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.
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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/citologíaRESUMEN
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.
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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/métodosRESUMEN
Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.
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Pez Cebra , Animales , Retina/citología , Retina/metabolismo , Fenotipo , Proliferación Celular , Regeneración , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Células Madre/citología , Células Madre/metabolismo , Cinética , Regeneración Nerviosa/fisiologíaRESUMEN
The unsuitable deformation stimulus, harsh urine environment, and lack of a regenerative microenvironment (RME) prevent scaffold-based urethral repair and ultimately lead to irreversible urethral scarring. The researchers clarify the optimal elastic modulus of the urethral scaffolds for urethral repair and design a multilayered PVA hydrogel scaffold for urethral scar-free healing. The inner layer of the scaffold has self-healing properties, which ensures that the wound effectively resists harsh urine erosion, even when subjected to sutures. In addition, the scaffold's outer layer has an extracellular matrix-like structure that synergizes with adipose-derived stem cells to create a favorable RME. In vivo experiments confirm successful urethral scar-free healing using the PVA multilayered hydrogel scaffold. Further mechanistic study shows that the PVA multilayer hydrogel effectively resists the urine-induced inflammatory response and accelerates the transition of urethral wound healing to the proliferative phase by regulating macrophage polarization, thus providing favorable conditions for urethral scar-free healing. This study provides mechanical criteria for the fabrication of urethral tissue-engineered scaffolds, as well as important insights into their design.
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Módulo de Elasticidad , Hidrogeles , Andamios del Tejido , Uretra , Cicatrización de Heridas , Andamios del Tejido/química , Animales , Hidrogeles/química , Ingeniería de Tejidos/métodos , Ratones , Regeneración , Cicatriz/patología , Masculino , Microambiente Celular , Ratas Sprague-Dawley , Células Madre/citologíaRESUMEN
During embryonic development, Wnt signaling influences both proliferation and sensory formation in the cochlea. How this dual nature of Wnt signaling is coordinated is unknown. In this study, we define a novel role for a Wnt-regulated gene, Mybl2, which was already known to be important for proliferation, in determining the size and patterning of the sensory epithelium in the murine cochlea. Using a quantitative spatial analysis approach and analyzing Mybl2 loss-of-function, we show that Mybl2 promoted proliferation in the inner sulcus domain but limited the size of the sensory domain by influencing their adjoining boundary position via Jag1 regulation during development. Mybl2 loss-of-function simultaneously decreased proliferation in the inner sulcus and increased the size of the sensory domain, resulting in a wider sensory epithelium with ectopic inner hair cell formation during late embryonic stages. These data suggest that progenitor cells in the inner sulcus determine boundary formation and pattern the sensory epithelium via MYBL2.
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Proliferación Celular , Cóclea , Proteína Jagged-1 , Células Madre , Animales , Cóclea/embriología , Cóclea/citología , Cóclea/metabolismo , Ratones , Epitelio/embriología , Epitelio/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Regulación del Desarrollo de la Expresión Génica , Vía de Señalización Wnt , Tipificación del Cuerpo/genética , Transactivadores/metabolismo , Transactivadores/genética , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/citología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestines without a cure. Current therapies suppress inflammation to prevent further intestinal damage. However, healing already damaged intestinal epithelia is still an unmet medical need. Under physiological conditions, Lgr5+ intestinal stem cells (ISCs) in the intestinal crypts replenish the epithelia every 3-5 days. Therefore, understanding the regulation of Lgr5+ ISCs is essential. Previous data suggest vitamin D signaling is essential to maintain normal Lgr5+ ISC function in vivo. Our recent data indicate that to execute its functions in the intestines optimally, 1,25(OH)2D requires high concentrations that, if present systemically, can cause hypercalcemia (i.e., blood calcium levels significantly higher than physiological levels), leading to severe consequences. Using 5-bromo-2'-deoxyuridine (BrdU) to label the actively proliferating ISCs, our previous data suggested that de novo synthesized locally high 1,25(OH)2D concentrations effectively enhanced the migration and differentiation of ISCs without causing hypercalcemia. However, although sparse in the crypts, other proliferating cells other than Lgr5+ ISCs could also be labeled with BrdU. This current study used high-purity Lgr5+ ISC lines and a mouse strain, in which Lgr5+ ISCs and their progeny could be specifically tracked, to investigate the effects of de novo synthesized locally high 1,25(OH)2D concentrations on Lgr5+ ISC function. Our data showed that 1,25(OH)2D at concentrations significantly higher than physiological levels augmented Lgr5+ ISC differentiation in vitro. In vivo, de novo synthesized locally high 1,25(OH)2D concentrations significantly elevated local 1α-hydroxylase expression, robustly suppressed experimental colitis, and promoted Lgr5+ ISC differentiation. For the first time, this study definitively demonstrated 1,25(OH)2D's role in Lgr5+ ISCs, underpinning 1,25(OH)2D's promise in IBD therapy.
