RESUMEN

The challenge of treating corneal scarring through keratoplasties lies in the limited availability of donor tissue. Various studies have shown the therapeutic use of cultivated corneal stromal stem cells (CSSCs) to mitigate tissue inflammation and suppress fibrosis and scar tissue formation in preclinical corneal wound models. To develop CSSC therapy for clinical trials on patients with corneal scarring, it is necessary to generate clinical-grade CSSCs in compliant to Good Manufacturing Practice (GMP) regulations. This chapter elucidates human CSSC isolation, culture, and cryopreservation under GMP-compliant conditions. It underscores quality assessment encompassing morphological traits, expression of stemness markers, anti-inflammatory activity, and keratocyte differentiation potency.

Asunto(s)

Técnicas de Cultivo de Célula , Diferenciación Celular , Sustancia Propia , Humanos , Técnicas de Cultivo de Célula/métodos , Sustancia Propia/citología , Separación Celular/métodos , Criopreservación/métodos , Células Madre/citología , Células Madre/metabolismo , Células Cultivadas , Biomarcadores , Células del Estroma/citología

RESUMEN

The pathological mechanisms of cataract remain largely unknown due to the lack of appropriate in vitro cellular models. We developed a stable in vitro system, namely, a "fried egg" differentiation method to generate functional lentoid bodies (LBs) from induced pluripotent stem cells (iPSCs). The iPSCs-derived LBs exhibited crystalline lens-like morphology and a transparent structure, and expressed lens-specific markers. TEM examination and optical analysis further demonstrated that it has the same cell arrangement structure and magnifying ability as lens.

Asunto(s)

Diferenciación Celular , Células Madre Pluripotentes Inducidas , Cristalino , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Cristalino/citología , Cristalino/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Catarata/patología

RESUMEN

Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.

Asunto(s)

Células Madre , Malla Trabecular , Malla Trabecular/metabolismo , Malla Trabecular/citología , Animales , Ratones , Humanos , Células Madre/citología , Células Madre/metabolismo , Regeneración , Glaucoma/terapia , Trasplante de Células Madre/métodos , Secretoma , Modelos Animales de Enfermedad , Glaucoma de Ángulo Abierto/terapia , Células Cultivadas , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Técnicas de Cultivo de Célula/métodos

RESUMEN

Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.

Asunto(s)

Técnicas de Cultivo de Célula , Diferenciación Celular , Células Madre Pluripotentes Inducidas , Organoides , Epitelio Pigmentado de la Retina , Humanos , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Organoides/citología , Organoides/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Cultivadas

