Extracellular vesicles (EVs) have been described as important mediators of cell communication, regulating several physiological processes, including tissue recovery and regeneration. In the kidneys, EVs derived from stem cells have been shown to support tissue recovery in diverse disease models and have been considered an interesting alternative to cell therapy. For this purpose, however, several challenges remain to be overcome, such as the requirement of a high number of EVs for human therapy and the need for optimization of techniques for their isolation and characterization. Moreover, the kidney’s complexity and the pathological process to be treated require that EVs present a heterogeneous group of molecules to be delivered. In this review, we discuss the recent advances in the use of EVs as a therapeutic tool for kidney diseases. Moreover, we give an overview of the new technologies applied to improve EVs’ efficacy, such as novel methods of EV production and isolation by means of bioreactors and microfluidics, bioengineering the EV content and the use of alternative cell sources, including kidney organoids, to support their transfer to clinical applications. Stem cells have been widely investigated in preclinical and clinical studies for therapeutic purposes, including kidney disease, due to their plasticity and self-renewing and proliferative capacity . The rolIn general, EVs can be classified into three main subtypes based on their biogenesis, size and content: exosomes, microvesicles and apoptotic bodies. Exosomes are derived from the endosomal network, ranging from 30 to 100 nm in diameter, and are released to the extracellular space after fusion of multivesicular bodies (MVBs) structures with the plasma membrane . Since eIn addition to the common molecules present in the EV subtypes, those derived from stem cells possess a unique composition of bioactive molecules responsible for their regenerative properties. This was confirmed by omics approaches to characterize the RNA and protein profiles of EVs ,9,10,11.Despite the advances in understanding the role of EVs in cell communication and their regenerative capacity in kidney disease, their clinical application still requires further efforts. For this, new strategies are being developed, combining in-depth stem cell biology knowledge, new methods in EV isolation and changes in EV composition see . Here, wThe biological effects of EVs are given by their cargo, for which the molecular composition is directly related to the cell origin. Mesenchymal stromal cells (MSCs) represent one of the main sources of therapeutic EVs in kidney disease models . AlthougBMMSCs are adult nonhematopoietic stromal cells that have been widely used as a source for therapeutic EVs in various disease models over the last years ,46,47,48New strategies to improve BMMSC-EVs have focused on their biodistribution. This is an important element of their therapeutic efficacy as clearance is rapidly mediated by the innate immune system, reducing their availability for damaged tissues . An inteThe combination of BMMSC-EVs administration with other treatments can also lead to improvements in kidney recovery. The use of pulsed focused ultrasound (pFUS), a short-duration high-intensity pulse of sound waves, led transiently to the release of local inflammatory and chemo-attractive signals . The comAdipose tissue has emerged as an alternative source of MSCs, requiring minimally invasive procedures and having a higher yield in the MSCs isolation (about 2500-fold higher) . BesidesRecently, ADMSC-EVs have been described to support kidney recovery by modulating the inflammatory response in a sepsis-induced AKI model. Administration of these EVs led to the increase in Sirtuin 1 expression and reduced NF-kB levels, resulting in animal mortality decrease . Further2 pressure, mimicking physiological conditions, led to increased EV secretion with improved biological effects [New strategies have been applied to improve the therapeutic potential of ADMSC-EVs. The maintenance of ADMSCs under low O effects . AdminisSeveral reports have shown that PDMSCs can originate from different parts of the placenta, such as the amniotic membrane, chorionic plate, decidua parietalis and umbilical cord. The effectiveness of PDMSC-EVs to promote kidney cell proliferation, improve kidney function, reduce fibrosis and support angiogenesis has been reported ,53. ReceKidney tissue presents a progenitor-like cell population that may be responsible for tissue maintenance and regeneration after damage ,55. ThesHuman Liver Stem Cells (HLSCs) have been described as a specific population isolated from normal adult human liver, capable of generating insulin-producing islet-like structures and of supporting the regeneration of liver parenchyma . The regSince the generation of iPSCs by the use of the reprogramming factors , different protocols have explored their ability to indefinitely proliferate in a dish and differentiate into almost all the cell types . Such a The study and production of EVs for kidney therapeutic purposes has mainly been performed in two-dimensional (2D) cell cultures. Although this is a simple and effective approach for collecting EVs, it does not reproduce the spatial organization of the native microenvironment and may alter biomechanical and biochemical cues that influence cell behavior . For insEVs from kidney organoids may also advance their use in therapy. Urinary EVs mainly originate from kidney epithelium and have been suggested as a method of intra-nephron communication among cells throughout the urinary tract . In effeThe capacity of EVs to transfer bioactive molecules led to the development of new therapeutic strategies based on the modification of their cargo . DiffereThere are two main approaches for bioengineering EVs: the direct and the indirect method . The dirThe strategy based on the indirect approach promotes changes in the cell of origin that will posteriorly result in the secretion of EVs enriched with a specific molecule. The cellular reprogramming of MSCs through genetic modifications is one of the main approaches used to improve EVs’ therapeutic effects . The oveThe new advances in gene-editing technologies, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) nucleases, have brought the possibility to accurately promote modifications in target genomic loci, allowing correction of mutations, regulating transcription and promoting changes in the epigenome . Due to The large amount of EVs required for preclinical and clinical applications is challenging. For clinical tests, a patient would need approximately 100 μg of EV/kg of body weight for each treatment. This requires expansion upscaling and prolonged maintenance of cells ,97. HencThe use of bioreactors has more recently been introduced as a possible solution to maximize the surface area for cell growth and, therefore, increase the capacity and efficiency of EV production. Bioreactor flasks offer a similar format to T-flask cultures, but can be designed to concentrate conditioned medium upon culture. Human embryonic kidney cells were cultured using this system, in the presence of a semi-permeable membrane that allowed continuous diffusion of nutrients and at the same time the accumulation of EVs . BioreacFurthermore, new dynamic systems that allow continuous perfusion-based cultures have been developed to enable simple media exchange and cell/conditioned medium separation. Such systems can support cultures over an extended period with high productivity. A common example of such systems are hollow-fiber bioreactors, which are 3D systems that use multiple parallel semi-permeable capillary membranes to allow the transfer of nutrients to and waste products from the cells seeded on the extracapillary space . Using tIt is worth mentioning that cell expansion in bioreactors may result in phenotypic alterations due to physicochemical differences compared to planar flask cultures, such as cell-to-microcarrier binding, mass transfer and shear stress produced by agitation (impellers) and oxygen sparging . These aThe isolation methods currently applied are based on the EVs’ physical and biochemical characteristics, varying through size, density, electrical charges and surface membrane composition see  116,117,145,146.Classical ultracentrifugation was the first and most commonly used method for EV isolation . This prThe different sizes of EVs have been used for designing novel isolation protocols. Examples such as the ultrafiltration system and tangential flow filtration (TFF) use membranes with a defined molecular weight cut-off ranging from 10 to 100 kDa and have been used to isolate EVs from urine-derived cell culture medium ,126. AnoAnother approach in EV isolation is the affinity-based technique, targeting specific proteins at the EV surface membranes. In this respect, immunoaffinity is most broadly used, which relies on the application of antibodies to sort a group of EVs that presents specific surface proteins . A similFurthermore, advances in microfluidics allowed the development of new platforms to efficiently isolate EVs from body fluids on a larger scale ,145,146.The current advances in EVs studies hold a great promise for the treatment of kidney diseases. The bottleneck in the translation to clinical applications has been widened by the new achievements in the manufacturing and isolation of EVs, allowing large-scale productions that can be easily transported and stored for long periods (long shelf life). Besides, the possibility of bioengineering EVs allows the designing of personalized treatments based on the patient’s kidney pathophysiological aspects. Future challenges concern the standardization of EVs production and the development of methods to control the quality and safety of therapeutic EVs. Such achievements will contribute to translate EVs into the clinic as a diagnostic tool and therapeutic strategy in the treatment of kidney diseases.


