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This study aims to identify the relationship among job challenge, learning agility, job autonomy, and job performance based on job demand-resources model. In particular, this study examined the mediating role of learning agility in the relationship between Job challenge and job performance, which has rarely been conducted. Furthermore, the moderating effect of job autonomy as job resource was explored. The data collected were from army officers and noncommissioned officers (NCO) of the Korean Army (N = 425). We hypothesized that there is a positive relationship among Job challenge and learning agility, Job challenge and job performance, the mediating role of learning agility in the relationship between Job challenge and job performance, and the moderating role of job autonomy. As predicted, we found that Job challenge was positively related to learning agility and job performance. We also found a mediating effect of learning agility on the relationship between Job challenge and job performance. Furthermore, job autonomy moderated the positive relationship between Job challenge and learning agility, although the moderating effect of job autonomy between Job challenge and job performance was not significant. Finally, the indirect relationship between Job challenge and job performance, via learning agility, was stronger for the higher job autonomy group than for the lower job autonomy group. The, theoretical and practical contributions and suggestions for future studies on learning agility and job characteristics are discussed.
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Decellularized extracellular matrix scaffold, widely utilized for organ engineering, often undergoes matrix decomposition after transplantation and produces byproducts that cause inflammation, leading to clinical failure. Here we propose a strategy using nano-graphene oxide to modify the biophysical properties of decellularized liver scaffolds. Notably, we demonstrate that scaffolds crosslinked with nano-graphene oxide show high resistance to enzymatic degradation via direct inhibition of matrix metalloproteinase activity and increased mechanical rigidity. We find that M2-like macrophage polarization is promoted within the crosslinked scaffolds, which reduces graft-elicited inflammation. Moreover, we show that low activities of matrix metalloproteinases, attributed to both nano-graphene oxide and tissue inhibitors of metalloproteinases expressed by M2c, can protect the crosslinked scaffolds against in vivo degradation. Lastly, we demonstrate that bioengineered livers fabricated with the crosslinked scaffolds remain functional, thereby effectively regenerating damaged livers after transplantation into liver failure mouse models. Overall, nano-graphene oxide crosslinking prolongs allograft survival and ultimately improves therapeutic effects of bioengineered livers, which offer an alternative for donor organs.
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Regeneração Hepática , Alicerces Teciduais , Camundongos , Animais , Fígado , Inflamação/metabolismo , Imunomodulação , Óxidos/metabolismo , Engenharia Tecidual , Matriz Extracelular/metabolismoRESUMO
We investigated the neuroprotective effects of deca nano-graphene oxide (daNGO) against reactive oxygen species (ROS) and inflammation in the human neuroblastoma cell line SH-SY5Y and in the 6-hydroxydopamine (6-OHDA) induced Parkinsonian rat model. An MTT assay was performed to measure cell viability in vitro in the presence of 6-OHDA and/or daNGO. The intracellular ROS level was quantified using 2',7'-dichlorofluorescein diacetate. daNGO showed neuroprotective effects against 6-OHDA-induced toxicity and also displayed ROS scavenging properties. We then tested the protective effects of daNGO against 6-OHDA induced toxicity in a rat model. Stepping tests showed that the akinesia symptoms were improved in the daNGO group compared to the control group. Moreover, in an apomorphine-induced rotation test, the number of net contralateral rotations was decreased in the daNGO group compared to the control group. By immunofluorescent staining, the animals in the daNGO group had more tyrosine hydroxylase-positive cells than the controls. By anti-Iba1 staining, 6-OHDA induced microglial activation showed a significantly decrease in the daNGO group, indicating that the neuroprotective effects of graphene resulted from anti-inflammation. In conclusion, nanographene oxide has neuroprotective effects against the neurotoxin induced by 6-OHDA on dopaminergic neurons. [BMB Reports 2023; 56(3): 202-207].