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Xiaozheng Tang (WXT), a traditional Chinese medicine (TCM) decoction, is effective for treating endometriosis. However, the effect of WXT on endometrium-derived mesenchymal stem cells (eMSCs) which play a key role in the fibrogenesis of endometriosis requires further elucidation. AIMS OF THE STUDY: The aim of this study was to clarify the potential mechanism of WXT in improving fibrosis in endometriosis by investigating the regulation of WXT on differentiation and paracrine of eMSCs. MATERIALS AND METHODS: The nude mice with endometriosis were randomly divided into model group, WXT group and mifepristone group. After 21 days of treatment, the lesion volume was calculated. Fibrosis in the lesions was evaluated by Masson staining and expression of fibrotic proteins. The differentiation of eMSCs in vivo was explored using a fate-tracking experiment. To further clarify the regulation of WXT on eMSCs, primary eMSCs from the ectopic lesions of endometriosis patients were isolated and characterized. The effect of WXT on the proliferation and differentiation of ectopic eMSCs was examined. To evaluate the role of WXT on the paracrine activity of ectopic eMSCs, the conditioned medium (CM) from ectopic eMSCs pretreated with WXT was collected and applied to treat ectopic endometrial stromal cells (ESCs), after which the expression of fibrotic proteins in ectopic ESCs was assessed. In addition, transcriptome sequencing was used to investigate the regulatory mechanism of WXT on ectopic eMSCs, and western blot and ELISA were employed to determine the key mediator. RESULTS: WXT impeded the growth of ectopic lesions in nude mice with endometriosis and reduced collagen deposition and the expression of fibrotic proteins fibronectin, collagen I, α-SMA and CTGF in the endometriotic lesions. The fate-tracking experiment showed that WXT prevented human eMSCs from differentiating into myofibroblasts in the nude mice. We successfully isolated eMSCs from the lesions of patients with endometriosis and demonstrated that WXT suppressed proliferation and myofibroblast differentiation of ectopic eMSCs. Moreover, the expression of α-SMA, collagen I, fibronectin and CTGF in ectopic ESCs was significantly down-regulated by the CM of ectopic MSCs pretreated with WXT. Combining the results of RNA sequencing, western blot and ELISA, we found that WXT not only reduced thrombospondin 4 expression in ectopic eMSCs, but also decreased thrombospondin 4 secretion from ectopic eMSCs. Thrombospondin 4 concentration-dependently upregulated the expression of collagen I, fibronectin, α-SMA and CTGF in ectopic ESCs, indicating that thrombospondin 4 was a key mediator of WXT in inhibiting the fibrotic process in endometriosis. CONCLUSION: WXT improved fibrosis in endometriosis by regulating differentiation and paracrine signaling of eMSCs. Thrombospondin 4, whose release from ectopic eMSCs is inhibited by WXT, may be a potential target for the treatment of endometriosis.

Asunto(s)

Diferenciación Celular , Medicamentos Herbarios Chinos , Endometriosis , Endometrio , Fibrosis , Células Madre Mesenquimatosas , Ratones Desnudos , Comunicación Paracrina , Endometriosis/tratamiento farmacológico , Endometriosis/patología , Endometriosis/metabolismo , Femenino , Animales , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Comunicación Paracrina/efectos de los fármacos , Humanos , Diferenciación Celular/efectos de los fármacos , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Endometrio/patología , Ratones , Células Cultivadas , Adulto , Modelos Animales de Enfermedad

RESUMEN

Traditional bioreactor systems involve the use of three-dimensional (3D) scaffolds or stem cell aggregates, limiting the accessibility to the production of cell-secreted biomolecules. Herein, we present the use a pulse electromagnetic fields (pEMFs)-assisted wave-motion bioreactor system for the dynamic and scalable culture of human bone marrow-derived mesenchymal stem cells (hBMSCs) with enhanced the secretion of various soluble factors with massive therapeutic potential. The present study investigated the influence of dynamic pEMF (D-pEMF) on the kinetic of hBMSCs. A 30-min exposure of pEMF (10V-1Hz, 5.82 G) with 35 oscillations per minute (OPM) rocking speed can induce the proliferation (1 × 105 â†’ 4.5 × 105) of hBMSCs than static culture. Furthermore, the culture of hBMSCs in osteo-induction media revealed a greater enhancement of osteogenic transcription factors under the D-pEMF condition, suggesting that D-pEMF addition significantly boosted hBMSCs osteogenesis. Additionally, the RNA sequencing data revealed a significant shift in various osteogenic and signaling genes in the D-pEMF group, further suggesting their osteogenic capabilities. In this research, we demonstrated that the combined effect of wave and pEMF stimulation on hBMSCs allows rapid proliferation and induces osteogenic properties in the cells. Moreover, our study revealed that D-pEMF stimuli also induce ROS-scavenging properties in the cultured cells. This study also revealed a bioactive and cost-effective approach that enables the use of cells without using any expensive materials and avoids the possible risks associated with them post-implantation.