Stress is broadly defined as the non-specific biological response to changes in homeostatic demands and is mediated by the evolutionarily conserved neuroendocrine networks of the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Activation of these networks results in transient release of glucocorticoids (cortisol) and catecholamines (epinephrine) into circulation, as well as activation of sympathetic fibers innervating end organs. These interventions thus regulate numerous physiological processes, including energy metabolism, cardiovascular physiology, and immunity, thereby adapting to cope with the perceived stressors. The developmental trajectory of the stress-axis is influenced by a number of factors, including the gut microbiome, which is the community of microbes that colonizes the gastrointestinal tract immediately following birth. The gut microbiome communicates with the brain through the production of metabolites and microbially derived signals, which are essential to human stress response network development. Ecological perturbations to the gut microbiome during early life may result in the alteration of signals implicated in developmental programming during this critical window, predisposing individuals to numerous diseases later in life. The vulnerability of stress response networks to maladaptive development has been exemplified through animal models determining a causal role for gut microbial ecosystems in HPA axis activity, stress reactivity, and brain development. In this review, we explore the evolutionary significance of the stress-axis system for health maintenance and review recent findings that connect early-life microbiome disturbances to alterations in the development of stress response networks. This allegory has often been applied to societal and cultural politics; however, it further extends relevancy to that of biological life and the maturation of its complex and multifaceted physiological systems. The developmental origins of diseases are often best viewed using an evolutionary lens to examine the underpinnings of when the affected physiological systems originated, as well as how and why they have been adaptively selected. Understanding biological systems from their inception provides insights into malfunctions that have occurred under modern environmental conditions.William Shakespeare’s “The Tempest” is famed for its literary citation, From an evolutionary perspective, physiological stress response systems have always been indispensable for organisms to appropriately evaluate the stochastic or unpredictable aspects of their environments and adapt accordingly to maintain homeostasis and ensure their survival. Therefore, the broad concepts of stress and homeostasis are interwoven, whereby homeostasis is the maintenance of relatively stable internal bodily compartments in the face of changing external conditions by using feedback mechanisms to vary internal activities and minimize deviations from established physiological set points. Stress, by contrast, perturbs homeostasis, and stress responses are the physiological cascade of events that occurs when an organism attempts to re-establish homeostatic norms in the face of perceived threats. The stress response, therefore, has clear and fundamental adaptive advantages, and evidence has shown the molecules and peptides that regulate physiological responses to stress have remained remarkably conserved for over 500 million years of vertebrate evolution .Similarly, immunity has existed for hundreds of millions of years as a vital physiological system that protects the host from internal and external dangers to infections and changes in homeostasis . TherefoHowever, despite the adaptive utility of acute stress and immune responses , chronicAn evolutionary mismatch that has gained attention in the past decade is the loss of diversity and keystone species from the communities of microbes in the human gut (the microbiota) and their genetic material (the microbiome), which is hypothesized to be at the root of the increased psychological-  and immune-  related chronic diseases . The eveAll organisms, including humans, continually encounter internal and external changes in the environment  which may trigger a stress response that overturns their internal rhythms and homeostasis. Sufficient deviations from established set points constitute a “stress” to the organism, which are addressed by various bodily responses to allow the animal to overcome the threat and re-establish homeostasis. Thus, the concept of stress is closely related to the concept of homeostasis, whereby the latter represents an established optimal state for key biological parameters, while the former is a state that arises when homeostasis is disrupted .Perception of a stressor (conscious or not) is communicated to the body via the central nervous system (CNS), which acts both neuronally for an immediate reaction, and hormonally for a delayed, prolonged response . TherefoPrimary responses involve two biochemical axes that elicit cellular changes in the adrenal gland when a “threat” is perceived - one through the fast-acting sympathetic nervous system (SNS) and the other through the slow-acting hypothalamic-pituitary-adrenal (HPA) axis. Both axes of the primary response are readily activated, and their actions are designed to prioritize vital bodily functions that prepare the organism for rapid engagement against the stressor .Following perceived threats, SNS responses are immediately propelled through efferent sympathetic nerve fibers that result in the circulatory release of catecholamines (epinephrine (EPI) and norepinephrine (NE)) from innervated cholinergic receptors on chromaffin cells in the adrenal medulla. EPI and NE are also immediately released from fibers that directly innervate other bodily organs, such as the heart. The release of catecholamines directly into tissues and circulation have immediate and potent effects on respiration and cardiovascular systems , blood oThe HPA axis is the second primary stress response axis, and involves the delayed release of glucocorticoids (GC)  from the adrenal cortex. Following a stressful stimulus, the hypothalamus responds to elevated NE by secreting corticotropin-releasing hormone (CRH) from neurosecretory neurons in the paraventricular nucleus (PVN) of the hypothalamus to influence the rapid release of adrenocorticotropic hormone (ACTH) from the anterior pituitary into circulation . ACTH thSecondary responses involve changes in plasma and tissue ion and metabolite levels, modulation of immune responses, and general physiological adjustments , while tThe categorization of stress into these defined levels is dependent on many factors, including the type or intensity of a stressor, the age and sex of an individual, as well as the organism’s own perception of the threat to its homeostasis . For exaSex differences are another important factor that affects the HPA response to stress in both humans and animals. In general, HPA-related stress molecule levels are higher in female rodents and rise faster than in males following HPA axis stimulation . Female The origin of the vertebrate stress axis is centered on an ancient neuroendocrine circuit  that has remained remarkably conserved over vertebrate evolution, despite the emergence of phylogenetically distinct classes of animals  occupying a vast range of habitats over that same period . Its pivRecently, there is an increasing awareness that colonization with gut microbes also plays an important role in the appropriate early-life development and functioning of the HPA axis in host species , and raiThe precise mechanisms by which regions of the brain recognize microbial signals from the gut have only recently been investigated, with promising candidate pathways discussed in more detail in later sections of this review. However, on the whole, a clear understanding of the bi-directional communications that exist between the gut and the brain continues to elude researchers. Given the symbiotic history of microbes and vertebrates , it is nUnderstanding the complex relationship between hosts and microbiomes from an evolutionary perspective helps to illuminate the influence the microbiome has had on the physiology of vertebrate hosts  and viceBased on the findings of these and other related studies, it is thought that animals who harbor microbial communities with specific metabolic properties or functional abilities are better able to resist and respond to environmental perturbations . This woThe influence of the microbiome on the development of the adaptive immune system in vertebrates is another example where the host and microbiome mutually benefit. For example, adaptive immunity mediates tolerance of the gut microbiome through IgA by driving perpetual surface structure modification in microbes that ultimately results in reduced proinflammatory signaling by immune factors in the host, and maintains a non-inflammatory connection to commensal gut microbes . Thus, aThe development of a robust HPA axis system has clear adaptive advantages for animals to manage various stressors. Coordination with other physiological systems is a critical aspect of the stress response to appropriately deploy necessary resources and efficiently maintain homeostasis . The welVertebrate immune responses are broadly categorized into the innate  and adaptive  immune systems. The splitting of immune functions into two branches occurred with the emergence of jawless vertebrate agnathans  500 millions of years before present (mybp), and has continued to diversify throughout vertebrate evolutionary radiation . The innThe connections between stress and immune systems are complex, multifaceted, and have deep evolutionary underpinnings that shaped their coordinated and bidirectional communications . The immNeurotransmitters, neuropeptides, and hormones of the SNS and HPA stress axes orchestrate a careful and balanced integration between stress responses and immune activity, and there is a plethora of evidence that the molecular machinery required to respond to these stress-derived molecules are present in immune cells . SimilarThe goal of this bi-directional communication is to co-regulate and coordinate responses to maintain homeostasis. For example, HPA-derived molecules, like CRH, are both immunosuppressive as a hypothalamic regulator of anti-inflammatory GCs , but canThe ubiquitous expression of GRs on various immune cell types mediates GCs influence on cell phenotype and function , includiAlthough the immunosuppressive and anti-inflammatory actions of GCs cover both acute and chronic exposure scenarios, the beneficial outcomes  are mostly reserved for short-term scenarios, with detrimental pathologies occurring over the long-term, such as increased infections, prolonged wound-healing, and decreased antibody production following vaccination . Stress-While the HPA axis and immune system exhibit remarkable evolutionary conservation, differences arise when we examine the ontogeny of the HPA axis during gestation across species. For example, the placenta of anthropoid primates, including humans, has evolved the unique ability to produce CRH, forming a cooperative maternal-fetal-placental endocrine unit that is not present in less-derived mammals . Placent4–5isomerase (3β-HSD)  . Apart f(3β-HSD) , fetal 3(3β-HSD) . This in(3β-HSD) , suggest(3β-HSD) . Finally(3β-HSD) .Development of the fetal HPA axis relies on maternal and placental signals via the maternal-fetal-placental unit, which represents a complex endocrine network, with the placenta acting as an integration site for signals between the mother and fetus . Unlike Placental 11β-HSD2 expression fluctuates over gestation and differs with fetal sex, with low levels during early and late gestation, and in pregnancies with