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Neuroblastoma , Fármacos Neuroprotetores , Doença de Parkinson , Humanos , Ratos , Animais , Espécies Reativas de Oxigênio/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Apoptose , Oxidopamina/toxicidade , Fármacos Neuroprotetores/farmacologia , Linhagem Celular Tumoral , Neuroblastoma/metabolismoRESUMO
Glioblastoma is considered one of the most aggressive and dangerous brain tumors. However, treatment of GBM has been still challenged due to blood-brain barrier (BBB). BBB prevents that the chemotherapeutic molecules are extravasated to brain. In this study, sonosensitive liposome encapsulating doxorubicin (DOX) was developed for enhancement of GBM penetration in combination with focused ultrasound (FUS) and microbubbles. Upon ultrasound (US) irradiation, microbubbles induce cavitation resulting in the tight junction of BBB endothelium to temporarily open. In addition, the composition of sonosensitive liposome was optimized by comparison of sonosensitivity and intracellular uptake to U87MG cells. The optimal sonosensitive liposome, IMP301-DC, resulted 123.9 ± 38.2 nm in size distribution and 98.2 % in loading efficiency. Related to sonosensitivity of IMP301-DC, US-triggered release ratio of doxorubicin was 69.2 ± 12.3 % at 92 W/cm2 of US intensity for 1 min. In the in vivo experiments, the accumulation of DiD fluorescence probe labeled IMP301-DC-shell in the brain through the BBB opening was increased more than two-fold compared to that of Doxil-shell, non-sonosensitive liposome. US exposure significantly increased GBM cytotoxicity of IMP301-DC. In conclusion, this study demonstrated that IMP301-DC could serve as an alternative solution to enhance the penetration to GBM treatment via BBB opening by non-invasive FUS combined with microbubbles.
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Lipossomos , Microbolhas , Barreira Hematoencefálica/efeitos da radiação , Encéfalo , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacologia , PolietilenoglicóisRESUMO
Alzheimer's disease (AD) is one of the progressive neurodegenerative diseases characterized by ß-amyloid (Aß) production and Phosphorylated-Tau (p-Tau) protein in the cerebral cortex. The precise mechanisms of the cause, responsible for disease pathology and progression, are not well understood because there are multiple risk factors associated with the disease. Viral infection is one of the risk factors for AD, and we demonstrated that Zika virus (ZIKV) infection in brain organoids could trigger AD pathological features, including Aß and p-Tau expression. AD-related phenotypes in brain organoids were upregulated via endoplasmic reticulum (ER) stress and unfolded protein response (UPR) after ZIKV infection in brain organoids. Under persistent ER stress, activated-double stranded RNA-dependent protein kinase-like ER-resident (PERK) triggered the phosphorylation of Eukaryotic initiation factor 2 (eIF2α) and then BACE, and GSK3α/ß related to AD. Furthermore, we demonstrated that pharmacological inhibitors of PERK attenuated Aß and p-Tau in brain organoids after ZIKV infection.
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BACKGROUND AND OBJECTIVES: Brain organoids have the potential to improve our understanding of brain development and neurological disease. Despite the importance of brain organoids, the effect of vascularization on brain organoids is largely unknown. The objective of this study is to develop vascularized organoids by assembling vascular spheroids with cerebral organoids. METHODS AND RESULTS: In this study, vascularized spheroids were generated from non-adherent microwell culture system of human umbilical vein endothelial cells, human dermal fibroblasts and human umbilical cord blood derived mesenchymal stem cells. These vascular spheroids were used for fusion with iPSCs induced cerebral organoids. Immunostaining studies of vascularized organoids demonstrated well organized vascular structures and reduced apoptosis. We showed that the vascularization in cerebral organoids up-regulated the Wnt/ß-catenin signaling. CONCLUSIONS: We developed vascularized cerebral organoids through assembly of brain organoids with vascular spheroids. This method could not only provide a model to study human cortical development but also represent an opportunity to explore neurological disease.
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Mitochondrial dysfunction is associated with familial Alzheimer's disease (fAD), and the accumulation of damaged mitochondria has been reported as an initial symptom that further contributes to disease progression. In the amyloidogenic pathway, the amyloid precursor protein (APP) is cleaved by ß-secretase to generate a C-terminal fragment, which is then cleaved by γ-secretase to produce amyloid-beta (Aß). The accumulation of Aß and its detrimental effect on mitochondrial function are well known, yet the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) contributing to this pathology have rarely been reported. We demonstrated the effects of APP-CTFs-related pathology using induced neural stem cells (iNSCs) from AD patient-derived fibroblasts. APP-CTFs accumulation was demonstrated to mainly occur within mitochondrial domains and to be both a cause and a consequence of mitochondrial dysfunction. APP-CTFs accumulation also resulted in mitophagy failure, as validated by increased LC3-II and p62 and inconsistent PTEN-induced kinase 1 (PINK1)/E3 ubiquitin ligase (Parkin) recruitment to mitochondria and failed fusion of mitochondria and lysosomes. The accumulation of APP-CTFs and the causality of impaired mitophagy function were also verified in AD patient-iNSCs. Furthermore, we confirmed this pathological loop in presenilin knockout iNSCs (PSEN KO-iNSCs) because APP-CTFs accumulation is due to γ-secretase blockage and similarly occurs in presenilin-deficient cells. In the present work, we report that the contribution of APP-CTFs accumulation is associated with mitochondrial dysfunction and mitophagy failure in AD patient-iNSCs as well as PSEN KO-iNSCs.