Asunto(s)

Reactores Biológicos , Campos Electromagnéticos , Células Madre Mesenquimatosas , Osteogénesis , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Perfilación de la Expresión Génica , Proliferación Celular , Diferenciación Celular , Células Cultivadas , Transcriptoma

RESUMEN

Extracellular matrix (ECM) stiffness is a major driver of stem cell fate. However, the involvement of the three-dimensional (3D) genomic reorganization in response to ECM stiffness remains unclear. Here, we generated comprehensive 3D chromatin landscapes of mesenchymal stem cells (MSCs) exposed to various ECM stiffness. We found that there were more long-range chromatin interactions, but less compartment A in MSCs cultured on stiff ECM than those cultured on soft ECM. However, the switch from compartment B in MSCs cultured on soft ECM to compartment A in MSCs cultured on stiff ECM included genes encoding proteins primarily enriched in cytoskeleton organization. At the topologically associating domains (TADs) level, stiff ECM tends to have merged TADs on soft ECM. These merged TADs on stiff ECM include upregulated genes encoding proteins enriched in osteogenesis, such as SP1, ETS1, and DCHS1, which were validated by quantitative real-time polymerase chain reaction and found to be consistent with the increase of alkaline phosphatase staining. Knockdown of SP1 or ETS1 led to the downregulation of osteogenic marker genes, including COL1A1, RUNX2, ALP, and OCN in MSCs cultured on stiff ECM. Our study provides an important insight into the stiff ECM-mediated promotion of MSC differentiation towards osteogenesis, emphasizing the influence of mechanical cues on the reorganization of 3D genome architecture and stem cell fate.

Asunto(s)

Diferenciación Celular , Matriz Extracelular , Células Madre Mesenquimatosas , Osteogénesis , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/genética , Matriz Extracelular/metabolismo , Diferenciación Celular/genética , Humanos , Células Cultivadas , Animales

RESUMEN

In clinical settings, addressing large bone defects remains a significant challenge for orthopedic surgeons. The use of genetically modified bone marrow mesenchymal stem cells (BMSCs) has emerged as a highly promising approach for these treatments. Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a multifunctional secreted glycoprotein, the role of which remains unclear in human hBMSCs. This study used various experimental methods to elucidate the potential mechanism by which SCUBE3 influences osteogenic differentiation of hBMSCs in vitro. Additionally, the therapeutic efficacy of SCUBE3, in conjunction with porous GeLMA microspheres, was evaluated in vivo using a mouse bone defect model. Our findings indicate that SCUBE3 levels increase significantly during early osteogenic differentiation of hBMSCs, and that reducing SCUBE3 levels can hinder this differentiation. Overexpressing SCUBE3 elevated osteogenesis gene and protein levels and enhanced calcium deposition. Furthermore, treatment with recombinant human SCUBE3 (rhSCUBE3) protein boosted BMP2 and TGF-ß expression, activated mitophagy in hBMSCs, ameliorated oxidative stress, and restored osteogenic function through SMAD phosphorylation. In vivo, GELMA/OE treatment effectively accelerated bone healing in mice. In conclusion, SCUBE3 fosters osteogenic differentiation and mitophagy in hBMSCs by activating the BMP2/TGF-ß signaling pathway. When combined with engineered hydrogel cell therapy, it could offer valuable guidance for the clinical management of extensive bone defects.

Asunto(s)

Proteína Morfogenética Ósea 2 , Diferenciación Celular , Células Madre Mesenquimatosas , Mitofagia , Osteogénesis , Transducción de Señal , Factor de Crecimiento Transformador beta , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Animales , Mitofagia/fisiología , Ratones , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Células Cultivadas , Masculino