females , enablinBeyond the prenatal period, the development of the HPA axis continues into early life, with stressors and environmental exposures continuing to influence its maturational trajectory either positively or negatively . The effThe gut microbiome represents a complex ecosystem with significant functional capacity, as these microorganisms encode a significant amount of genetic material beyond that of the human genome . This geThe composition of the gut microbiota in early life is primarily determined by the ecological frameworks of selection and priority effects. Along the length of the gastrointestinal tract, environmental factors such as pH, motility, temperature, and nutrient availability select for the consortium of microbes that occupy a given ecological niche, with the majority of microbes residing within the colon . In paraIn humans, initial colonization of the infant gastrointestinal tract begins following rupture of the amniotic sac, as the placenta is a sterile environment, with the exception of uncommon pre-labor pathogens . During Colonization patterns during these first few years are influenced by a number of prenatal and postnatal factors, including maternal health , gestatiSubstantial research efforts have sought to define the patterns of microbial colonization occurring in the first years of life , in an aLactobacillus dominance , and other neuroactive molecules  derived by commensal gut microorganisms and the host . MultiplPioneering studies in the 1990s showed that microbes could not only respond to vertebrate hormones and neuromodulators , but werSerotonin (5-HT), for example, is a critical biogenic indoleamine that is synthesized from the amino acid, tryptophan. It functions as a neurotransmitter in the brain and is well-documented to regulate mood and cognition . In the Lactobacillus and Bifidobacterium  , where Gs adults , includiApart from LPS, other microbiota-derived constituents, like cell wall peptidoglycans, can translocate beyond the BBB during postnatal development in healthy mice and activate neuronal pattern-recognition receptors (PRRs), like nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and peptidoglycan recognition protein 2 (pglyrp2), both of which are involved in neurodevelopmental processes . NOD2 isGut bacteria convert host-indigestible dietary fibers  into SCFAs  through fermentation in the gut. These microbial-derived metabolites are essential for host health and are utilized for many physiological functions, including epigenetic changes in chromatin structure , gut funFaecalibacterium and Coprococcus bacteria were consistently associated with higher quality of life indicators, and were depleted in those with depression, even after correcting for antidepressants  mice have altered phenotypic proportions with more cells resembling the ramified, surveying phenotype, and had globally defective immune responses to infection challenges . These aicroglia . Similaricroglia . DespiteEarly-life environmental exposures play a critical role in the development and functional programming of central neural circuitry, including the HPA axis , with seBifidobacterium infantis. Interestingly, this exaggerated response was partially rescued following colonization of GF animals only at an early developmental time point. The absence of a rescue effect with microbial colonization at later stages implied a critical window exists during which the microbiome is able to influence the developmental programming of the HPA axis. Subsequent studies in GF animals have revealed similar findings, including elevated CRH mRNA in the hypothalamus and reduced GR mRNA expression in the hippocampus, indicative of greater HPA axis activity and reduced potential for negative feedback inhibition  and hippocampus . InvestiLachnospiraceae and Ruminococcaceae are widely reported, often decreasing following a stressor , relative abundance, and specific taxa being reported across numerous studies. However, inconsistencies in these shifts are common, likely due to differences in the degree and timing of stress imparted during stress paradigms, rodent strains, and animal housing conditions. Commonly observed trends include decreased alpha-diversity and shifts in the relative abundance of the dominant phyla, Bacteroidetes and Firmicutes, with increases in potentially pathogenic and facultative anaerobic Proteobacteria . These cstressor . Taxa bestressor , found istressor  and healstressor , suggestfovibrio , reductiermansia , and incellaceae  are commtabolism  and is otabolism , whereasammation , and redammation . TogetheWhile animal studies have revealed a causal relationship between the early-life gut microbiome and prenatal or postnatal stress exposures, the extrapolation of these findings to the mother-infant relationship and lifelong health trajectories in humans is constrained by the available data in humans and by the greater complexity of the human situation. Distinct differences exist between the study designs and data obtained in animal versus human studies, and while animal models are a valuable tool to establish proof of principle and examine relationships in the absence of confounding factors, they do not comprehensively reflect human data. At this time, human data exploring this subject area are limited and narrow in scope. Furthermore, it must be recognized that stress-microbiome associations in humans are embedded within social, institutional, cultural and economic structures that inform and shape the nature of these associations. For conclusive statements on the relationship between prenatal or postnatal stress, the infant microbiome, and stress response programming to be made in humans, comprehensive cohort studies including a broad array of factors that may contribute to or be protective against prenatal or postnatal stress and microbiome alterations are essential, such as paternal stress, caregiving practices , family dynamics, social environment, and economic status.Lactobacillus, one of the dominant taxa in the healthy vaginal microbiota that colonizes infants during birth  and Lactobacillus farciminis, and prebiotics, polydextrose (PDX) and galacto-oligosaccharide (GOS), have beneficial effects following stress insults, including reducing HPA axis reactivity to stress, CRH expression, and intestinal permeability in females; increasing HPA axis negative feedback inhibition through increased expression of GR, MR and CRH receptors; and ameliorating anxiety-like behavior and gene expression alterations in corticolimbic brain regions in males , and stress-immune-microbiome interactions may provide valuable insights into strategies to reduce or prevent undesirable health outcomes in infants exposed to significant early-life stress, or microbial alterations. The influence of kangaroo care and tactile stimulation on the microbiota and stress have been evaluated independently, with evidence of benefits for these practices on desired health outcomes . HoweverVO and EM equally conceptualized, researched, and authored the manuscript with editorial assistance from GG and M-CA. All authors approved the final manuscript.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.


To describe a novel technique for the reconstruction of geometrically complex defects of the midface using an osteotomized folded scapular tip-free flap.Five patients underwent maxillectomy with defects disrupting two or more of the following facial axes: orbital, nasofacial, and palatal axes. Patients underwent primary reconstruction using an angular artery-based scapular tip-free flap with an osteotomy to fold the flap. Harvest techniques, including placement of osteotomies, folding and plating, surgical esthetic, and functional outcomes, are presented.Osteotomies placed in the scapular tip-free flap allowed folding of the osseous flap and improved restoration of all three facial axes with a single flap. In one patient, the tip of the scapula was used to reconstruct the nasofacial axis, while the body and lateral border were used to reconstruct the palate. In four patients, the tip of the scapula was used to reconstruct the orbital axis, while the body and lateral border were used to reconstruct the nasofacial axis. Patients had successful oronasal separation, healed wounds withstanding adjuvant therapy, satisfactory orbital positioning and facial projection, preserved masticatory surfaces and opportunity for dental implants.The midface is geometrically complex and is one of the most challenging head and neck sites to reconstruct. Ablative defects in this area can disrupt facial axes resulting in poor esthetic and functional outcomes. This study demonstrates the reconstructive advantages of a novel osteotomized folded scapular tip-free flap. Due to the complex anatomy and variable effects on form and function, midfacial reconstruction is the most challenging head and neck reconstruction. Common goals in midfacial reconstruction include restoring oronasal separation, supporting the orbit, obtaining a healed wound in preparation for possible adjuvant therapy, reestablishing facial contour, and allowing for functional dentition. The reconstructive ladder includes the use of obturator prostheses, local, pedicled and free flaps. Bone reconstruction establishes long-term facial contour, supporting the facial envelope and globe, and has become standard for complex maxillary reconstruction.Loss of midfacial structures due to tumor extirpation, trauma, or congenital defects can have significant functional and aesthetic consequences,4. Previous studies have demonstrated the utility of the scapula tip for midfacial reconstruction–8. The scapula tip-free flap has advantages over the fibula or iliac crest related to the chimeric nature of the subscapular vascular system allowing for multiple tissue types, long vascular pedicle, ability to perform extirpation and flap harvest concurrently, acceptable donor site morbidity, and excellent versatility in rebuilding 3-dimentional bone structures of the craniofacial skeleton,6,8,9. The scapula tip can be placed horizontally to reconstruct the palatoalveolar complex or vertically to restore the zygomaticomaxillary and nasomaxillary buttresses,10. However, some authors report difficulty orienting the scapula tip bone to simultaneously reconstruct the vertical and horizontal buttresses in complex maxillary defects.The radial forearm, fibula, iliac crest, and scapula osteocutaneous free flap have been described for use in boney reconstruction of maxillary defects. However few have described this technique and surgical outcomes in midfacial reconstruction. Here, we present five cases of complex midfacial reconstruction performed with scapular tip-free flap with an osteotomy to allow for folding of the flap and simultaneous restoration of multiple facial axes.Osteotomizing the scapular tip-free flap has been described in mandibular reconstruction. Free flap harvest was performed concurrently with tumor ablation. The anatomy of the subscapular arterial system, when viewed from a semidecubitous position, is shown in This study was approved by the University of Michigan IRB (HUM 00116093). Patients underwent surgical resection of tumors, which included maxillectomy without orbital exenteration. Maxillectomy defects differed but resulted in the disruption of two or more of the following facial axes: orbital, nasofacial, and palatal axes . In all Open osteotomies were performed with a “green stick” fracture method to maintain continuity of the periosteum to ensure adequate blood supply to the distal segments. After measurement of the boney defect in both planes, the scapula is harvested in the standard fashion. The subscapularis muscle and infraspinatus muscle are cut sharply off the scapula to allow for a more precise osteotomy. There is no extra length of bone