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In vitro platforms for studying the human brain have been developed, and brain organoids derived from stem cells have been studied. However, current organoid models lack three-dimensional (3D) vascular networks, limiting organoid proliferation, differentiation, and apoptosis. In this study, we created a 3D model of vascularized spheroid cells using an injection-molded microfluidic chip. We cocultured spheroids derived from induced neural stem cells (iNSCs) with perfusable blood vessels. Gene expression analysis and immunostaining revealed that the vascular network greatly enhanced spheroid differentiation and reduced apoptosis. This platform can be used to further study the functional and structural interactions between blood vessels and neural spheroids, and ultimately to simulate brain development and disease.
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Técnicas de Cocultura/métodos , Dispositivos Lab-On-A-Chip , Neovascularização Fisiológica/fisiologia , Células-Tronco Neurais/citologia , Esferoides Celulares/citologia , Apoptose/fisiologia , Vasos Sanguíneos/fisiologia , Diferenciação Celular/fisiologia , Humanos , Engenharia TecidualRESUMO
BACKGROUND: Human mesenchymal stem cells (hMSCs) therapy has recently been considered a promising treatment for atopic dermatitis (AD) due to their immunomodulation and tissue regeneration ability. In our previous studies, we demonstrated that hMSCs alleviate allergic inflammation in murine AD model by inhibiting the activation of mast cells and B cells. Also our phase I/IIa clinical trial showed clinical efficacy and safety of hMSCs in moderate-to-severe adult AD patients. However, hMSCs therapy against atopic dermatitis have had poor results in clinical field. Therefore, we investigated the reason behind this result. We hypothesized that drug-cell interaction could interfere with the therapeutic efficacy of stem cells, and investigated whether coadministration with pimecrolimus, one of the topical calcineurin inhibitors, could influence the therapeutic potential of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in AD. METHODS: hUCB-MSCs were subcutaneously injected to AD-induced mice with or without pimecrolimus topical application. To examine whether pimecrolimus influenced the immunomodulatory activity of hUCB-MSCs, hUCB-MSCs were treated with pimecrolimus. RESULTS: Pimecrolimus disturbed the therapeutic effect of hUCB-MSCs when they were co-administered in murine AD model. Moreover, the inhibitory functions of hUCB-MSCs against type 2 helper T (Th2) cell differentiation and mast cell activation were also deteriorated by pimecrolimus treatment. Interestingly, we found that pimecrolimus decreased the production of PGE2, one of the most critical immunomodulatory factors in hUCB-MSCs. And we demonstrated that pimecrolimus downregulated COX2-PGE2 axis by inhibiting nuclear translocation of NFAT3. CONCLUSIONS: Coadministration of pimecrolimus with hMSCs could interfere with the therapeutic efficacy of hMSCs in atopic dermatitis, and this is the first study that figured out the interaction of hMSCs with other drugs in cell therapy of atopic dermatitis. Therefore, this study might give rise to improvement of the clinical application of hMSCs therapy and facilitate the widespread application of hMSCs in clinical field.
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Dermatite Atópica , Células-Tronco Mesenquimais , Animais , Ciclo-Oxigenase 2 , Dermatite Atópica/tratamento farmacológico , Humanos , Camundongos , Tacrolimo/análogos & derivados , Tacrolimo/farmacologiaRESUMO
Three-dimensional (3D) bioprinting is a promising technology to establish a 3D in vitro hepatic model that holds great potential in toxicological evaluation. However, in current hepatic models, the central area suffers from hypoxic conditions, resulting in slow and weak metabolism of drugs and toxins. It remains challenging to predict accurate drug effects in current bioprinted hepatic models. Here, we constructed a hexagonal bioprinted hepatic construct and incorporated a spinning condition with continuous media stimuli. Under spinning conditions, HepG2 cells in the bioprinted hepatic construct exhibited enhanced proliferation capacity and functionality compared to those under static conditions. Additionally, the number of spheroids that play a role in boosting drug-induced signals and responses increased in the bioprinted hepatic constructs cultured under spinning conditions. Moreover, HepG2 cells under spinning conditions exhibited intensive TGFß-induced epithelial-to-mesenchymal transition (EMT) and increased susceptibility to acetaminophen (APAP)-induced hepatotoxicity as well as hepatotoxicity prevention by administration of N-acetylcysteine (NAC). Taken together, the results of our study demonstrate that the spinning condition employed during the generation of bioprinted hepatic constructs enables the recapitulation of liver injury and repair phenomena in particular. This simple but effective culture strategy facilitates bioprinted hepatic constructs to improve in vitro modeling for drug effect evaluation.
