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Tissue-derived extracellular vesicles (EVs) are emerging as pivotal players to maintain organ homeostasis, which show promise as a next-generation candidate for medical use with extensive source. However, the detailed function and therapeutic potential of tissue EVs remain insufficiently studied. Here, through bulk and single-cell RNA sequencing analyses combined with ultrastructural tissue examinations, we first reveal that in situ liver tissue EVs (LT-EVs) contribute to the intricate liver regenerative process after partial hepatectomy (PHx), and that hepatocytes are the primary source of tissue EVs in the regenerating liver. Nanoscale and proteomic profiling further identify that the hepatocyte-specific tissue EVs (Hep-EVs) are strengthened to release with carrying proliferative messages after PHx. Moreover, targeted inhibition of Hep-EV release via AAV-shRab27a in vivo confirms that Hep-EVs are required to orchestrate liver regeneration. Mechanistically, Hep-EVs from the regenerating liver reciprocally stimulate hepatocyte proliferation by promoting cell cycle progression through Cyclin-dependent kinase 1 (Cdk1) activity. Notably, supplementing with Hep-EVs from the regenerating liver demonstrates translational potential and ameliorates insufficient liver regeneration. This study provides a functional and mechanistic framework showing that the release of regenerative Hep-EVs governs rapid liver regeneration, thereby enriching our understanding of physiological and endogenous tissue EVs in organ regeneration and therapy.

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AMP-activated protein kinase (AMPK), a crucial regulatory kinase, monitors energy levels, conserving ATP and boosting synthesis in low-nutrition, low-energy states. Its sensitivity links microenvironmental changes to cellular responses. As the primary support structure and endocrine organ, the maintenance, and repair of bones are closely associated with the microenvironment. While a series of studies have explored the effects of specific microenvironments on bone, there is lack of angles to comprehensively evaluate the interactions between microenvironment and bone cells, especially for bone marrow mesenchymal stem cells (BMMSCs) which mediate the differentiation of osteogenic lineage. It is noteworthy that accumulating evidence has indicated that AMPK may serve as a hub between BMMSCs and microenvironment factors, thus providing a new perspective for us to understand the biology and pathophysiology of stem cells and bone. In this review, we emphasize AMPK's pivotal role in bone microenvironment modulation via ATP, inflammation, reactive oxygen species (ROS), calcium, and glucose, particularly in BMMSCs. We further explore the use of AMPK-activating drugs in the context of osteoarthritis and osteoporosis. Moreover, building upon the foundation of AMPK, we elucidate a viewpoint that facilitates a comprehensive understanding of the dynamic relationship between the microenvironment and bone homeostasis, offering valuable insights for prospective investigations into stem cell biology and the treatment of bone diseases.

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Inter-organ communication through hormones, cytokines and extracellular vesicles (EVs) has emerged to contribute to the physiological states and pathological processes of the human body. Notably, the liver coordinates multiple tissues and organs to maintain homeostasis and maximize energy utilization, with the underlying mechanisms being unraveled in recent studies. Particularly, liver-derived EVs have been found to play a key role in regulating health and disease. As an endocrine organ, the liver has also been found to perform functions via the secretion of hepatokines. Investigating the multi-organ communication centered on the liver, especially in the manner of EVs and hepatokines, is of great importance to the diagnosis and treatment of liver-related diseases. This review summarizes the crosstalk between the liver and distant organs, including the brain, the bone, the adipose tissue and the intestine in noticeable situations. The discussion of these contents will add to a new dimension of organismal homeostasis and shed light on novel theranostics of pathologies.

