures and tubular structures, accounted for 11.08 ± 3.3% of total podocytes and 7.5 ± 1.9% of all proximal tubular cells in mice. The results of intravenous injection of CD133+CD24+PDX− cells reduced proteinuria and improved chronic glomerular damage of SCID mice suffering from nephrotic syndrome [Experiments in rat and mouse models of tubular or glomerular damage suggested a therapeutic effect of adult RSPCs of different types and sites of origin. The mechanism of RSPCs involved in kidney repair is associated to integrate into nephrons and is in parallel to a paracrine mechanism based on renoprotective molecules. The activated RSPCs would differentiate along a particular cell lineage and replace the damaged kidney cells. During acute kidney injury, the LRCs in the papilla migrated to the upper papilla and formed a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury . The eve in vivo , 203, anobserved , 31, 204syndrome . Shen etsyndrome . In addisyndrome , 199. Hosyndrome .+/CD73+ progenitors induce erythropoietin under hypoxia and inhibit prolyl pydroxylase [+ renal progenitor cells contributed to the protection against fibrosis, limiting the presence of pro-inflammatory in SCID mice with AKI [A variety of cytokine/growth factors including IL-15, endothelial growth factor (EGF), and TNF-α that are secreted by RSPCs were detected, supporting a paracrine mechanism, similar to that described for MSCs might also contribute to systemic tissue repair , 208. Buroxylase . Aggarwawith AKI . Recentlwith AKI . Hishikawith AKI . Also, twith AKI . Since tDespite the important role played in the repair and regeneration of damaged kidney tissue, a study showed that the dysregulated proliferation of renal progenitor cells generates hyperplastic glomerular lesions, scarring, and nephron loss eventually leading to degenerative disease such as crescentic nephritis or collapsing glomerulopathy .Many researchers believe the presence of renal stem/progenitor cells, but a debate in this field is still going on and some different theories have been put forward. Kusaba et al. have showed that fully differentiated epithelial cells accomplish proximal tubule repair through reversible dedifferentiation and proliferation, without any contribution from the preexisting intratubular SC or progenitor population. In their study, injury to proximal epithelial tubule induced expression of markers of putative epithelial SCs in human kidney . What is more, no dilution of fate marker was observed when mice with completely labeled kidneys were subjected to injury and repair, indicating that unlabeled progenitors do not contribute to kidney repair . Berger Stem cells have shown great potential in kidney injury repair and disease treatment, but some pressing issues remain to be resolved before the clinical application. ESCs have limited clinical application due to ethical issues regarding their origin, tumorigenicity after transplantation. As the donor cells most likely do not originate from the recipient patient, immunological rejection of the cells differentiated from ESCs upon allogeneic cell transplantation remains a major challenge for cell therapy. Simultaneous administration of immunosuppressive drugs can aid in overcoming these problems but can induce serious side effects . It has Despite considerable body of evidence in in vitro supports the usefulness of MSCs, controversies about the use of SCs still persist. In clinical trials, the improvement of renal function and structure induced by MSCs in clinical trials is not as obvious as expected. The main reasons for the limited clinical efficacy were the low engraftment, the poor survival rate, and the impaired paracrine capacity of injected cells in vivo. The majority of grafted MSCs might be trapped in the lungs, liver, and spleen. For those cells engrafted into the target tissues, the harsh microenvironment still would induce apoptosis in over 90% of transplanted MSCs in 1 week. To improve the beneficial effects of MSC transplantation, the efficient strategy of administration of MSC needs to be discussed. A recent meta-analysis of MSC administration in kidney disease revealed that cell administration > 1 day after injury yielded greater therapeutic value than within 24 h of injury . Liu et The immune rejection of stem cell injection is another challenge that hinders the clinical application of stem cell-based therapy. The allogeneic cells derived from hESCs will be robustly immune rejected by the recipient due to the direct activation of T cells by the foreign MHCs. While the patient-specific iPSCs share the same genetic background, and their derivatives might have a better chance to be immune tolerated, the genomic instability and abnormal epigenetics associated with iPSCs can induce immunogenicity in some cells of their derivatives . MSCs haWhatever the sources or the approaches utilized such as ESCs, iPSCs, MSCs, or RSPCs, it is widely acceptable that there is an enormous hope underlying the usage of SC therapy as a clinically viable alternative for kidney diseases. Recent advances in cell-based therapy using various cell sources have demonstrated great promise towards restoring normal kidney functions in the AKI, DN, and CKD models, etc. The kidney organoids induced by stem cells provide the authentic and practical models for investigating kidney development and disease and progressing understanding about tissue regeneration, drug screening, and disease modeling. Although the stem cells from different sources show great promise as therapeutic agents for kidney diseases, the amount of research