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Biomimética , Bioimpressão/instrumentação , Proliferação de Células , Fígado/patologia , Modelos Biológicos , Impressão Tridimensional/estatística & dados numéricos , Engenharia Tecidual , Acetaminofen/toxicidade , Acetilcisteína/farmacologia , Analgésicos não Narcóticos/toxicidade , Sequestradores de Radicais Livres/farmacologia , Células Hep G2 , Humanos , Hidrogéis , Técnicas In Vitro , Fígado/efeitos dos fármacos , Alicerces Teciduais/química , Testes de ToxicidadeRESUMO
Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.
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Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Canais de Cátion TRPP/genética , Células A549 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Células-Tronco Mesenquimais/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Canais de Cátion TRPP/metabolismo , Transfecção , TransgenesRESUMO
Acute pancreatitis is an acute inflammatory process in the pancreas that is common in dogs. This study was designed to compare cytokines between healthy dogs and dogs with suspected acute pancreatitis. For the canine cytokine antibody array, three healthy dogs and three dogs with suspected acute pancreatitis were included. Interleukin (IL)-2, IL-6, IL-10, GM-CSF, and TNF-α were not detected in either group based on the results. Conversely, IL-8 (p = 0.035), Monocyte Chemoattractant Protein-1 (MCP)-1 (p = 0.0138), Receptor for Advanced Glycation Endproducts (RAGE) (p = 0.0079), and stem cell factor (SCF) (p = 0.034) were significantly increased in dogs with suspected acute pancreatitis. However, vascular endothelial growth factor (VEGF) (p = 0.6971) did not differ significantly between groups. For the canine serum Enzyme-Linked Immunosorbent Assay (ELISA), eight healthy dogs and eight dogs with suspected acute pancreatitis were included. ELISA revealed that IL-8 (p < 0.0001), MCP-1 (p < 0.0001), RAGE (p = 0.006), and SCF (p = 0.0002) were all significantly upregulated in the experimental group. We confirmed multiple patterns of cytokines in suspected acute pancreatitis of dogs via canine cytokine antibody array using a small quantity of serum. After this procedure, we reevaluated the cytokines, which were significantly increased in dogs with suspected acute pancreatitis, by ELISA, with more samples. Through this study, we confirmed that MCP-1, RAGE, and SCF were newly suggested factors in dogs with suspected acute pancreatitis.
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While the neuropathological characteristics of Niemann-Pick disease type C (NPC) result in a fatal diagnosis, the development of clinically available therapeutic agent remains a challenge. Here we propose graphene quantum dots (GQDs) as a potential candidate for the impaired functions in NPC in vivo. In addition to the previous findings that GQDs exhibit negligible long-term toxicity and are capable of penetrating the blood-brain barrier, GQD treatment reduces the aggregation of cholesterol in the lysosome through expressed physical interactions. GQDs also promote autophagy and restore defective autophagic flux, which, in turn, decreases the atypical accumulation of autophagic vacuoles. More importantly, the injection of GQDs inhibits the loss of Purkinje cells in the cerebellum while also demonstrating reduced activation of microglia. The ability of GQDs to alleviate impaired functions in NPC proves the promise and potential of the use of GQDs toward resolving NPC and other related disorders.
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Grafite , Doença de Niemann-Pick Tipo C , Pontos Quânticos , Autofagia , Humanos , Lisossomos , Doença de Niemann-Pick Tipo C/tratamento farmacológicoRESUMO
Brain organoids have emerged as a novel model system for neural development, neurodegenerative diseases, and human-based drug screening. However, the heterogeneous nature and immature neuronal development of brain organoids generated from pluripotent stem cells pose challenges. Moreover, there are no previous reports of a three-dimensional (3D) hypoxic brain injury model generated from neural stem cells. Here, we generated self-organized 3D human neural organoids from adult dermal fibroblast-derived neural stem cells. Radial glial cells in these human neural organoids exhibited characteristics of the human cerebral cortex trend, including an inner (ventricular zone) and an outer layer (early and late cortical plate zones). These data suggest that neural organoids reflect the distinctive radial organization of the human cerebral cortex and allow for the study of neuronal proliferation and maturation. To utilize this 3D model, we subjected our neural organoids to hypoxic injury. We investigated neuronal damage and regeneration after hypoxic injury and reoxygenation. Interestingly, after hypoxic injury, reoxygenation restored neuronal cell proliferation but not neuronal maturation. This study suggests that human neural organoids generated from neural stem cells provide new opportunities for the development of drug screening platforms and personalized modeling of neurodegenerative diseases, including hypoxic brain injury.