RESUMEN

Systemic sclerosis (SSc) is a life-threatening autoimmune disease characterized by widespread fibrosis in the skin and several internal organs. Nudix Hydrolase 21 (NUDT2 or CFIm25) downregulation in fibroblasts is known to play detrimental roles in both skin and lung fibrosis. This study aims to investigate the upstream mechanisms that lead to NUDT21 repression in skin fibrosis. We identified transforming growth factor ß (TGFß1) as the primary cytokine that downregulated NUDT21 in normal skin fibroblasts. In the bleomycin-induced dermal fibrosis model, consistent with the peak activation of TGFß1 at the late fibrotic stage, NUDT21 was downregulated at this stage, and delayed NUDT21 knockdown during this fibrotic phase led to enhanced fibrotic response to bleomycin. Further investigation suggested TGFß downregulated NUDT21 through microRNA (miRNA) 181a and 181b induction. Both miR-181a and miR-181b were elevated in bleomycin-induced skin fibrosis in mice and primary fibroblasts isolated from SSc patients, and they directly targeted NUDT21 and led to its downregulation in skin fibroblasts. Functional studies demonstrated that miR-181a and miR-181b inhibitors attenuated bleomycin-induced skin fibrosis in mice in association with decreased NUDT21 expression, while miR-181a and miR-181b mimics promoted bleomycin-induced fibrosis. Overall, these findings suggest a novel role for miR-181a/b in SSc pathogenesis by repressing NUDT21 expression.

Asunto(s)

Bleomicina , Fibroblastos , Fibrosis , MicroARNs , Esclerodermia Sistémica , Piel , MicroARNs/genética , MicroARNs/metabolismo , Animales , Humanos , Ratones , Fibrosis/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/inducido químicamente , Bleomicina/toxicidad , Bleomicina/efectos adversos , Piel/patología , Piel/metabolismo , Femenino , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Ratones Endogámicos C57BL , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Células Cultivadas , Regulación hacia Abajo

RESUMEN

Parkinson's disease (PD) is characterized by astrocyte activation and disruptions in circadian rhythm. Within the astrocyte population, two distinct reactive states exist: A1 and A2. A1 astrocytes are associated with neurotoxicity and inflammation, while A2 astrocytes exhibit neuroprotective functions. Our investigation focused on the role of REV-ERBα, a member of the nuclear receptor superfamily and a key regulator of the circadian clock, in astrocyte activation. We observed that REV-ERBα expression in A1 astrocytes was reduced to one-third of its normal level. Notably, activation of REV-ERBα prompted a transformation of astrocytes from A1 to A2. Mechanistically, REV-ERBα inhibition was linked to the classical NF-κB pathway, while it concurrently suppressed the STAT3 pathway. Furthermore, astrocytes with low REV-ERBα expression were associated with dopaminergic neurons apoptosis. Intriguingly, the opposite effect was observed when using a REV-ERBα agonist, which mitigated astrocyte activation and reduced dopaminergic neuron damage by 50%. In summary, our study elucidates the pivotal role of REV-ERBα in modulating astrocyte function and its potential implications in PD pathogenesis.

Asunto(s)

Astrocitos , Neuronas Dopaminérgicas , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Animales , Neuronas Dopaminérgicas/metabolismo , Ratones , Células Cultivadas , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Apoptosis , Ratones Endogámicos C57BL , Transducción de Señal

RESUMEN

The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.

Asunto(s)

Astrocitos , Corteza Cerebral , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Ratones Noqueados , Transducción de Señal , Animales , Astrocitos/metabolismo , Ratones , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Apirasa/genética , Apirasa/metabolismo , Células Cultivadas , Adenosina Trifosfato/metabolismo , Medios de Cultivo Condicionados , Adenosina/metabolismo , Adenosina/análogos & derivados , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2Y/genética , Ratones Endogámicos C57BL , Antígenos CD

RESUMEN

Eczema herpeticum (EH) is a disseminated severe herpes simplex virus type 1 (HSV-1) infection that mainly occurs in a subset of patients suffering from atopic dermatitis (AD). EH is complex and multifaceted, involving immunological changes, environmental influences, and genetic aberrations. Certain genetic variants of the thymic stromal lymphopoietin (TSLP) may predispose to develop severe HSV-1-induced eczema. Therefore, we investigated the impact of TSLP on HSV-1 infection. TSLP encodes for two distinct forms: a long-form (lfTSLP), primarily associated with type 2 immunity, and a short-form (sfTSLP) with anti-inflammatory and antimicrobial properties. While sfTSLP reduced HSV-1 infectibility in human primary keratinocytes (HPK), lfTSLP did not. In HPK treated with sfTSLP, HSV-1 gene expression, and replication decreased, while virion binding to cells and targeting of incoming capsids to the nucleus were not diminished compared to untreated cells. sfTSLP caused only minor changes in the expression of innate immunity cytokines, and its inhibition of HSV-1 infection did not require de novo protein synthesis. Time window experiments indicated a different antiviral mechanism than LL-37. sfTSLP showed the strongest antiviral effect when administered to HPK before or after inoculation with HSV-1, and outperformed the inhibitory potential of LL-37 under these conditions. Our data show that sfTSLP has antiviral functions and promotes repression of the HSV-1 infection in HPK.