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Receptores Acoplados a Proteínas G , Células Madre , Vitamina D , Animales , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Células Madre/efectos de los fármacos , Células Madre/citología , Vitamina D/farmacología , Vitamina D/análogos & derivados , Vitamina D/metabolismo , Ratones , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Regeneración/efectos de los fármacos , Ratones Endogámicos C57BL , Colitis/metabolismo , Colitis/inducido químicamente , Colitis/patología , Intestinos/efectos de los fármacosRESUMEN
BACKGROUND: Fibrosis with unrelieved chronic inflammation is an important pathological change in keloids. Mitochondrial autophagy plays a crucial role in reducing inflammation and inhibiting fibrosis. Adipose stem cell-derived exosomes, a product of adipose stem cell paracrine secretion, have pharmacological effects, such as anti-inflammatory and antiapoptotic effects, and mediate autophagy. Therefore, this study aims to investigate the function and mechanism of adipose stem cell exosomes in the treatment of keloids. METHOD: We isolated adipose stem cell exosomes under normoxic and hypoxic condition to detect their effects on keloid fibroblast proliferation, migration, and collagen synthesis. Meanwhile, 740YPDGFR (PI3K/AKT activator) was applied to detect the changes in autophagic flow levels and mitochondrial morphology and function in keloid fibroblasts. We constructed a human keloid mouse model by transplanting human keloid tissues into six-week-old (20-22 g; female) BALB/c nude mice, meanwhile, we applied adipose stem cell exosomes to treat the mouse model and observed the retention and effect of ADSC exosomes in vivo. RESULTS: ADSC exosomes can inhibit the PI3K/AKT/mTOR signaling pathway. The exosomes of ADSCs decreased the inflammatory level of KFs, enhanced the interaction between P62 and LC3, and restored the mitochondrial membrane potential. In the human keloid mouse model, ADSC exosomes can exist stably, promote mitochondrial autophagy in keloid tissue, improve mitochondrial morphology, reduce inflammatory reaction and fibrosis. Meanwhile, At the same time, the exosomes derived from hypoxic adipose stem cells have played a more effective role in both in vitro and in vivo experiments. CONCLUSIONS: Adipose stem cell exosomes inhibited the PI3K/AKT/mTOR pathway, activated mitochondrial autophagy, and alleviated keloid scars.
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Autofagia , Exosomas , Queloide , Mitocondrias , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Queloide/metabolismo , Queloide/terapia , Queloide/patología , Exosomas/metabolismo , Exosomas/trasplante , Animales , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Ratones , Mitocondrias/metabolismo , Femenino , Ratones Endogámicos BALB C , Ratones Desnudos , Tejido Adiposo/metabolismo , Tejido Adiposo/citología , Células Madre/metabolismo , Células Madre/citología , Proliferación Celular , Fibroblastos/metabolismoRESUMEN
In the companion Perspective 'Past and future of human developmental biology' (Hopwood, 2024), historian Nick Hopwood proposes that the field of human developmental biology has gone through periods of attention and neglect. Development invited researchers from the field to respond to this idea. In this article, published to coincide with the 10th anniversary of Development's 'From Stem Cells to Human Development' meeting, researchers from eight countries comment on how they believe their local legal, political, regulatory, societal and technological frameworks are influencing the field's trajectory.