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Lesões Encefálicas/patologia , Hipóxia Encefálica/patologia , Modelos Biológicos , Neurônios/patologia , Organoides/patologia , Adulto , Biomarcadores/metabolismo , Córtex Cerebral/patologia , Humanos , Oxigênio/metabolismoRESUMO
Liver tissue engineering offers a promising strategy for liver failure patients. Since transplantation rejection resulting in vessel thrombosis is regarded as a major hurdle, vascular reconstruction is one of indispensable requirements of whole organ engineering. Here we demonstrated a novel strategy for reconstruction of a vascularized bioengineered human liver (VBHL) using decellularized liver scaffolds in an efficient manner. First we achieved fully functional endothelial coverage of scaffolds by adopting the anti-CD31 aptamer as a potent coating agent for re-endothelialization. Through an ex vivo human blood perfusion that recapitulates the blood coagulation response in humans, we demonstrated significantly reduced platelet aggregation in anti-CD31 aptamer coated scaffolds. We then produced VBHL constructs using liver parenchymal cells and nonparenchymal cells, properly organized into liver-like structures with an aligned vasculature. Interestingly, VBHL constructs displayed prominently enhanced long-term liver-specific functions that are affected by vascular functionality. The VBHL constructs formed perfusable vessel networks in vivo as evidenced by the direct vascular connection between the VBHL constructs and the renal circulation. Furthermore, heterotopic transplantation of VBHL constructs supported liver functions in a rat model of liver fibrosis. Overall, we proposed a new strategy to generate transplantable bioengineered livers characterized by highly functional vascular reconstruction.
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Células Endoteliais , Alicerces Teciduais , Animais , Engenharia Biomédica , Humanos , Fígado , Ratos , Engenharia TecidualRESUMO
Recent studies on developing three-dimensional (3D) brain organoids from stem cells have allowed the generation of in vitro models of neural disease and have enabled the screening of drugs because these organoids mimic the complexity of neural tissue. Niemann-Pick disease, type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in the NPC1 or NPC2. The pathological features underlying NPC are characterized by the abnormal accumulation of cholesterol in acidic compartments, including late endosomes and lysosomes. Due to the inaccessibility of brain tissues from human NPC patients, we developed NPC brain organoids with induced neural stem cells from NPC patient-derived fibroblasts. NPC organoids exhibit significantly reduced size and proliferative ability, which are accompanied by accumulation of cholesterol, impairment in neuronal differentiation, and autophagic flux and dysfunction of lysosomes; therefore, NPC organoids can recapitulate the main phenotypes of NPC patients. Furthermore, these pathological phenotypes observed in NPC organoids were reversed by treatment with valproic acid and HPBCD, which are known to be an effective treatment for several neurodegenerative diseases. Our data present patient-specific phenotypes in 3D organoid-based models of NPC and highlight the application of this model to drug screening in vitro.
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Encéfalo/patologia , Modelos Biológicos , Células-Tronco Neurais/patologia , Doença de Niemann-Pick Tipo C/patologia , Organoides/patologia , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Niemann-Pick Tipo C/genética , Organoides/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido Valproico/farmacologiaRESUMO
While graphene and its derivatives have been suggested as a potential nanomedicine in several biomimetic models, their specific roles in immunological disorders still remain elusive. Graphene quantum dots (GQDs) may be suitable for treating intestinal bowel diseases (IBDs) because of their low toxicity in vivo and ease of clearance. Here, GQDs are intraperitoneally injected to dextran sulfate sodium (DSS)-induced chronic and acute colitis model, and its efficacy has been confirmed. In particular, GQDs effectively prevent tissue degeneration and ameliorate intestinal inflammation by inhibiting TH1/TH17 polarization. Moreover, GQDs switch the polarization of macrophages from classically activated M1 to M2 and enhance intestinal infiltration of regulatory T cells (Tregs). Therefore, GQDs effectively attenuate excessive inflammation by regulating immune cells, indicating that they can be used as promising alternative therapeutic agents for the treatment of autoimmune disorders, including IBDs.