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Non-alcoholic fatty liver disease (NAFLD), a chronic liver condition and metabolic disorder, has emerged as a significant health issue worldwide. D-mannose, a natural monosaccharide widely existing in plants and animals, has demonstrated metabolic regulatory properties. However, the effect and mechanism by which D-mannose may counteract NAFLD have not been studied. In this study, network pharmacology followed by molecular docking analysis was utilized to identify potential targets of mannose against NAFLD, and the leptin receptor-deficient, genetically obese db/db mice was employed as an animal model of NAFLD to validate the regulation of D-mannose on core targets. As a result, 67 targets of mannose are predicted associated with NAFLD, which are surprisingly centered on the mechanistic target of rapamycin (mTOR). Further analyses suggest that mTOR signaling is functionally enriched in potential targets of mannose treating NAFLD, and that mannose putatively binds to mTOR as a core mechanism. Expectedly, repeated oral gavage of supraphysiological D-mannose ameliorates liver steatosis of db/db mice, which is based on suppression of hepatic mTOR signaling. Moreover, daily D-mannose administration reduced hepatic expression of lipogenic regulatory genes in counteracting NAFLD. Together, these findings reveal D-mannose as an effective and potential NAFLD therapeutic through mTOR suppression, which holds translational promise.

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Type H vessels couple angiogenesis with osteogenesis, while sympathetic cues regulate vascular and skeletal function. The crosstalk between sympathetic nerves and type H vessels in bone remains unclear. Here, we first identify close spatial connections between sympathetic nerves and type H vessels in bone, particularly in metaphysis. Sympathoexcitation, mimicked by isoproterenol (ISO) injection, reduces type H vessels and bone mass. Conversely, beta-2-adrenergic receptor (ADRB2) deficiency maintains type H vessels and bone mass in the physiological condition. In vitro experiments reveal indirect sympathetic modulation of angiogenesis via paracrine effects of mesenchymal stem cells (MSCs), which alter the transcription of multiple angiogenic genes in endothelial cells (ECs). Furthermore, Notch signaling in ECs underlies sympathoexcitation-regulated type H vessel formation, impacting osteogenesis and bone mass. Finally, propranolol (PRO) inhibits beta-adrenergic activity and protects type H vessels and bone mass against estrogen deficiency. These findings unravel the specialized neurovascular coupling in bone homeostasis and regeneration.

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The blood vessel system is essential for skin homeostasis and regeneration. While the heterogeneity of vascular endothelial cells has been emergingly revealed, whether a regeneration-relevant vessel subtype exists in skin remains unknown. Herein, a specialized vasculature in skin featured by simultaneous CD31 and EMCN expression contributing to the regeneration process is identified, the decline of which functionally underlies the impaired angiogenesis of diabetic nonhealing wounds. Moreover, enlightened by the developmental process that mesenchymal condensation induces angiogenesis, it is demonstrated that mesenchymal stem/stromal cell aggregates (CAs) provide an efficacious therapy to enhance regrowth of CD31+ EMCN+ vessels in diabetic wounds, which is surprisingly suppressed by pharmacological inhibition of extracellular vesicle (EV) release. It is further shown that CAs promote secretion of angiogenic protein-enriched EVs by proteomic analysis, which directly exert high efficacy in boosting CD31+ EMCN+ vessels and treating nonhealing diabetic wounds. These results add to the current knowledge on skin vasculature and help establish feasible strategies to benefit wound healing under diabetic condition.

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Psychological stress emerges to be a common health burden in the current society for its highly related risk of mental and physical disease outcomes. However, how the quickly-adaptive stress response process connects to the long-observed organismal alterations still remains unclear. Here, we investigated the profile of circulatory extracellular vesicles (EVs) after acute stress (AS) of restraint mice by phenotypic and proteomic analyses. We surprisingly discovered that AS-EVs demonstrated significant changes in size distribution and plasma concentration compared to control group (CN) EVs. AS-EVs were further characterized by various differentially expressed proteins (DEPs) closely associated with biological, metabolic and immune regulations and were functionally important in potentially underlying multiple diseases. Notably, we first identified the lipid raft protein Stomatin as an essential biomarker expressed on the surface of AS-EVs. These findings collectively reveal that EVs are a significant function-related liquid biopsy indicator that mediate circulation alterations impinged by psychological stress, while also supporting the idea that psychological stress-associated EV-stomatin can be used as a biomarker for potentially predicting acute stress responses and monitoring psychological status. Our study will pave an avenue for implementing routine plasma EV-based theranostics in the clinic.