needed as the greenstick fracture of the scapula does not put the muscular attachments on tension, and there is still excellent blood flow from the angular artery to both segments. Depending on patient preference, either the left or right scapula can be used with minimal change in the pedicle and/or boney geometry. For defects where the scapular tip recreates the orbital axis, the pedicle comes off the front of the face of the maxilla and over the mandible. For defects where the scapular tip recreates the nasofacial axis or palatal axis, the pedicle comes off posteriorly and is brought through the parapharyngeal space.The demographic information, defect description, and classification of patients who underwent osteotomized folded scapula tip-free flap are included in , and an osteotomy was created [A 50-year-old woman diagnosed with osteosarcoma of the left maxillary sinus was treated with neoadjuvant chemotherapy followed by maxillectomy without orbital exenteration. The surgical defect included the left hemipalate, premaxilla, alveolar, zygomatic and frontal processes of the maxilla, and a portion of the orbital rim, disrupting the nasofacial and palatal axes . Reconst created . The sca created . The tip created . Follow-A 64-year-old man diagnosed with squamous cell carcinoma of the left maxillary sinus was treated with maxillectomy without orbital exenteration with a surgical defect including the orbital floor and rim and superior half of the maxilla, including the frontal and zygomatic processes. This resulted in disruption of the orbital and nasofacial axes . The palA 29-year-old woman with osteosarcoma of the left maxilla was treated with neoadjuvant chemotherapy followed by maxillectomy without orbital exenteration and extensive buccal space resection. The surgical defect was a total maxillectomy including the left orbital floor and rim, premaxilla, alveolar and frontal processes of the maxilla, and the left hemipalate, disrupting the orbital, nasofacial and palatal axes. A reconstruction plate was placed along the orbital rim across the maxilla. The scapular tip was harvested as above, and an osteotomy was created to allow for folding. The tip of the scapula was used to reconstruct the orbital floor, while the body and lateral border were used to reconstruct the nasofacial axis, similar to Sixty-year-old man with squamous cell carcinoma of the right maxillary sinus treated with right maxillectomy without orbital exenteration. The surgical defect included the orbital floor and rim and superomedial aspect of the maxilla, including the frontal process and piriform aperture. The lateral buttress and palate remained intact. A reconstruction plate was placed along the inferior orbital rim from the nasal bones to the lateral orbital rim. The scapular tip was harvested as above, and an osteotomy was created to allow for folding. The tip of the scapula was used to reconstruct the orbital floor, while the body and lateral border were used to reconstruct the nasofacial axis, similar to A 58-year-old man with deeply invasive basal cell carcinoma of the right facial skin and maxilla underwent maxillectomy without orbital exenteration, including facial skin. The surgical defect included the right orbital rim and floor, total maxillectomy including the alveolar and frontal processes of the maxilla, and the maxillary alveolus, disrupting the orbital, nasofacial, and palatal axes. A reconstruction plate was placed along the orbital rim. The scapular tip was harvested as above, and an osteotomy was created to allow for folding. The tip of the scapula was used to reconstruct the orbital floor, while the body and lateral border were used to reconstruct the nasofacial axis, similar to Reconstruction of midfacial defects is one of the most challenging in the head and neck due to complex anatomy and variable effects on form and function. Ablative defects in this area can reduce orbital and nasal support, loss of facial projection and significant tooth-bearing segments, and formation of oronasal or oroantral fistulae. An osteotomized and folded scapula tip-free flap can be successfully used for complex midfacial reconstruction.. The osteocutaneous radial forearm has limited bone stock for significant defects but can be successfully used in smaller maxillary defects. The fibula-free flap was historically considered the primary reconstructive option for complex midface reconstruction, given that it provides the longest bone stock and reliable bone for dental rehabilitation. To reconstruct multiple planes of the maxilla, the fibula-free flap can be layered; however, this results in complicated pedicle geometry and may predispose to flap complications and failure,19.Alternative vascularized bone flaps include iliac crest, osteocutaneous radial forearm, and fibula free flap. The opinions on the superiority of one flap over another vary and are dependent on surgeon and institutional preference. Published literature indicates that the iliac crest free flap has been largely abandoned given donor site morbidity, including hernia development. Case series at multiple institutions have indicated that osteotomies and dental implants can be used successfully in scapular tip-free flaps for mandibular reconstruction, although there is limited data on these techniques in maxillary reconstruction,20,21.Free flaps based on the subscapular arterial system provide the most versatile of the osseous flaps given the option for separate segments of bone, multiple skin paddles, and significant volume to fill the defect. The scapular tip-free flap from the angular artery provides a long pedicle length and has morphologic similarity to the palateHere, we illustrate surgical techniques for osteotomizing an angular artery-based scapular tip-free flap for complex midfacial reconstruction. In very different maxillary defects, we describe how this technique can be used to simultaneously restore multiple planes of the 3-dimensional midface. All patients had excellent aesthetic and functional outcomes.In conclusion, an angular artery-based scapula tip-free flap with an osteotomy can be used to reconstruct complex midfacial defects with excellent aesthetic and functional outcomes. This is a unique surgical technique that allows for reconstruction of the horizontal and vertical buttresses of the midface with a single flap. This reconstructive option allows for oronasal separation, restoration of orbital support and facial contour, and functional dentition and mastication.


Vogt-Koyanagi-Harada disease (VKH) is a rare multisystemic inflammatory autoimmune disorder. Glaucoma secondary to VKH frequently occurs during the recurrent phase of anterior uveitis; however, acute angle-closure glaucoma (ACG) secondary to both VKH and human immunodeficiency virus (HIV) infection has rarely been reported. We describe a case of secondary acute ACG involving VKH, characterized by sudden vision loss, moderately elevated intraocular pressure (IOP), shallow anterior chamber, and fully or partially closed angle, in an HIV-infected patient. Both VKH and HIV infection contributed to the occurrence of ACG due to the leakage and forward rotation of the ciliary body, as well as choroidal effusion. The deterioration of IOP and serous macular detachment were observed after initial corticosteroid therapy. Visual acuity and IOP were improved with subretinal fluid absorption after continued corticosteroid therapy. Understanding the response of IOP and serous macular detachment after corticosteroid therapy is important for clinical practice. Human immunodeficiency virus (HIV) is a lentivirus that infects cells of the human immune system, causing its dysfunction . In 2017Certain patients diagnosed with VKH can develop acute angle-closure glaucoma (ACG), with ACG as the first symptom, including a sudden or gradual increment in intraocular pressure (IOP), shallow anterior chamber, and aqueous flare often accompanied by choroiditis and optic disc oedema . HoweverA 55-year-old man infected with HIV complained of pain in both eyes, headache, and epicranium ache for 2 days with an episode of fever (38.5°C). In addition, he did not have any systemic symptoms of HIV infection. The patient had normal best-corrected visual acuity (BCVA) for both eyes . His symptoms mainly included bilateral conjunctival oedema and superficial scleral hyperaemia without corneal oedema. Shallow peripheral anterior chamber depth (PACD) was evaluated for both eyes on slit-lamp microscopy, which was about one-quarter of the corneal thickness at the 6 o'clock position. In addition, his IOPs were 19 and 21 mmHg in the right and left eye, respectively. Central corneal thickness was 505 mm in the right eye and 494 mm in the left eye. Moreover, ultrasound biomicroscopy (UBM) revealed shallow anterior chambers  and angle closure in both eyes . Therefo+ T-cell count was 296 cells/μl.Unfortunately, the patient's BCVA decreased to 0.15 and 0.12 in the right and the left eye, respectively, 1 day later with keratic precipitate (dust type), which indicated acute uveitis. We realized that the previous UBM had already revealed both leakage and forward rotation of the ciliary body. Multifocal serous retinal detachments with hyperaemic optic discs were detected in both eyes through dilated pupils. Moreover, typical “multiple lake” hyperfluorescence and optic disc leakage in the late phase were also observed via fundus fluorescein angiography . In addiAfter the administration of 105 mg prednisone once a day for 1 week, the IOPs of the right and left eye increased to 23 and 26 mmHg, respectively, with the aggravation of the serous retinal detachment . After cThis study was reviewed and approved by the Institutional Review Board of the First Hospital of China Medical University, and written informed consent was obtained from the participant for the publication of this case report .In this study, we present a rare case of secondary ACG involving VKH and HIV infection. Acute ACG as the first ocular manifestation of VKH is not common, and its pathogenesis may be acute inflammatory oedema, damage to the blood-aqueous humor barrier in the ciliary body, or the forward rotation of the iris and lens . In addiTo our knowledge, glucocorticoids are the first-line agents for the treatment of VKH. Glucocorticoid therapy administered during the acute phase slows down the chronic process and effectively prevents its onset in many patients. The combination of glucocorticoids and immunosuppressive drugs can help in both early vision recovery and the effective suppression of inflammation . In thisIn this patient, the inflammation subsided after continued glucocorticoid treatment for 2 weeks, the acute inflammatory oedema of the ciliary body was reduced, the iris septum receded, and the anterior chamber angle opened . The serDue to their anti-inflammatory and immunosuppressive effects, glucocorticoids can reduce the synthesis of inflammatory cytokines, inhibit the accumulation of inflammatory cells at inflammatory sites, reduce capillary permeability, and destroy the blood-retinal barrier, thereby reducing exudation, inhibiting tissue swelling, and reducing exudate retention, improving oedema, and promoting absorption of the SRF .Increased autoimmunity and rheumatic diseases were observed in HIV-positive patients due to a disruption of the Th1/Th2 balance, stimulation of autoantibodies, chronic immune activation, immune confusion caused by HIV molecular mimicry, and increased cytotoxic T-cell response and immune dysregulation . The comThis case study has some limitations; we missed information about the HIV status of the patient, including the viral load. In addition, we did not follow-up on the patient's HIV treatment to investigate its effect on the progress of VKH and secondary ACG. Finally, the UBM results from the follow-up were lost, and we had to use a slit-lamp microscope to evaluate the opening of the PACD.In summary, this is the first report of secondary ACG in a patient with VKH and HIV infection. Both VKH and HIV infection contributed to the occurrence of ACG due to the leakage and forward rotation of the ciliary body and choroidal effusion. In clinical practice, it is important to understand the response of IOP and serous macular detachment after corticosteroid therapy.