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by loss of motor neurons and degeneration of neuromuscular junctions. To improve disease progression, previous studies have suggested many options that have shown beneficial effects in diseases, especially stem cell therapy. In this study, we used repeated intramuscular transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) and observed positive effects on muscle atrophy and oxidative stress. In an in vivo study, motor function, body weight and survival rate were assessed, and skeletal muscle tissues were analyzed by western blotting and immunohistochemistry. After intramuscular transplantation, the hUCB-MSCs survived within the skeletal muscle for at least 1 week. Transplantation ameliorated muscle atrophy and the rate of neuromuscular degeneration in skeletal muscle through reductions in intracellular ROS levels. Both expression of skeletal muscle atrophy markers, muscle atrophy F-box (MAFbx)/atrogin1 and muscle RING finger 1 (MuRF1), were also reduced; however, the reductions were not significant. Moreover, transplantation of hUCB-MSCs improved protein synthesis and inhibited the iNOS/NO signaling pathway through AMPK activation. Our results suggest that repeated intramuscular transplantation of hUCB-MSCs can be a practical option for stem cell therapy for ALS.
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Esclerose Lateral Amiotrófica/terapia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sistema de Sinalização das MAP Quinases , Transplante de Células-Tronco Mesenquimais/métodos , Destreza Motora , Superóxido Dismutase-1/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intramusculares , Camundongos , Camundongos Transgênicos , Atrofia Muscular/terapia , Mioblastos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Although the generation of ETV2-induced endothelial cells (iECs) from human fibroblasts serves as a novel therapeutic strategy in regenerative medicine, the process is inefficient, resulting in incomplete iEC angiogenesis. Therefore, we employed chromatin immunoprecipitation (ChIP) sequencing and identified molecular mechanisms underlying ETV2-mediated endothelial transdifferentiation to efficiently produce iECs retaining appropriate functionality in long-term culture. We revealed that the majority of ETV2 targets in human fibroblasts are related to vasculature development and signaling transduction pathways, including Rap1 signaling. From a screening of signaling pathway modulators, we confirmed that forskolin facilitated efficient and rapid iEC reprogramming via activation of the cyclic AMP (cAMP)/exchange proteins directly activated by cAMP (EPAC)/RAP1 axis. The iECs obtained via cAMP signaling activation showed superior angiogenesis in vivo as well as in vitro. Moreover, these cells could form aligned endothelium along the vascular lumen ex vivo when seeded into decellularized liver scaffold. Overall, our study provided evidence that the cAMP/EPAC/RAP1 axis is required for the efficient generation of iECs with angiogenesis potential.
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AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Fatores de Transcrição/metabolismo , Reprogramação Celular/genética , Expressão Ectópica do Gene , Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Fatores de Transcrição/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Although human mesenchymal stem cells (hMSCs) hold considerable promise as an alternative therapeutic reagent for allergic disorders including atopic dermatitis (AD), the strategy for enhancing hMSC-based therapy remains challenging. We sought to investigate whether preconditioning with mast cell (MC) granules could enhance the therapeutic efficiency of human umbilical cord blood-derived MSCs (hUCB-MSCs) against AD. Methods: AD was experimentally induced in NC/Nga mice by repeated applications of 4% sodium dodecyl sulfate (SDS) and dermatophagoides farinae (Df) extract, and preconditioned hUCB-MSCs were subcutaneously injected. The therapeutic effect was determined by gross examination and additional ex vivo experiments performed using blood and skin samples to determine the resolution of allergic inflammation. To explore the underlying mechanisms, several co-culture assays with primary isolated immune cells and wound closure assays were conducted. Results: Pretreatment of MC granules enhanced the therapeutic effects of hUCB-MSCs by attenuating the symptoms of AD in an experimental animal model. MC granule-primed cells suppressed the activation of major disease-inducing cells, MCs and B lymphocytes more efficiently than naïve cells both in vitro and in vivo. Histamine-mediated upregulation of the COX-2 signaling pathway was shown to play a crucial role in suppression of the allergic immune response by MC-pretreated hUCB-MSCs. Moreover, MC pretreatment improved the wound healing ability of hUCB-MSCs. Conclusions: Our findings indicate that pre-exposure to MC granules improved the therapeutic effect of hUCB-MSCs on experimental AD by resolving the allergic immune reaction and accelerating the tissue regeneration process more efficiently than naïve cells, suggesting a potential enhancement strategy for stem cell-based therapy.