currently available is insufficient to achieve the docking of different kidney diseases with the most suitable transplanted stem cells. But interestingly, because of their immunomodulatory properties, MSCs may be the most promising candidates for stem cell therapy of autoimmune-related kidney diseases. In addition, MSCs obtained from autologous sources in sufficient amounts allow early treatment of patients affected by AKI. There is a need to carry out appropriately designed experimental studies to verify long-term safety of these therapies and to examine the risks of fibrosis, maldifferentiation or malignancy, and AEs of systemic immune suppression. Only the results of larger, well-powered rigorously designed clinical trials will assist in determining the real clinical efficacy of SC therapy in kidney diseases. Additionally, there are more factors that should be taken into consideration in the clinical application of SC therapy, such as appropriate selection of cell types, number of cells required, and appropriate route of administration. These combined efforts favor the translation of experimental results into clinical practice in the near future.


Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation. As addressed by a recent position paper of the International Society for Extracellular Vesicle (ISEV) In this work, by comprehensively analyzing existing exosome isolation techniques, we provide suggestions and insights for future exosome separation methods and related applications. In addition, from the perspective of exosome-based diagnosis and therapeutics, we also emphasize the issue of quantitative exosome and microvesicle separation.Table 1). In this section, by analyzing principles, procedures, and advantages and disadvantages of individual techniques, we provide a panoramic view of current exosome isolation strategies. This overview not only facilitates the optimization of exosome isolation strategies in different applications, but also provides new outlooks for the development of novel devices and approaches for efficient exosome isolation.Exosomes are nano-sized extracellular vesicles distributed through vastly complex body fluids, which makes high-yield exosome isolation challenging Importantly, as we will discuss in Section 3, although vesicles prepared by current approaches are commonly denoted as exosomes, it should be noted that the term “exosome” is often used improperly in published articles or clinical trials. Apart from exosomes, the “exosome samples” prepared via current techniques also include a great number of non-exosome vesicles such as microvesicles, apoptotic bodies and ectosomes With the capacity to generate centrifugal forces as high as 1,000,000 ×g , ultracentrifugation is an optimal process for separating small particles including bacteria, viruses, and cellular organelles. As such, ultracentrifugation readily translates to exosome isolation and has contributed to many pioneering exosome explorations Differential ultracentrifugation, also referred to as simple ultracentrifugation or the pelleting method, is the most commonly reported strategy for exosome separation (45.7%) Due to ease of use, little technical expertise requirement, and compatibility with large volume preparation without complicated sample pre-treatment, differential ultracentrifugation has been widely employed over the past 30 years to isolate exosomes from various sources such as cell culture medium, serum, saliva, urine, and cerebrospinal fluid Fortunately, to further improve the exosome isolation efficiency of this classical separation technique, various types of centrifugation strategies have been developed during the past two centuries through the exploration of the different physical properties of objects. Among these strategies, a widely used method is density-gradient centrifugation, which separates particles by density . Historically, the density-gradient-based centrifugation method has been commonly used in hematological study for the separation of subpopulations of blood cells, due to the differences in density of different cell types -1In 1937, Linderstorm-Lang discovered that after centrifugation in a density-gradient tube, objects of a particular density would remain suspended in medium of a similar density 29However, such commonly used isopycnic ultracentrifugation depends entirely on the density difference between different solutes in samples. Although this method effectively separates exosomes from common contaminants such as protein aggregates, this method cannot separate extracellular vesicles with similar buoyant density (but different size) to exosomes  Despite various advantages and wide application, ultracentrifugation does have its shortcomings. For instance, although gradient ultracentrifugation is capable of purifying exosomes with minimal contamination, the processing volume of this method is limited by the thin loading zone Similar to conventional filtration methods, ultrafiltration uses an ultrafine Nano-membrane with different MWCO (molecular weight cut-off) to isolate extracellular vesicles from clinical samples or cell culture medium and differentiate between exosomes and co-vesicles by size Based on this principle, several simplified and easy-to-use ultrafiltration devices have been recently developed to facilitate the fast preparation of exosomes with yield comparable to that of the ultracentrifugation method Over the past decade, due to high efficiency (minutes for ultrafiltration vs up to 16h for ultracentrifugation) and simplicity , ultrafiltration gained increasing popularity, representing