Asunto(s)

Citocinas , Herpesvirus Humano 1 , Queratinocitos , Linfopoyetina del Estroma Tímico , Humanos , Citocinas/metabolismo , Queratinocitos/virología , Queratinocitos/inmunología , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/genética , Células Cultivadas , Replicación Viral , Erupción Variceliforme de Kaposi/virología , Erupción Variceliforme de Kaposi/inmunología , Herpes Simple/virología , Herpes Simple/inmunología , Herpes Simple/genética , Inmunidad Innata

RESUMEN

Obstructive uropathy is a common kidney disease caused by elevated hydrostatic pressure (HP), but relevant molecular and cellular mechanisms have not yet been well understood. In this study, we ex vivo investigated the effects of elevated HP on human renal epithelial cells (HREpCs). Primary HREpCs were subjected to 100 cmH2O HP for 8 or 48 h. Then, the cells were cultured without HP stimulation for another 24 h or 72 h. Cell morphology showed almost no change after 8h HP treatment, but exhibited reversible elongation after 48h HP treatment. HP treatment for 8 h increased the expression of TGFB1 and VEGFA but decreased the expression of CSF2 and TGFB2. On the other hand, HP treatment for 48 h downregulated the expression of CSF2, TGFB2, PDGFB, VEGFA, and VEGFB, while upregulated the expression of TGFB3. Interestingly, all changes induced by 48 h HP treatment were detected more severe compared to 8 h HP treatment. In conclusion, elongated ex vivo HP loading to renal epithelial cells induces reversible changes on cell morphology and disturbs the expression of several growth factors, which provides novel mechanistic insight on elevated HP-caused kidney injury such as obstructive uropathy.

Asunto(s)

Células Epiteliales , Presión Hidrostática , Riñón , Humanos , Células Epiteliales/metabolismo , Riñón/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Células Cultivadas , Factor A de Crecimiento Endotelial Vascular/metabolismo , Regulación de la Expresión Génica , Factor de Crecimiento Transformador beta1/metabolismo

RESUMEN

Purpose: Metabolic defects in the retinal pigment epithelium (RPE) underlie many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells. Methods: We profiled nutrient use of various human RPE cells, including differentiated and dedifferentiated fetal RPE (fRPE), induced pluripotent stem cell-derived RPE (iPSC RPE), Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE, CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and ARPE-19 cell lines using Biolog Phenotype MicroArray Assays. Results: Differentiated fRPE cells and healthy iPSC RPE cells can use 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose their epithelial phenotype through dedifferentiation, nutrient use becomes restricted to 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can use 37 nutrients; however, compared to cSFD RPE and healthy iPSC RPE, they are unable to use lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased use of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. Dedifferentiated ARPE-19 cells grown in traditional culture media cannot use lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation toward an epithelial phenotype, restoring the ability to use these nutrients. Conclusions: Epithelial phenotype confers metabolic flexibility to healthy RPE for using various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the potentially important roles of nutrient availability and use in RPE differentiation and diseases.