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Institutional Review Board of the First Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.RH conceived of and designed the study, acquired the data, responsible for administrative, technical, material support, and supervised the study. RH and XB developed the methodology, analyzed and interpreted the data, wrote, reviewed, and revised the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the Beijing Bethune Charitable Foundation (No. AF-OG-03-1.1-03).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Retinal degenerative diseases, such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy or glaucoma, represent the main causes of a decreased quality of vision or even blindness worldwide. However, despite considerable efforts, the treatment possibilities for these disorders remain very limited. A perspective is offered by cell therapy using mesenchymal stem cells (MSCs). These cells can be obtained from the bone marrow or adipose tissue of a particular patient, expanded in vitro and used as the autologous cells. MSCs possess potent immunoregulatory properties and can inhibit a harmful inflammatory reaction in the diseased retina. By the production of numerous growth and neurotrophic factors, they support the survival and growth of retinal cells. In addition, MSCs can protect retinal cells by antiapoptotic properties and could contribute to the regeneration of the diseased retina by their ability to differentiate into various cell types, including the cells of the retina. All of these properties indicate the potential of MSCs for the therapy of diseased retinas. This view is supported by the recent results of numerous experimental studies in different preclinical models. Here we provide an overview of the therapeutic properties of MSCs, and their use in experimental models of retinal diseases and in clinical trials. The retina is a highly specialized structure composed of several layers of morphologically and functionally different cell types. The individual layers are mutually interconnected and their primary function is to capture a light signal via the photoreceptors and to convert it into electrical impulses. These impulses are relayed to ganglion cells and then pass through the optic nerve into the visual cortex of the brain.Individual retinal layers have an irreplaceable role in the capture and transduction of light signals. A disease or damage to any particular cell layer has a negative impact on the surrounding cell types and is reflected by the impairment of the vision. The progression of the retinal damage results in the development of retinal degenerative disorders. Although the exact etiology, causes and starting mechanisms of these diseases are mostly unknown, many factors, such as oxidative stress, light-induced damage, chemical insults, vascular defects, cytokine imbalance, damage of blood–retinal barrier and infiltration with immune cells or aging, have been suggested to contribute to the development of retinal degeneration ,2,3. IrrRetinal diseases are a heterogenous and multifactorial group of light-threatening disorders, which include age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), pediatric Stargardt’s macular dystrophy, glaucoma and many other similar forms. Although retinal diseases have various causes and different etiologies, a common characteristic is the death or dying of the specialized retinal cells and the loss of integrity of the retina or the degeneration of the photoreceptors; and this process then results in a visual impairment and ultimately in blindness.VEGFA and HTRA1), have been identified in the retina with this type of disease [In several of these disorders, including AMD, the earliest changes observed are caused by a loss of the cells of the retinal pigment epithelium (RPE), which play a major role in photoreceptor nutrition and in the maintenance of homeostasis. The degeneration of RPE and photoreceptors is the main cause of AMD which may have its onset in choroidal neovascularization or in accumulation of amorphous deposits. Both these causes lead to alterations in the retina and to the impairment of its functions. Early AMD is characterized by the appearance of soft drusen and pigmentary changes in the RPE, which can progress into two forms of advanced AMD—dry and wet AMD. Both of these forms result in the loss of central vision . AMD is  disease . Alterna disease .To date, the treatment options for retinal diseases have been very limited. In the advanced stages, laser photocoagulation remains the main method of treatment for DR. Other therapeutic approaches are represented by a vitrectomy or different microsurgery interventions, which involve complicated surgery and are highly invasive procedures. Recently, less invasive treatment of some forms of retinal degenerative diseases has been based on the administration of inhibitors of vascular endothelial growth factor (VEGF) or other drugs. However, these inhibitors only induce short-term effects and just slow down the progression of the disease. Therefore, the need for a safe and less invasive approach to prevent development and to treat these sight-threatening manifestations of retinal diseases is vital.Since the loss of specialized retinal cells and local inflammatory reactions are the main causes contributing to the progression of retinal degenerative diseases, the inhibition of inflammation and a support for the surviving retinal cells appear to be prospective approaches to manage these diseases. Recent studies have indicated that various types of stem cells could contribute, by paracrine effects, to the support of the survival of the residual retinal cells, and to the inhibition of inflammation . A theraMSCs are multipotent stem cells which can be obtained relatively easily in a sufficient amount from various types of tissues and expanded in vitro for autologous application. It has been shown that MSCs retain their differentiation potential during their in vitro expansion, and that they can be differentiated into different cell types including cells expressing RPE or photoreceptor cell markers ,15,16. SMSCs currently represent the most frequently studied type of adult stem cells. Originally, these cells were described as a population of bone marrow-derived cells that adhere to plastic and form fibrocyte-like colonies . They haThe immunomodulatory properties of MSCs are mediated by multiple mechanisms including regulation by direct cell-to-cell contact, the production of various immunomodulatory molecules, the negative effects on antigen-presenting cells or the activation of regulatory T cells (Tregs). The complexity of the immunoregulatory effects of MSCs is also evident from the observation that MSCs inhibited lymphocyte proliferation induced by mitogens and alloantigens by different mechanisms . In geneBcl-2, Bax, p53) associated with apoptosis. Any damage in the retina attracts the cells of the immune system which produce chemokines and cytokines, and thus potentiate inflammatory and apoptotic reactions. Therefore, the inhibition of a local inflammatory reaction and attenuation of apoptosis might be promising approaches to alleviate and inhibit the development of retinal injury. In this respect, MSCs by their immunoregulatory, anti-inflammatory and antiapoptotic properties could also be a promising therapeutic tool for developing retinal disorders [Degenerative and inflammatory reactions in the diseased retina are regularly associated with a locally enhanced production of a variety of cytokines. These molecules can be produced either by inflammatory immune cells or by the activated cells of the retina . It has isorders ,61. We hisorders .Ngf, Gdnf and Il-6 was enhanced in differentiated MSCs, which suggests a higher potential of differentiated MSCs for the regeneration of diseased retinal tissue. It was demonstrated that the supernatants from light-injured retina significantly promote the secretion of neurotrophic factors by MSCs and slow down the process of apoptosis in damaged retinal cells [MSCs are potent producers of various growth and trophic factors. Some of these factors are produced by MSCs constitutively, while others are only secreted after activation with proinflammatory cytokines, mitogens or other signals. It has been suggested that the production of growth factors and their paracrine action are the main mechanisms of the therapeutic action of MSCs. Among the growth factors which are produced by MSCs and that could contribute to retinal regeneration are hepatocyte growth factor (HGF), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), insulin-like growth factor-1 (IGF-1), pigment epithelium growth factor (PEGF), fibrocyte growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), angiopoietin-1, erythropoietin, VEGF and TGF-β ,64,65,66al cells . Anotheral cells . All theOne of the characteristics of stem cells is the ability to differentiate or even transdifferentiate into different cell types. With regards to differentiation into ocular cells, relatively extensive data exist about the differentiation of MSCs and other stem cells into cornea-like cells ,70,71,72Rho and Rlbp genes [The ability of different types of stem cells and MSCs to differentiate into retinal cells has been reviewed by Salehi et al. . For exabp genes . The sambp genes ,78,79. Sbp genes , which pIn our study, to simulate the environment of the damaged retina, we cultured mouse BM-MSCs with the retinal cell extract and with supernatant from Concanavalin A-stimulated mouse spleen cells. MSCs cultured for 7 days in such conditions differentiated to cells expressing retinal cell markers such as rhodopsin, S antigen, retinaldehyde binding protein, calbindin 2, recoverin and retinal pigment epithelium 65 . InterfeIn addition to the ability of MSCs to produce several growth, immunoregulatory or neurotrophic factors, MSCs release various types of extracellular vesicles (EVs). These particles encapsulate different functional molecules which could support the survival of cells ,84. For Furthermore, mitochondrial transfer has been described as additional mechanism which MSCs can use to support anti-inflammatory conditions and cell survival ,88. SincFinally, the ability of MSCs to fuse with other cell types has been documented in various models ,91. TherAbundant experimental data demonstrate the beneficial therapeutic effects of MSCs on retinal diseases ,93,94. I3) for the destruction of RPE cells that mimic progression of macular degeneration [N-methyl D-aspartic acid (NMDA) or N-methyl-N-nitrosourea (MNU) that induces apoptosis and the selective degeneration of ganglion cells and photoreceptors that are processes resembling hereditary RP or glaucoma [Although several questions about the clinical use of MSCs still remain unanswered, for the purpose of great interest to use stem cells for the treatment of currently incurable retinal diseases, the first clinical trials using MSCs have been initiated ,101. Howneration ,103, or glaucoma ,105. Theglaucoma ,107,108.Although stem cell therapy is safe, as shown in both animal studies and clinical trials ,25, therFirst at all, there is a heterogeneity of individual MSC samples, based on differences in the cell source, isolation and culture procedure. MSCs are used at different time intervals after their isolation