around 5.4% of the currently used exosome isolation methods Apart from vesicle clogging and trapping, the co-presence of nanoparticles with sizes comparable to those of exosomes presents another limitation of ultrafiltration In 1955, Grant H.L and Colin R.R invented a size-based separation technique termed size-exclusion chromatography (SEC) to isolate solutes of different molecular weights by passing aqueous solution through a column made of starch and water .In terms of exosome-based therapeutic application and functional studies, perhaps the most appealing feature of SEC is its ability to preserve the natural biological activities of the separated exosomes 67Despite various advantages, the SEC method also faces several challenges. According to a recent comparison study, exosomes prepared via SEC column commonly displayed wider size distribution, especially in the lower size range, suggesting the existence of contaminants with sizes similar to those of exosomes such as proteins aggregates and lipoproteins. To eliminate such contaminants, in the 2013 ISEV conference, Gardiner proposed an exosome isolation strategy by combining ultrafiltration and SEC Along with the size-dependent exosome isolation strategies discussed above, polymer-induced precipitation presents another commonly used strategy for exosome isolation. Analogous to ethanol-mediated nucleic acid precipitation, highly hydrophilic polymers interact with water molecules surrounding the exosomes to create a hydrophobic micro-environment, resulting in exosome precipitation .Existing polymer-based exosome precipitation methods generally employ PEG with molecular weights from 6000 to 20000 Da 75Typically, polymer precipitation-based exosome isolation is characterized by high yield. As shown in a recent comparison study using urinary samples, polymer precipitation achieved the highest yield of exosome and genetic contents  for subsequent profiling analysis, compared to differential ultracentrifugation and ultrafiltration methods ATPS has been widely used to separate various substances including cells, proteins, and metal ions The observation that some proteins and receptors that are common in all exosomes, regardless of their origin For effective immunoaffinity-based exosome isolation, antibodies need to be fixed on a solid surface for exosome separation. Over the past few years, matrices including chromatography, beads, plates, and various types of microfluidic apparatuses have been used Plates and microchips are also popular matrices on which to develop immunoaffinity-based exosome separation systems, in addition to the commonly used magnetic beads. For example, by using microplate, an anti-CD9 antibody-based system has been devised to capture and quantify exosomes from various types of mediums such as urine and blood 2+-dependent Tim4 protein, which specifically binds to phosphatidylserine, a protein highly expressed on the exosome surface 2+2+ through the addition of elution buffers containing Ca2+ chelators such as EDTA. Under such a gentle Ca2+ chelator treatment, the natural state of exosomes can be preserved. In another case, researchers from the Korea Advanced Institute of Science and Technology developed an Exosome-specific Dual-patterned Immune-filtration chip for specific exosome capture by introducing a chemically cleavable linker 3,3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) between the antibody (anti-CD63) and the solid immobilization surface Even though immunoaffinity-based exosome isolation ensures high-purity exosome isolation with an easy procedure, the non-neutral pH and non-physiological elution buffers (to separate exosomes from antibodies) associated with this method could irreversibly affect the biological function of the collected exosomes. The denatured exosome samples, although generally acceptable for diagnosis purposes (via assessing genetic and protein contents of exosome), are not favorable for exosome-based functional studies and various therapeutic applications .Although immunoaffinity allows separation of a specific subpopulation of exosomes, at the same time it raises concerns about isolation of only the specific populations of exosomes that possess the antibody-recognized proteins. Considering the vast number of heterogeneous properties of exosomes in body fluids, this would result in an analytical bias  39Taking this into account, apart from protein markers, other substances universally expressed on exosome membrane have been targeted, such as the saccharide chains  overexpressed on exosomal membranes , another option is to employ cheaper and more stable antibody substitutes, such as aptamer technologies. Aptamers, which are short single-stranded DNA or RNA sequences, can specifically recognize and bind to their targets with high affinity and specificity in a manner similar to antibodies in vitro chemical synthesis and exhibit several advantages such as low batch-to-batch variation, easy scaling up and down for different applications, extended shelf life, low or no immunogenicity, low production cost and easy chemical modification to improve binding properties 2+ and K2+) to the formation of specific three-dimensional structure of aptamers, the binding capacity of aptamers can be easily remodulated under mild conditions Even though antibody products possess distinct advantages, the high costs related to antibody development and production as well as their perishability significantly compromises their application, especially for large scale exosome preparation. To counteract these problems, apart from