Asunto(s)

Diferenciación Celular , Células Madre Pluripotentes Inducidas , Fenotipo , Epitelio Pigmentado de la Retina , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Diferenciación Celular/fisiología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Cultivadas , Línea Celular

RESUMEN

The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co-culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell-cell interactions, act as the active components, improving graft maturation in this co-culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC-only culture), co-culture (ACL remnant cells and BMSCs, CM) and co-culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC-EV) and co-culture (CM-EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF-ß)-, vascular endothelial growth factor (VEGF)-, collagen synthesis- and tenogenesis-related genes. However, CM-induced effects were reversed by the CM ∆ EVs treatment. CM-EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC-EV and non-EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis-, TGF-ß-, VEGF- and tenogenesis-related genes in both cell types.

Asunto(s)

Ligamento Cruzado Anterior , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Técnicas de Cocultivo , Vesículas Extracelulares , Células Madre Mesenquimatosas , Vesículas Extracelulares/metabolismo , Animales , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Conejos , Ligamento Cruzado Anterior/citología , Ligamento Cruzado Anterior/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Células Cultivadas , Regulación de la Expresión Génica , Comunicación Celular , Factor de Crecimiento Transformador beta/metabolismo , Masculino

RESUMEN

OBJECTIVES: Chronic rejection remains the leading cause of progressive decline in graft function. Accumulating evidence indicates that macrophages participate in chronic rejection dependent on CD40-CD40L. The FOS family members are critical in inflammatory and immune responses. However, the mechanisms underlying the role of FOS family members in chronic rejection remain unclear. In this study, we aimed to elucidate the role and underlying mechanisms of FOS-positive macrophages regulated by CD40 that mediate chronic allograft rejection. MATERIALS AND METHODS: We downloaded publicly accessible chronic rejection kidney transplant single-cell sequencing datasets from the gene expression omnibus database. Differentially expressed genes between the CD40hi and CD40low macrophage chronic rejection groups were analyzed. We established a chronic rejection mouse model by using CTLA-4-Ig. We treated bone marrow-derived macrophages with an anti-CD40 antibody. We assessed expression of the FOS family by flow cytometry, real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. We identified altered signaling pathways by using RNA sequencing analysis. We detected DNA specifically bound to transcription factors by using ChIP-sequencing, with detection of the degree of graft fibrosis and survival. RESULTS: FOS was highly expressed on CD40hi macrophages in patients with chronic transplantrejection. Mechanistically, we showed that CD40 activated NF-κB2 translocation into the nucleus to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant.We showed thatNF-κB2 regulated c-Fos and FosB expression by binding to the c-fos and fosb promoter regions. Inhibition of c-Fos/activator protein-1 decreased graft fibrosis and prolonged graft survival. CONCLUSIONS: CD40 may activate transcription factor NF-κB2 translocation into the nucleus of macrophages to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant. Inhibition of c-Fos/activator protein-1 decreased grafts fibrosis and prolonged graft survival.

Asunto(s)

Antígenos CD40 , Modelos Animales de Enfermedad , Rechazo de Injerto , Trasplante de Corazón , Macrófagos , Proteínas Proto-Oncogénicas c-fos , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Antígenos CD40/metabolismo , Antígenos CD40/genética , Células Cultivadas , Enfermedad Crónica , Bases de Datos Genéticas , Fibrosis , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Rechazo de Injerto/genética , Supervivencia de Injerto , Trasplante de Corazón/efectos adversos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Factor de Transcripción AP-1/metabolismo