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The teeth are vertebrate-specific, highly specialized organs performing fundamental functions of mastication and speech, the maintenance of which is crucial for orofacial homeostasis and is further linked to systemic health and human psychosocial well-being. However, with limited ability for self-repair, the teeth can often be impaired by traumatic, inflammatory, and progressive insults, leading to high prevalence of tooth loss and defects worldwide. Regenerative medicine holds the promise to achieve physiological restoration of lost or damaged organs, and in particular an evolving framework of developmental engineering has pioneered functional tooth regeneration by harnessing the odontogenic program. As a key event of tooth morphogenesis, mesenchymal condensation dictates dental tissue formation and patterning through cellular self-organization and signaling interaction with the epithelium, which provides a representative to decipher organogenetic mechanisms and can be leveraged for regenerative purposes. In this review, we summarize how mesenchymal condensation spatiotemporally assembles from dental stem cells (DSCs) and sequentially mediates tooth development. We highlight condensation-mimetic engineering efforts and mechanisms based on ex vivo aggregation of DSCs, which have achieved functionally robust and physiologically relevant tooth regeneration after implantation in animals and in humans. The discussion of this aspect will add to the knowledge of development-inspired tissue engineering strategies and will offer benefits to propel clinical organ regeneration.

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Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

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Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

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Glioma-associated oncogene homolog 1 (Gli1) marks a subpopulation of endogenous mesenchymal stem cells (MSCs) characterized by perivascular location. Here, we present an optimized immunofluorescence staining protocol to identify resident Gli1+ MSCs in fixed/frozen bone sections from LacZ transgenic mice. This protocol describes the preparation of fixed/frozen tissue sections and the use of LacZ immunofluorescent staining for the in vivo characterization of endogenous MSCs, regarding their specific identity and specialized niches, and is applicable to LacZ-expressing cells of diverse organs. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

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[This corrects the article DOI: 10.7150/thno.23620.].

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Poor healing of cutaneous wounds is a common medical problem in the field of traumatology. Due to the intricate pathophysiological processes of wound healing, the use of conventional treatment methods, such as chemical molecule drugs and traditional dressings, have been unable to achieve satisfactory outcomes. Within recent years, explicit evidence suggests that mesenchymal stem cells (MSCs) have great therapeutic potentials on skin wound healing and regeneration. However, the direct application of MSCs still faces many challenges and difficulties. Intriguingly, exosomes as cell-secreted granular vesicles with a lipid bilayer membrane structure and containing specific components from the source cells may emerge to be excellent substitutes for MSCs. Exosomes derived from MSCs (MSC-exosomes) have been demonstrated to be beneficial for cutaneous wound healing and accelerate the process through a variety of mechanisms. These mechanisms include alleviating inflammation, promoting vascularization, and promoting proliferation and migration of epithelial cells and fibroblasts. Therefore, the application of MSC-exosomes may be a promising alternative to cell therapy in the treatment of cutaneous wounds and could promote wound healing through multiple mechanisms simultaneously. This review will provide an overview of the role and the mechanisms of MSC-derived exosomes in cutaneous wound healing, and elaborate the potentials and future perspectives of MSC-exosomes application in clinical practice.

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Innervation and extracellular vesicle secretion co-exist in the local tissue microenvironment for message transfer, but whether they are interconnected to regulate organ homeostasis remains unknown. Sympatho-adrenergic activation is implicated in stress-induced depression and leads to bone loss, but the mechanisms and therapeutics are incompletely elucidated. Here, it is revealed that sympathetic neurostress through the ß1/2 -adrenergic receptor (ß1/2-AR) signaling triggers the transcription response of a microRNA, miR-21, in osteoblasts, which is transferred to osteoclast progenitors via exosomes for dictating osteoclastogenesis. After confirming that miR-21 deficiency retards the ß1/2-AR agonist isoproterenol (ISO)-induced osteopenia, it is shown that the pharmacological inhibition of exosome release by two clinically-relevant drugs, dimethyl amiloride and omeprazole, suppresses osteoblastic miR-21 transfer and ameliorates bone loss under both ISO and chronic variable stress (CVS)-induced depression conditions. A targeted delivery approach to specifically silence osteoblastic miR-21 is further applied, which is effective in rescuing the bone remodeling balance and ameliorating ISO- and CVS-induced osteopenias. These results decipher a previously unrecognized paradigm that neural cues drive exosomal microRNA communication to regulate organ homeostasis and help to establish feasible strategies to counteract bone loss under psychological stresses.