and different doses of cells are used. It has been documented that a longer cell culture duration has an impact on MSC morphology, secretory potential and migratory properties ,110. TheThere is still controversy about in vivo survival of in vitro cultivated MSCs. Although MSCs are considered immune privileged cells which do not express costimulatory molecules and MHC Class II molecules, in the presence of some cytokines they can express these molecules and become a target for immune cells. Without respect to these observations, numerous studies suggested a long-term survival of allogeneic or even xenogeneic MSCs in immunocompetent recipients. In contrast to these studies, Eggenhofer et al.  claimed Another unresolved issue is the fate and immunological functions of MSCs after their transfer into the inflammatory environment of the diseased retina. There is a possibility that immunosuppressive MSCs, transferred into an environment where there are proinflammatory cytokines present, can turn out into a cell population supporting the development of aggressive proinflammatory Th17 cells.The route of MSC administration is also very important. After the intravenous injection of MSCs, only a small proportion of the delivered cells can be found in the eye (our preliminary observations). Therefore, for the treatment of the retina, the intraocular delivery of cells appears more effective. Using experimental models, especially based on small animals, the intravitreal injection of MSCs is the most common way. However, in the healthy eye, only a few cells can be detected in the vitreous cavity, and the occurrence of side effects after such delivery of MSCs was reported . TherefoTo study the mechanisms of retinal diseases and to validate new therapeutic approaches for these diseases, various experimental models resembling different types of retinal damage have been established and tested. These models are based on pharmacological interventions which induce the degeneration of specialized retinal cells, or utilize mutant animal strains, genetically modified recipients or various mechanical damages or injuries . We revi3. NaIO3 is a chemical which selectively induces the degeneration and death of RPE cells. It was shown in vitro that the exposure of human RPE cell line ARPE-19 to NaIO3 induces the activation of inflammasome, changes the expression of molecules involved in the apoptosis, induces cell dysfunctions resembling conditions in AMD and finally causes RPE cell death [3 [3 increases the expression of genes for proinflammatory cytokines IL-1α and IL-6 or for proapoptotic Bax and p53 molecules in cultured mouse retinal explants. This increase was inhibited in the presence of mouse BM-MSCs, or by using a supernatant obtained after the cultivation of MSCs .AMD is characterized by a progressive degeneration of the RPE and photoreceptors, and this process represents the major cause of visual impairment and irreversible blindness in the elderly population. Numerous experimental models have been established to study the individual steps of AMD progression. Transgenic experimental animal models provide systems to explore the cellular and molecular mechanisms of this disease. Some advantages are offered by laser-induced models. Other approaches are based on the application of pharmacological agents inducing pathological changes in the retina. One of the well-established pharmacological models is based on the systemic or a local administration of NaIOll death ,118. In 3 in vivo causes a rapid degeneration of the RPE cells and consequent damage to the outer nuclear layer. Increased levels of mRNA for Htra-1 a C3, the genes associated with the development of AMD, were detected in the retina of the NaIO3-treated recipients [3 thus provides a suitable experimental model for study of the late phase of nonvascular AMD called geographic atrophy. The subretinal delivery of NaIO3 in rats causes the formation of an atrophic area characterized by the degeneration of RPE cells and photoreceptors [3 in rabbits after vitrectomy induced retinal atrophy and diffused outer retinal degeneration [3 protected the RPE layer, photoreceptors and other nuclear cells from the damage [3-induced retinal damage. Since the RPE cells are the first damaged cell type in the progression of AMD, the protective effect of MSCs may be a promising option for the treatment of this condition.The intravenous or intraperitoneal application of NaIOcipients ,119,120.eceptors . The intneration . In these damage . Gong ete damage  showed tDR represents a common complication of diabetes which is caused by hyperglycemia and by injury in retinal microvasculature and neurons. This disorder represents one of the leading causes of blindness globally. Despite the high prevalence of DR and extensive research, the treatment options for this disease are still strongly limited. Various experimental animal models have been established for the study of treatment possibilities. These models have been generated by a selective inbreeding or genetic modifications, the feeding of a galactose diet, or by a pharmacological induction using streptozotocin. This chemical selectively damages the β cells of the pancreas, increases blood glucose level and decreases the number of ganglion cells. To date, various animal models have been used to test the possibilities of treating DR by the application of stem cells . In the In models of streptozotocin-induced diabetes and DR, the application of MSCs had a positive effect on the retinal architecture. For example, Ezquer et al.  showed tAkitaIns2) mice were created by a point mutation in the insulin-2 gene and represent a spontaneous type-l diabetes model. It was shown that hyperglycemia in these mice causes neurodegenerative effects in the retina resulting in retinal thickness [Akita mice during the progression of the diseases [Akita mice improved vascular permeability and vision in this model of nonproliferative DR. Similar results were also obtained after the application of the conditioned medium from human A-MSCs which were pretreated with TNF-α and IFN-γ. The conditioned medium from A-MSCs also reduced the retinal expression of GFAP, the gene associated with neuroinflammation [In addition to models of pharmacologically induced diabetes, several studies used models of spontaneously or genetically induced diabetes. These models were described in detail by Robinson et al.  and Lai hickness . In addidiseases . The theammation . The expammation ,136. Locammation .PDE6b gene which is, under physiological conditions, important for the signal transmission. In addition, it was shown that activated microglia with proinflammatory polarization occur in the rd1 retina [PDE gene for rod-phosphodiesterase. This mutation causes the degeneration of photoreceptors and other retinal cell types [Mrfp gene, which is expressed in the RPE layer of cells [Merkt gene, causing photoreceptor damage and an increase in microglial activation in the retina, resulting in inherited retinal degeneration [RP is a group of inherited neurodegenerative diseases characterized by a loss of photoreceptor cells, leading to visual impairment and eventually to blindness. The experimental models  of this disease are based on the use of spontaneous or genetically induced degeneration of the photoreceptors, and on the administration of chemicals inducing degeneration of the retinal cells ,138,139.1 retina . It is all types . Moreovell types . Anotherof cells . In addineration ,145. Somneration . The admneration . Moreoveneration . DecreasN-methyl-N-nitrosourea (MNU). A single systemic administration of MNU causes retinal degeneration in various species [Another approach to imitate the retinal degeneration observed in RP is based on the administration of  species . Deng et species  showed tThus, as in the case of other experimental models of retinal degeneration, the positive therapeutic effects of MSC therapy were also demonstrated in RP models.Glaucoma is a heterogenous group of eye diseases mainly caused by increased intraocular pressure and characterized by the progressive loss of retinal ganglion cells. So far, numerous experimental models have been established to study this disease. They include the intracameral injection of microbeads, laser photocoagulation, episcleral vein cauterization, the injection of hyaluronic acid and various models based on genetically modified rodents ,152,153.A trabecular meshwork regeneration observed after intraocular administration of BM-MSCs was described in a model of a laser-induced retinal damage model ,158. TheAltogether, the animal models indicated positive therapeutic effects of MSC-based therapies in various types of retinal diseases. Selected experimental models where MSCs were used are summarized in Currently, several clinical trials are in progress to test the potential of MSCs for the treatment of retinal degenerative diseases. Most of these studies are in the phase 1 or 2 focused on the safety of MSC application. The first finished studies showed that the administration of MSCs is not associated with serious complications ,165. MorThe application of MSCs appears safe and no serious treatment-related problems were observed in the eyes of patients with AMD, RP or retinal vascular occlusion six months after the administration of autologous BM-MSCs in the phase 1 testing. An improvement in visual function was also noted, but as the studies were designed to assess the safety of the treatment, it was not possible to definitely confirm whether this improvement was caused by the MSC application . SimilarHowever, further clinical trials with a higher number of patients and a longer follow-up are still needed to evaluate the efficacy of MSC therapy. It will also be necessary to evaluate the benefits and advantages of autologous MSCs and the transplantation of stem cells obtained from another source, such as Wharton’s jelly or the umbilical cord, or the use of a conditioned medium or exosomes. Selected clinical trials with MSCs for retinal degenerative disorders and their results are shown in Sight-threatening retinal degenerative diseases represent the main cause of visual impairment or even blindness worldwide. Despite great endeavors, there is still a lack of effective therapeutic approaches to stop or even cure these disorders. A prospective option has recently been offered by stem cell therapy. Experimental data from numerous animal models and using different types of stem cells offer promising results. Although the data from the experimental models are encouraging, numerous questions about the use of stem cell therapy have to be resolved to make this therapy more effective and safe . NeverthOne of the most important issues associated with MSC-based therapy, the safety of MSC administration, has been tested in numerous preclinical studies and clinical trials. Various experimental models and clinical studies using an intravitreal administration of MSCs demonstrated the safety of this therapy without any undesirable side effects ,101. AnoFinally, the immunoregulatory action of MSCs could strongly depend on the cytokine environment . The infAlthough there are still many issues to be addressed before the final approval of MSC therapy for retinal degenerative diseases, the results obtained so far in preclinical animal models and in clinical trials are promising and encouraging. Therefore, the stem cell-based therapy offers a prospective option, especially for the patients without alternative therapeutic options. However, before the definitive expansion of the clinical use of MSC-based therapies, several questions, such as the sources of MSCs, the conditions of the in vitro propagation of MSCs, the routes of the cell applications or the possibility of the use of MSC products have to be answered and carefully verified.