RESUMEN

OBJECTIVES: Mesenchymal stem cells are gaining attention in medicine because of their anti-inflammatory and immunosuppressive properties. Inflammatory conditions can modulate immune responses in mesenchymal stem cells.We investigated the expression of long noncoding RNAs (RMRP, MALT1, NKILA,THRIL, and Linc-MAF-4) in humanWharton jelly mesenchymal stem cells primed with polyinosinicpolycytidylic acid. MATERIALS AND METHODS: Mesenchymal stem cells were isolated from human Wharton jelly by the explant method. To determine the stem nature of the cells, we performed a differentiation test on bone and fat cells. We used flow cytometry analysis to determine surface markers. Umbilical cord mesenchymal stem cells (1 × 105) were cultured in T75 culture flasks in Dulbecco's modified Eagle medium containing 10% fetal bovine serum. After cells reached approximately 80% confluency, cells were exposed to 50 µg/mL of polyinosinic-polycytidylic acid, a Toll-like receptor 3 ligand, for 24, 48, and 72 hours. The control group were cells not exposed to polyinosinic-polycytidylic acid. Real-time polymerase chain reaction evaluated RMRP, MALAT1, NKILA, THRIL, and Linc-MAF-4 long noncoding RNAs. RESULTS: We observed significantly increased expression of NKILA inWharton jelly mesenchymal stem cells stimulated with polyinosinic-polycytidylic acid at 72 hours compared with expression level in the control group (P < .001). CONCLUSIONS: Results indicated that a potential mechanism by which the Toll-like receptor 3 ligand improves immunosuppression of mesenchymal stem cells can be attributed to the regulatory role of long noncoding RNAs, possibly through increased expression of anti-inflammatory long noncoding RNAs such as NKILA.

Asunto(s)

Diferenciación Celular , Células Madre Mesenquimatosas , Poli I-C , ARN Largo no Codificante , Receptor Toll-Like 3 , Gelatina de Wharton , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/genética , Gelatina de Wharton/citología , Células Cultivadas , Poli I-C/farmacología , Diferenciación Celular/efectos de los fármacos , Factores de Tiempo , Regulación de la Expresión Génica , Osteogénesis/efectos de los fármacos

RESUMEN

Mesenchymal stem cell derived extracellular vesicles (MSC EVs) are paracrine modulators of macrophage function. Scientific research has primarily focused on the immunomodulatory and regenerative properties MSC EVs derived from bone marrow. The dental pulp is also a source for MSCs, and their anatomical location and evolutionary function has primed them to be potent immunomodulators. In this study, we demonstrate that extracellular vesicles derived from dental pulp stem cells (DPSC EVs) have pronounced immunomodulatory effect on primary macrophages by regulating the NFκb pathway. Notably, the anti-inflammatory activity of DPSC-EVs is enhanced following exposure to an inflammatory stimulus (LPS). These inhibitory effects were also observed in vivo. Sequencing of the naïve and LPS preconditioned DPSC-EVs and comparison with our published results from marrow MSC EVs revealed that Naïve and LPS preconditioned DPSC-EVs are enriched with anti-inflammatory miRNAs, particularly miR-320a-3p, which appears to be unique to DPSC-EVs and regulates the NFκb pathway. Overall, our findings highlight the immunomodulatory properties of DPSC-EVs and provide vital clues that can stimulate future research into miRNA-based EV engineering as well as therapeutic approaches to inflammation control and disease treatment.

Asunto(s)

Pulpa Dental , Vesículas Extracelulares , Inmunomodulación , Inflamación , FN-kappa B , Pulpa Dental/citología , Pulpa Dental/inmunología , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/inmunología , Humanos , Animales , Inflamación/inmunología , Inflamación/metabolismo , FN-kappa B/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , MicroARNs/genética , Lipopolisacáridos/farmacología , Ratones , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/inmunología , Células Cultivadas , Transducción de Señal , Células Madre/inmunología , Células Madre/metabolismo , Masculino