combined application with other methods as previously suggested 102in situ diagnosis  nanointerface Nonspecific binding is a big issue for microfluidic-based immunoaffinity isolation as the method is incompatible with extra blocking and washing steps. This is different than conventional bead- or plate- based immunoaffinity approaches, where nonspecific binding between non-exosome vesicles and the exosome-specific antibodies ) can be efficiently eliminated via stringent blocking and washing. In recent years, the advances in nanotechnology have provided valuable opportunities to address this problem. For instance, Ramanathan et al. presented a powerful microfluidic system for high-specific exosome capturing and analysis, facilitated by the tunable alternating current electrohydrodynamic (ac-EHD) mediated nanoscale lateral fluid flow  technique As discussed in Section 1.5, although immunoaffinity-based exosome isolation allows easy exosome isolation, this method is limited by isolation of only specific subset of exosomes, high cost, and difficulty in maintaining the natural structure of exosomes. Size-dependent microfluidic isolation and contact-free separation strategies are two types of the most successful instances to address this problem. Next, we will discuss these two types of instruments to demonstrate recently developed examples of microfluidic devices.The first size-dependent microfluidic system discussed is the well-documented Exosome Total Isolation Chip (ExoTIC) In addition to antibody and size-dependent microfluidics, rapid developments in microfabrication technology have enabled researchers to explore contact-free particle sorting mechanisms , for efficient, scalable, and high-quality exosome isolation.In recent years, the unique migration pattern of particles in non-Newtonian viscoelastic fluids has attracted great interests. As documented, the elastic lift force created by a viscoelastic medium flow was able to control and manipulate the position of particles in a size-dependent manner Figure A. Indeedin situ electronic manipulation, which facilitated the isolation of exosomes with preferred sizes Under the pressure of ultrasonic waves, particles with different mechanical properties  experience differential radiation forces In addition to elastic lift force and acoustic force, the simplicity of electroactive strategies  had also been explored for developing contact-free exosome isolation microfluidic systems Although the application of exosomes has been used in diverse therapeutic purposes such as drug delivery, novel cell-free vaccine development and regenerative medicine, there has been an emphasis on their potential for disease diagnosis, especially in non-invasive cancer liquid biopsy in situ non-invasive cancer detection To facilitate post-separation exosome imaging, Ashcroft and colleagues produced a novel immuno-microfluidic device featuring a mica channel surface It should be noted that the analytical sensitivity of reported “real-time on-chip exosome analysis” (including detection limit and response time) primarily depends on the specificity and binding capacity of the selected antibodies (for ELISA assay) as well as the sensitivity and compatibility of the utilized equipment. Therefore, such features of the analytical module need to be carefully investigated when designing real-time exosome analysis microfluidic devices.As discussed in Section 1, the basic and applied researches of exosomes have been obstructed mainly by two issues Extracellular vesicles consist of mainly microvesicles and exosomes. In theory, the concentration of any component in the extracellular vesicle can be achieved by the isolation of another component. In practice, efforts aiming at efficient “exosome and microvesicle separation” have relied mainly on obtaining purified exosomes via identifying highly specific exosome biomarkers. This is due to (1) the lack of understanding of microvesicles and 2) the great potential of exosomes displayed in both basic research and theranostic applications in recent years. Theoretically, this strategy is feasible as the exosome proteins (including nucleic acids) are not a random combination of cell fragments but are integrated by a strict protein sorting mechanism to maintain their stable protein expression  the greaIn the face of this reality, we believe it is necessary for all scientists engaged in extracellular vesicle-related work to consider the following questions: (1) Is it a mistake to rely solely on the identification of novel exosome markers for exosome and microvesicle separation? (2) Are 100% purified exosomes and microvesicle components necessary for current basic and clinical research? (3) What is the key speed-limiting factor for current exosome studies?Is isolating 100% purified exosomes necessary for current exosome studies? In fact, the key obstacle facing current exosome studies is not the impurity of exosome samples, but the lack of information about the proportion of exosomes and microvesicles in the collected “exosome” samples. This is understandable. From the perspective of exosome-based diagnosis and basic investigations, based on enriched exosome samples, rational experimental controls and optimized statistical models, as long as the composition of the studied samples could be accurately determined, the reliability of the assays could be ensured. For example, in functional studies conducted via siRNA-based gene regulation, it is unlikely, and not necessary, to completely inhibit the gene of interest. In general, an inhibition rate of around 80% is considered sufficient