With the development of sequencing technology, the research on medicinal plants is no longer limited to the aspects of chemistry, pharmacology, and pharmacodynamics, but reveals them from the genetic level. As the price of next-generation sequencing technology becomes affordable, and the long-read sequencing technology is established, the medicinal plant genomes with large sizes have been sequenced and assembled more easily. Although the review of plant genomes has been reported several times, there is no review giving a systematic and comprehensive introduction about the development and application of medicinal plant genomes that have been reported until now. Here, we provide a historical perspective on the current situation of genomes in medicinal plant biology, highlight the use of the rapidly developing sequencing technologies, and conduct a comprehensive summary on how the genomes apply to solve the practical problems in medicinal plants, like genomics-assisted herb breeding, evolution history revelation, herbal synthetic biology study, and geoherbal research, which are important for effective utilization, rational use and sustainable protection of medicinal plants. Medicinal plants, in the simple definition, are plants that can be used for medicinal purposes; in the detailed definition, are plants that have been verified and used for a long time as traditional medicines, have been found to have medicinal value in modern research, or contain medicinal ingredients in them. And they can provide the essential resources for human life, such as drugs, nourishment, condiments, and medicinal oil. They also uncovered and promoted the evolution of nature, animals, and humans. The foundation of all life is the genetic code. Therefore, access to the primary DNA sequence and how genes are encoded within the genome has become a basic resource in biology . The genRegarding plant genome sequencing methods and strategies, radical changes have taken place in the past 5 years, and medicinal plant genome sequencing is no exception. Previous reviews summarized the status of sequenced plant genomes before 2012 , the staIn this manuscript, we conducted a systematic review of medicinal plants genome research. Moreover, the genome situation, sequencing technology development, and application of medicinal plant genomes are discussed. This review provides a historical perspective on the current situation of genomes in medicinal plant biology and highlights the use of rapidly developing sequencing technologies in plant biology. Challenges in genomics for medicinal plants are improved to some extent by long-read sequencing technologies regarding the current limitations. Multiple omics methods are integrated to make better use of medicinal plant genome data and to solve practical problems meeting in the breeding and medical fields. We also conduct a comprehensive summary of the application of medicinal plant genomes, to promote the studies of important questions in plant biology, like genomics-assisted herb breeding, herbal synthetic biology, and geoherbal research, which are significant for securing the future of medicinal plants and their active compounds.The systematic literature search was performed by the following PRISMA guidelines . FirstlyPanax notoginseng (Chinese name: Sanqi) as an example, five versions of its genomes have been reported, the first two versions published in 2017 were sequenced by the next-generation sequencing (NGS) technology of Illumina platform , two gymnosperms, and one lycopodiophyta plant. The simplified phylogeny of the major clades of sequenced medicinal plants is also shown in We have made statistics on the medicinal plant genome articles over the years, and have a basic understanding of the general characteristics of the reported medicinal plant genomes. The comparison of size and repetitiveness ratio of these published medicinal plant genomes and their evolution relationship is shown in It has been said that plant genome reports were formulaic and lack biology significance, their descriptions mainly included the assembly, protein-coding genes, repeats, evolution analysis, some aspects of biology, usually with a focus on transcription factors and active compounds biosynthesis pathway . AccordiArtemisia annua is one of the most famous medicinal plants, while the discovery of artemisinin has won the 2015 Nobel Prize in Physiology or Medicine , midstream (FPS) and downstream (DBR2), and the other was to overexpress the expression of transcription factors like AaMYB2 that could regulate the expression of ADS, CYP71AV1, DBR2, and ALDH1 in artemisinin biosynthesis pathway, which could significantly improve artemisinin and dihydroartemisinic acid content, providing a new insight for increasing the supply of artemisinin from plant sources , 168 genes associated with stem thickness  and 33 genes associated with disease resistance trait (included genes encoding LRR receptor-like serine/threonine-protein kinases)  . These vGinkgo biloba is a living fossil without living relatives, which represents one of the four extant gymnosperm lineages . Its genome showed that LTR-RT insertions and two whole-genome duplications (WGD) events in evolution history contribute to the large genome size and long introns. In angiosperms, chromosomal breakages and fusions, as well as uneven gene loss, might occur to prevent a continuous growth in genome size  sequencing of Pacific Biosciences company. SMRT sequencing is run on cells, which have tiny wells called zero-mode waveguides (ZMWs). In each ZMWs, a DNA polymerase/template complex gets immobilized, and synthesizes a new DNA strand . Each inThe emergence of third-generation sequencing technology has made a great leap in sequencing read length and brought medicinal plant genome sequencing into a stage of rapid development. The strategy used in this stage is a combination of second- and third-generation sequencing technologies, which can ensure long read length, high throughput, and reasonable sequencing price at the same time. Medicinal plant genomes are large and have high-ratio repetitive elements, the frequently-used strategy is combining high coverage Illumina and low coverage PacBio SMRT or ONT data. Because third-generation sequencing can provide long-read sequences to increase the assembly accuracy and genome draft quality, but the price is relatively high, so Illumina platform is used to guarantee enough sequencing data. And this can make it possible to assemble large and complex medicinal plant genomes to the chromosome level. After these years of sequencing development, the medicinal plants not only can obtain draft genome relevant information and dig out target protein-coding genes, but also recognize the chromosome-level of the genome to discover the evolution, gene cluster’s function, repetitive elements effect, and so on.de novo sequencing of the genomes at the same time, and accordingly publish them at the same time. Then, with the continuous development of gene sequencing technology, we can obtain longer sequencing read lengths, so as to assemble more complete and accurate high-level genomes. Genomes assembled to the chromosome level are the current trend. The information that the genome gives us is no longer a contig or scaffold, but the chromosome and the position of a gene on the chromosome.We found that not only does the number of medicinal plant genomes sequenced continue to increase, but the number of medicinal plant genomes sequenced repeatedly is also increasing. Why? First of all, because the genomes of many medicinal plants have not been revealed yet, many teams are performing Momordica charantia (bitter gourd), Salvia miltiorrhiza (Danshen), Punica granatum (pomegranate), Panax notoginseng (Sanqi), Panax ginseng (Asian ginseng), etc. Bitter gourd and danshen have two reported versions of the genome. Bitter gourd completed the de novo assembly of the genome draft in 2017, as well as basic annotation and evolutionary analysis , which is a popular and nutritious fruit with medicinal properties, has three published genome versions (Panax notoginseng (Sanqi) is a well-known TCM whose genome research is sought after by scientists, and a total of five versions have been reported. The three recent versions are assembled to the chromosome level (P. notoginseng. Panax ginseng (Asian ginseng), reputed as the king of medicinal herbs, belongs to the same genus Panax, which also has two versions of reported genomes  genome sequencing revealed that a specialized metabolic pathway for the synthesis of 40-deoxyflavonebioactives evolved in the genus Scutellaria and found that the gene encoding a specific cinnamate coenzyme A ligase likely obtained its new function following recent mutations and that four genes encoding enzymes in the 40-deoxyflavone pathway are present as tandem repeats in the genome of Huangqin. Further analysis discovered that gene duplications, segmental duplication, gene amplification, and point mutations coupled to gene neo- and subfunctionalizations were involved in the evolution of 40-deoxyflavone synthesis in Scutellaria. These results not only provide significant insight into the evolution of specific flavone biosynthetic pathways in the mint family Lamiaceae but also facilitate the development of tools for enhancing bioactive productivity by molecular breeding in plants , to adapt to the environment alteration and explain the characters of plants. We summarized the WGD and WGT events of some representative species reported in the medicinal plant genome articles, and these situations are shown in P. ginseng, P. notoginseng, and E. senticosus belong to Araliaceae, and P. notoginseng is diploid, while P. ginseng and E. senticosus are tetraploid. Two rounds of WGD were discovered in these Araliaceae plants, the first round occurred around 29.6 Mya, P. ginseng, and E. senticosus both had the second round of WGD, which were found almost 2.2 Mya in P. ginseng and 13 Mya in E. senticosus, respectively. Additionally, these recent WGDs were discovered to contribute to the ability of P. ginseng to overwinter and E. senticosus to adapt to cold environment, enabling them to live and spread broadly through the cold area  and two major quantitative trait loci associated with hull thickness and color (Ccph1 and Ccph2), were discovered to be the potential identification loci for domestication. These findings will greatly facilitate and benefit the molecular breeding of coix and provide a powerful reference for the domestication and evolution of medicinal plants.Coix is a widely cultivated grass crop with high nutritional and medicinal value, which has been domesticated as early as the Neolithic era. However, its genetic research and breeding were hampered by the lack of a sequenced genome. Two chromosome-level genomes of coix have been reported simultaneously, which belong to elite cultivar Beijing  and wildaheishan , respectThe active components of medicinal plants with complex and diverse structures are the material basis for their medicinal effect, and it’s also an important source of new drug discovery. However, many medicinal plant materials often face a series of problems in the process of development and utilization, for example, the growth of many medicinal materials