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BACKGROUND: Hepatic steatosis is a big hurdle to treat type 2 diabetes (T2D). Fasting-mimicking diet (FMD) has been shown to be an effective intervention in dyslipidemia of T2D. However, fasting may impair the normal glucose metabolism. Human umbilical cord-derived mesenchymal stem cell (UC-MSC) transplantation has been discovered to regulate immune reactions and reduce hyperglycemia in diabetes. However, the effect of UC-MSCs on improving the lipid metabolism disorder is not quite satisfactory. We have investigated the efficacy comparison and interaction between FMD and UC-MSC infusion, aiming to establish effective T2D therapies and explore its mechanism. METHODS: C57/BL6 mice were fed with high-fat diet (HFD) to induce a diet-induced obese (DIO) mouse model. Leptin receptor-deficient (db/db) mice were used for follow-up experiments. DIO or db/db mice were divided into 4 groups: phosphate buffer saline (PBS), UC-MSCs, FMD, and UC-MSCs + FMD. At the end of the study period, mice were fasted and sacrificed, with the measurement of physiological and biochemical indexes. In addition, the fresh liver, skin, and white adipose tissue were analyzed by histology. RESULTS: FMD restored the lipid metabolism in DIO mice, whereas its capacity to rescue hyperglycemia was uncertain. Infusion of UC-MSCs was effective in T2D glycemic control but the impact on dyslipidemia was insufficient. Furthermore, both the glucose and the lipid alterations of DIO and db/db mice recovered after UC-MSCs combined with FMD. It was proved that UC-MSCs promoted FMD effects on ameliorating hyperglycemia and restoring the lipid metabolism in T2D mice, while FMD had little promotion effect on UC-MSCs. Mechanistically, we discovered that UC-MSC infusion significantly modulated systematic inflammatory microenvironment, which contributed to concerted actions with FMD. CONCLUSIONS: We established a strategy that combined UC-MSC infusion and FMD and was effective in treating T2D, which provided potential approaches for developing novel clinical T2D therapies.

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Photoreceptor apoptosis is recognized as one key pathogenesis of retinal degeneration, the counteraction of which represents a promising approach to safeguard visual function. Recently, mesenchymal stem cell transplantation (MSCT) has demonstrated immense potential to treat ocular disorders, in which extracellular vesicles (EVs), particularly exosomes, have emerged as effective ophthalmological therapeutics. However, whether and how MSCT protects photoreceptors against apoptotic injuries remains largely unknown. Here, we discovered that intravitreal MSCT counteracted photoreceptor apoptosis and alleviated retinal morphological and functional degeneration in a mouse model of photoreceptor loss induced by N-methyl-N-nitrosourea (MNU). Interestingly, effects of MSCT were inhibited after blockade of exosomal generation by GW4869 preconditioning. Furthermore, MSC-derived exosomal transplantation (EXOT) effectively suppressed MNU-provoked photoreceptor injury. Notably, therapeutic efficacy of MSCT and EXOT on MNU-induced retinal degeneration was long-lasting as photoreceptor preservance and retinal maintenance were detected even after 1-2 months post to injection for only once. More importantly, using a natural occurring retinal degeneration model caused by a nonsense mutation of Phosphodiesterase 6b gene (Pde6bmut), we confirmed that MSCT and EXOT prevented photoreceptor loss and protected long-term retinal function. In deciphering therapeutic mechanisms regarding potential exosome-mediated communications, we identified that miR-21 critically maintained photoreceptor viability against MNU injury by targeting programmed cell death 4 (Pdcd4) and was transferred from MSC-derived exosomes in vivo for functional regulation. Moreover, miR-21 deficiency aggravated MNU-driven retinal injury and was restrained by EXOT. Further experiments revealed that miR-21 mediated therapeutic effects of EXOT on MNU-induced photoreceptor apoptosis and retinal dysfunction. These findings uncovered the efficacy and mechanism of MSCT-based photoreceptor protection, indicating exosomal miR-21 as a therapeutic for retinal degeneration.