for most subsequent investigations. From the perspective of exosome-based drug development, according to the current drug approval system of most countries , unlike the high purity required for chemical compounds, the requirements for quality control and safety assessment of cell-derived compounds such as exosomes can be met as soon as (1) the exosome proportion is sufficiently high and (2) individual components of the preparation can be quantified and described Therefore, the key question of current exosome studies is how to effectively quantify the individual components of the collected exosome samples. However, we cannot achieve this goal by solely relying on exosome markers, as reflected in various immunoaffinity-based commercial exosome extraction reagents (using antibodies targeting exosome markers). Although they claim to be able to enrich exosomes, some crucial information, including the proportion (or purity) of exosomes in the extract, and the content of other components , is invariably absent. Surely, even if a perfect exosome marker with 100% specificity is available for exosome isolation (this would be very unlikely in practice), it still cannot guarantee that the remaining vesicles are all microvesicles, which may contain excess exosomes, apoptotic bodies or protein precipitates. Moreover, in the absence of understanding of the microvesicle, we run into issues in determining the specificity of the employed exosome marker.Since relying on biomarkers of only one component  cannot yield reliable quantitative information of individual components in the extracellular vesicle mixture, it seems that the only feasible method is to simultaneously employ both exosome and microvesicle markers. In fact, even if the applied biomarkers for exosome and microvesicle are not 100% specific, the respective markers of the two components combined with mathematical calculation  is also of great value to address the long-standing issue of quantitative exosome/microvesicle separation.For both diagnosis and therapeutic applications, researchers should carefully consider the strengths and weaknesses of the accessible strategies. As discussed previously, immunoaffinity capture promises selective isolation of highly purified exosomes of specific origins, or even subpopulations of exosomes from biological fluids. Therefore, in cases where diagnosis is scheduled for the subsequent investigations, immunoaffinity may present the most sensitive and specific method. Unfortunately, the current immunoaffinity method is compromised heavily by the lack of reliable markers for exosome isolation On the other hand, the therapeutic applications of exosomes are limited by the lack of an effective method to isolate high-quality exosomes in bulk Conversely, no microfluidic device has been readily applied in clinical applications, in spite of the remarkable advances achieved in recent years. Major roadblocks to clinical applications include standardization, scalability, and validation Furthermore, to facilitate the in-depth investigation of exosomes and their related biological functions, more efforts need to be made for the development of simultaneous exosome separation/quantification strategies and devices, to achieve not only efficient exosome isolation, but also real-time exosome quantification and analysis. Although several real-time exosome isolation/detection apparatuses have been reported, standardizing the analysis module for comparable and reliable readouts still represents a great challenge. In fact, compared to analysis, development of standardized exosome isolation methods constitutes an even harder task. Due to the heterogeneous features of biological samples, a reliable exosome separation technique suitable for every study is still not available. A bespoke selection of separation methods tailored for particular exosome-containing objects is imperative for high-quality exosome isolation and readout validation. As a result, future efforts may need to develop different exosome isolation standards to meet the particular properties of different types of biological samples and target particles  to be screened.The observation of exosome-mediated cell signaling provides a great opportunity for developing exosome-related basic and applied biomedical applications in various fields. Despite the revolutionary progress in exosome-based theranostic applications over the past few decades, there are still fundamental unanswered questions in the field. These questions address some of the hotspots of current biomedical research such as the secretory regulation mechanism of exosomes, exosomal content sorting mechanism and their intercellular transduction pathway. Various exosome separation strategies and devices have been suggested to facilitate the investigation of exosomes and their related biological functions. As comprehensively discussed in this work, standardization in exosome preparation such as specimen handling, isolation, and quantification has still not been established. Through studying the nature of particular samples and specific application settings, we believe careful selection of isolation techniques (or a combination of isolation techniques) will help investigators address many of the challenges faced in current exosome studies. In addition, we also believe that the exosome/microvesicle separation and quantification strategy (using both exosome and microvesicle markers) as suggested in this work can provide a plausible strategy to obtain accurate quantitative information for future exosome (and microvesicle)-related investigations.