is greatly affected by environmental factors; some rare herbs grow slowly and are difficult to grow by artificial cultivation; most of the active ingredients are low in content, complex in chemical structure and difficult in chemical synthesis; traditional methods of natural extraction or artificial chemical synthesis cannot meet the needs of scientific research and new drug development. Synthetic biology will be an effective way to resolve these problems.As high-throughput sequencing technology for genome and transcriptome studies have developed rapidly, using bioinformatics method and functional genomics approach to screen and identify enzyme-coding genes on specific secondary biosynthesis pathway from a large number of the original species of medicinal plants, which will greatly accelerate the analysis process of secondary biosynthesis pathway and lay a solid foundation for herbal synthetic biology research of medicinal plants.Tripterygium wilfordii genome is one of the typical examples. Because of the extremely low yield of triptolide extracted from T. wilfordii, its original plant cannot be grown on a large scale, and the current chemical synthesis route is limited to a yield of less than 1.64%. A more promising method to obtain more triptolide could be metabolic engineering, which can be realized via a synthetic biology strategy. However, it required elucidation of the triptolide biosynthesis pathway. Therefore, the sequencing of the T. wilfordii genome was completed, and cytochrome P450 TwCYP728B70 involved in triptolide biosynthesis was identified, accordingly, the triptolide content in the CYP728B70 overexpression line increased obviously (bviously . It’s imGeoherbs, controlled by genetic factors and affected by environmental conditions, are representative of high-quality medicinal materials. The utilization of sequencing technology and data can provide useful tools to elucidate the molecular mechanism of geoherbs. For the same medicinal plants in different areas, epigenomic studies of medicinal plants can be carried out to clarify the genetic variation of different production areas, especially the modification effect of different environments on the epigenome of medicinal material, including DNA methylation modification, small RNA sequencing analysis, chromatin immunoprecipitation analysis, and so on. In addition, microorganisms in soil are also important factors in the growth environment of geoherbs. Metagenomic analysis of soil microbial community can be sequenced to provide the basis for revealing the interaction between soil microorganisms and the growth of medicinal plants.Recently, 545 genomes of ginkgo trees sampled from 51 populations across the world were sequenced to identify three refugia in China and detect multiple cycles of population expansion and reduction along with glacial admixture between relict populations in the southwestern and southern refugia, and multiple anthropogenic introductions of ginkgo were proved to occur from eastern China into different continents. This study provides insight into the evolutionary history of ginkgo and helps to provide protection and utilization way for its valuable genomic resources .Because of the rapid development and progress of sequencing technology, more and more biosynthesis pathways of active ingredients from medicinal plants have been revealed. The early-stage was based on the mining from transcriptome data, and the later stage was based on the combined mining from genome and transcriptome data. Although transcriptome sequencing has so far occupied a major position in the research of biosynthesis pathways of medicinal ingredients, genome data can provide more important information, for example, it can reveal the evolution process of biosynthesis pathway genes, thereby efficiently synthesizing secondary metabolites with medicinal activity. In the opium poppy genome, a great discovery about a gene cluster including 15 genes was reported. Meanwhile, in its evolution process, the events like gene duplication, rearrangement, and fusion, could lead to the aggregation and co-expression of genes in the two metabolic pathways of noscapine and morphinan, so that it resulted in the formation of this supergene cluster, which could synergistically synthesize the medicinal ingredients in opium poppy . TherefoA. thaliana, Zea mays, and Solanum lycopersicum (It also provides new ideas for the application of medicinal plant genomes. Through the evolution process, gene duplications and neofunctionalization can generate gene clusters, which may relate to specialized metabolites, and this phenomenon has already been observed in several model plants, like persicum . In medipersicum , which cetc.The continuous emergence of high-quality genomes has made the application of comparative genomics analysis more and more extensive and in-depth, and it is also a powerful tool for researchers to dig out biological problems and explain biological phenomena . ComparaScutellaria baicalensis and Scutellaria barbata. The comparative genomic analysis of them showed the recent LTR may result in chromosomal rearrangement and expansion, and tandem duplication of paralogs after their speciation might contribute to the divergent evolution of flavonoid biosynthesis gene families, which provided a significant foundation for the evolution and chemodiversity studies in the Lamiaceae (One representative example of comparative genomics among different medicinal plant genome species can be amiaceae .Forsythia suspense. Genome-wide comparative analysis was then conducted for the 15 natural populations across its current distribution range. The results revealed that candidate genes associated with local adaptation were functionally correlated with heterogeneous environmental factors, and supported the hypothesis that adaptive differentiation should be highly obvious in the genes of signal crosstalk between different environmental variables, which gave insights into the fundamental genetic mechanisms of the local adaptation to climatic gradients in plant species (Moreover, a representative of comparative genomics among different populations in the same species can be  species .The use of medicinal plants has a long history and diverse application methods. Related works of research mainly focus on the discovery of chemical basis and the analysis of pharmacodynamic effect, but the understanding of medicinal plant genetic resources is relatively weak. Therefore, the research on the genome of medicinal plants should make use of the latest technologies and achievements of genomics, and integrate the studies of structural genome, functional genome, transcriptome, proteome, epigenome, metagenome, synthetic biology, metabolome, bioinformatics, and other relevant databases. Therefore, the essence of medicinal plants can be revealed, the relationship among genetic resources, chemical quality, and drug efficacy can be recognized.We are most concerned about the medicinal value of medicinal plants. The medicinal value is not only reflected in the content of their medicinal ingredients, but also the stability of the quality of their medicinal materials. Now medicinal plant genomes can be annotated to obtain protein-coding genes, especially biosynthesis genes of active ingredients, analyze their evolutionary history and domestication process, and discover genes that respond to environmental stresses to help improve their resistance and ability. However, the powerful ability of the medicinal plant group has not yet been manifested, and its ability to solve the difficulties in practical applications remains to be developed. How to use the information of the medicinal plant genome to transform and obtain excellent medicinal plant varieties has not yet been realized. Determining suitable model medicinal plants is of great significance to the research on the practical application of medicinal plant genomes. The determination of appropriate model medicinal plants is of great significance to the study of the genomics of medicinal plants. From the perspective of general biological characteristics, it usually should have the traits of a short age cycle, many offspring, and stable phenotype. As for genetic resources, the genome should be relatively small, easy to sequence, and genetic transformation is relatively easy. As for medicinal characteristics, it should be suitable for secondary metabolite biosynthesis and production research. Therefore, the establishment and improvement of a suitable model medicinal plant platform will greatly enhance the application value of medicinal plant genomes.The assembly of plant genomes is a challenging problem because of their high repetitiveness due to TEs, extreme genome sizes, and polyploid nature. With the development and emergence of long-read sequencing  and long1 (2 and Database of 10,000 Medicinal Plants.3 These databases summarize the medicinal plant genomes that have been reported at this stage or aim to build a biological big data platform for medicinal plants, linking the omics data, active ingredients, disease information, and other information to promote their modernization. All of the above indicate that the medicinal plant genome has entered the stage of big data association research from the stage of exploring the unknown. Moreover, because of the limitations of previous technologies and methods, the disclosed medicinal plant genome information is limited. If the obtained medicinal plant genome information is aggregated and shared through the database, this should be a huge treasure to be unearthed, which will prompt the research efficiency of medicinal plants.Moreover, thanks to the advancement and development of sequencing technology and bioinformatics algorithms, at least one hundred medicinal plant genomes have been obtained. How to use them thoroughly and effectively has attracted the attention of many institutions and researchers. In recent years, several databases of medicinal plant genomes have already been built, such as the Herbal Medicine Omics Database1 , 1K MediThanks to the invention of the long-read sequencing technology, the research on medicinal plant genomes has developed rapidly and is no longer limited by their huge genome size and high repetitive sequences. The number of genomes reported in the past 2 years has increased significantly, and the quality of genomes has also been greatly improved, most of which have been assembled to the chromosome level. Correspondingly, the sequencing strategy they adopted has also been continuously updated, making them more and more widely used, answering and solving many problems in scientific researches and practical applications, including herb breeding assistance, evolutionary history revealing, domestication process understanding, herb synthetic biology study, geoherbal research and comparative genome analysis, these are of great significance to the effective use and sustainable protection of medicinal plants, which can improve their research efficiency and promote their modern development.Q-QC planned the manuscript outline, wrote the draft, and created the figures and tables. YO, Z-YT, C-CL, Y-YZ, and C-SC proofread the manuscript. HZ supervised the study and revised the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.