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Senescence is closely related to the occurrence of retinal degeneration. Recent studies have shown that bone marrow mesenchymal stem cells (BMMSCs) have significant therapeutic effects on retinal degeneration, While BMMSCs suffer from functional decline in bone aging. Whether senescence affects BMMSCs therapy on retinal degeneration remains unknown. Here, we applied the previously established bone progeria animal model, the senescence-accelerated mice-prone 6 (SAMP6) strain, and surprisingly discovered that SAMP6 mice demonstrated retinal degeneration at 6 months old. Furthermore, BMMSCs derived from SAMP6 mice failed to prevent MNU-induced retinal degeneration in vivo. As expected, BMMSCs from SAMP6 mice exhibited impairment in the differentiation capacities, compared to those from the age-matched senescence-accelerated mice-resistant 1 (SAMR1) strain. Moreover, BMMSCs from SAMR1 mice counteracted MNU-induced retinal degeneration, with increased expression of the retina survival hallmark, N-myc downstream regulated gene 2 (NDRG2). Taken together, these findings reveal that bone progeria diminished the therapeutic effects of BMMSC on retinal degeneration.

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Mesenchymal stem/stromal cells (MSCs) reside in the perivascular niche and modulate tissue/organ homeostasis; however, little is known about whether and how their localization and function are linked. Particularly, whether specific MSC subsets couple with and regulate specialized vessel subtypes is unclear. Here, we show that Gli1+ cells, which are a subpopulation of MSCs couple with and regulate a specialized form of vasculature. The specific capillaries, i.e., CD31hiEMCNhi type H vessels, are the preferable vascular subtype which Gli1+ cells are adjacent to in bone. Gli1+ cells are further identified to be phenotypically coupled with type H endothelium during bone growth and defect healing. Importantly, Gli1+ cell ablation inhibits type H vessel formation associated with suppressed bone generation and regeneration. Mechanistically, Gli1+ cells initiate angiogenesis through Gli and HIF-1α signaling. These findings suggest a morphological and functional framework of Gli1+ cells modulating coupled type H vasculature for tissue homeostasis and regenerative repair.

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OBJECTIVES: Gli1+ cells have received extensive attention in tissue homeostasis and injury mobilization. The aim of this study was to investigate whether Gli1+ cells respond to force and contribute to bone remodelling. MATERIALS AND METHODS: We established orthodontic tooth movement (OTM) model to assess the bone response for mechanical force. The transgenic mice were utilized to label and inhibit Gli1+ cells, respectively. Additionally, mice that conditional ablate Yes-associated protein (Yap) in Gli1+ cells were applied in the present study. The tooth movement and bone remodelling were analysed. RESULTS: We first found Gli1+ cells expressed in periodontal ligament (PDL). They were proliferated and differentiated into osteoblastic cells under tensile force. Next, both pharmacological and genetic Gli1 inhibition models were utilized to confirm that inhibition of Gli1+ cells led to arrest of bone remodelling. Furthermore, immunofluorescence staining identified classical mechanotransduction factor Yap expressed in Gli1+ cells and decreased after suppression of Gli1+ cells. Additionally, conditional ablation of Yap gene in Gli1+ cells inhibited the bone remodelling as well, suggesting Gli1+ cells are force-responsive cells. CONCLUSIONS: Our findings highlighted that Gli1+ cells in PDL directly respond to orthodontic force and further mediate bone remodelling, thus providing novel functional evidence in the mechanism of bone remodelling and first uncovering the mechanical responsive property of Gli1+ cells.

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