RESUMEN

Purpose: Bone loss is a common complication of type 2 diabetes mellitus (T2DM). Circadian rhythms play a significant role in T2DM and bone remodeling. Eldecalcitol (ED-71), a novel active vitamin D analog, has shown promise in ameliorating T2DM. We aimed to investigate whether the circadian rhythm coregulator BMAL1 mediates the anti-osteoporotic effect of ED-71 in T2DM and its associated mechanisms. Methods: A T2DM mouse model was established using high-fat diet (HDF) and streptozotocin (STZ) injection, and blood glucose levels were monitored weekly. HE staining, Masson staining, and Micro-CT were performed to assess the changes in bone mass. IHC staining and IF staining were used to detect osteoblast status and BMAL1 expression and RT-qPCR was applied to detect the change of oxidative stress factors. In vitro, high glucose (HG) stimulation was used to simulate the cell environment in T2DM. RT-qPCR, Western blot, IF, ALP staining and AR staining were used to detect osteogenic differentiation and SIRT1/GSK3ß signaling pathway. DCFH-DA staining was used to detect reactive oxygen species (ROS) levels. Results: ED-71 increased bone mass and promoted osteogenesis in T2DM mice. Moreover, ED-71 inhibited oxidative stress and promoted BMAL1 expression in osteoblasts The addition of STL1267, an agonist of the BMAL1 transcriptional repressor protein REV-ERB, reversed the inhibitory effect of ED-71 on oxidative stress and the promotional effect on osteogenic differentiation. In addition, ED-71 facilitated SIRT1 expression and reduced GSK3ß activity. The inhibition of SIRT1 with EX527 partially attenuated ED-71's effects, whereas the GSK3ß inhibitor LiCl further enhanced ED-71's positive effects on BMAL1 expression. Conclusion: ED-71 ameliorates bone loss in T2DM by upregulating the circadian rhythm coregulator BMAL1 and promoting osteogenesis through inhibition of oxidative stress. The SIRT1/GSK3ß signaling pathway is involved in the regulation of BMAL1.

Asunto(s)

Factores de Transcripción ARNTL , Ritmo Circadiano , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Ratones Endogámicos C57BL , Osteogénesis , Regulación hacia Arriba , Animales , Factores de Transcripción ARNTL/metabolismo , Factores de Transcripción ARNTL/genética , Ratones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Osteogénesis/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Masculino , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ritmo Circadiano/efectos de los fármacos , Estreptozocina , Vitamina D/farmacología , Vitamina D/análogos & derivados , Dieta Alta en Grasa , Células Cultivadas

RESUMEN

PURPOSE: Epithelial-mesenchymal transition (EMT) is a crucial pathological process that contributes to proliferative vitreoretinopathy (PVR), and research indicates that factors present in the vitreous that target cells play pivotal roles in regulating EMT. Experimental studies have confirmed that rabbit vitreous (RV) promotes EMT in human retinal pigment epithelial (RPE) cells. The long noncoding RNA (lncRNA) MALAT1 has been implicated in EMT in various diseases. Thus, this study aimed to investigate the involvement of lncRNA MALAT1 in vitreous-induced EMT in RPE cells. METHODS: MALAT1 was knocked down in ARPE-19 cells by short hairpin RNA (shRNA) transfection. Reverse transcription PCR (RT‒PCR) was used to evaluate MALAT1 expression, and Western blotting analysis was used to measure the expression of EMT-related proteins. Wound-healing, Transwell, and cell contraction assays were conducted to assess cell migration, invasion, and contraction, respectively. Additionally, cell proliferation was assessed using the CCK-8 assay, and cytoskeletal changes were examined by immunofluorescence. RESULTS: MALAT1 expression was significantly increased in ARPE-19 cells cultured with RV. Silencing MALAT1 effectively suppressed EMT and downregulated the associated factors snail1 and E-cadherin. Furthermore, silencing MALAT1 inhibited the RV-induced migration, invasion, proliferation, and contraction of ARPE-19 cells. Silencing MALAT1 also decreased RV-induced AKT and P53 phosphorylation. CONCLUSIONS: In conclusion, lncRNA MALAT1 participates in regulating vitreous-induced EMT in human RPE cells; these results provide new insight into the pathogenesis of PVR and offer a potential direction for the development of antiproliferative drugs.

Asunto(s)

Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Proteínas Proto-Oncogénicas c-akt , ARN Largo no Codificante , Epitelio Pigmentado de la Retina , ARN Largo no Codificante/genética , Transición Epitelial-Mesenquimal/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/patología , Conejos , Animales , Células Cultivadas , Vitreorretinopatía Proliferativa/genética , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patología , Transducción de Señal , Regulación de la Expresión Génica , Western Blotting