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Biología Evolutiva , Humanos , Biología Evolutiva/tendencias , Biología Evolutiva/historia , Células Madre/citologíaRESUMEN
BACKGROUND: Influence on stem cells' angiogenesis and osteogenesis of NAD(P)H Quinone Dehydrogenase 1(NQO1) has been established, but its impact on dental pulp stem cells (DPSCs) is unexplored. An important strategy for the treatment of arteriosclerosis is to inhibit calcium deposition and to promote vascular repair and angiogenesis. This study investigated the function and mechanism of NQO1 on angiogenesis and osteogenesis of DPSCs, so as to provide a new ideal for the treatment of arteriosclerosis. METHODS: Co-culture of human DPSCs and human umbilical vein endothelial cells (HUVECs) was used to detect the angiogenesis ability. Alkaline phosphatase (ALP) activity, alizarin red staining (ARS), and transplantation of HA/tricalcium phosphate with DPSCs were used to detect osteogenesis. RESULTS: NQO1 suppressed in vitro tubule formation, migration, chemotaxis, and in vivo angiogenesis, as evidenced by reduced CD31 expression. It also enhanced ALP activity, ARS, DSPP expression and osteogenesis and boosted mitochondrial function in DPSCs. CoQ10, an electron transport chain activator, counteracted the effects of NQO1 knockdown on these processes. Additionally, NQO1 downregulated MAPK signaling, which was reversed by CoQ10 supplementation in DPSCs-NQO1sh. CONCLUSIONS: NQO1 inhibited angiogenesis and promoted the osteogenesis of DPSCs by suppressing MAPK signaling pathways and enhancing mitochondrial respiration.
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Pulpa Dental , Células Endoteliales de la Vena Umbilical Humana , Sistema de Señalización de MAP Quinasas , NAD(P)H Deshidrogenasa (Quinona) , Neovascularización Fisiológica , Osteogénesis , Humanos , Osteogénesis/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , Neovascularización Fisiológica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Pulpa Dental/citología , Pulpa Dental/metabolismo , Técnicas de Cocultivo , Células Madre/metabolismo , Células Madre/citología , Células Cultivadas , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Ubiquinona/metabolismo , Animales , Diferenciación Celular , AngiogénesisRESUMEN
Latest developments in the field of stem cell research and regenerative medicine compiled from publicly available information and press releases from non-academic institutions in March 2024.
Regenerative medicine industry news digest, March 2024.
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Medicina Regenerativa , Investigación con Células Madre , Medicina Regenerativa/tendencias , Medicina Regenerativa/métodos , Humanos , Células Madre/citologíaRESUMEN
Cell identities are defined by intrinsic transcriptional networks and spatio-temporal environmental factors. Here, we explored multiple factors that contribute to the identity of adipose stem cells, including anatomic location, microvascular neighborhood, and sex. Our data suggest that adipose stem cells serve a dual role as adipocyte precursors and fibroblast-like cells that shape the adipose tissue's extracellular matrix in an organotypic manner. We further find that adipose stem cells display sexual dimorphism regarding genes involved in estrogen signaling, homeobox transcription factor expression and the renin-angiotensin-aldosterone system. These differences could be attributed to sex hormone effects, developmental origin, or both. Finally, our data demonstrate that adipose stem cells are distinct from mural cells, and that the state of commitment to adipogenic differentiation is linked to their anatomic position in the microvascular niche. Our work supports the importance of sex and microvascular function in adipose tissue physiology.
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Adipocitos , Tejido Adiposo , Fibroblastos , Caracteres Sexuales , Células Madre , Animales , Femenino , Adipocitos/citología , Adipocitos/metabolismo , Masculino , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Fibroblastos/metabolismo , Fibroblastos/citología , Células Madre/metabolismo , Células Madre/citología , Ratones , Diferenciación Celular , Adipogénesis/genética , Ratones Endogámicos C57BL , Matriz Extracelular/metabolismo , HumanosRESUMEN
The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. Itm2a+ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a+ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a+ cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Sca1, and Gli1. Itm2a+ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of Itm2a+ P-SSCs resulted in defects in fracture healing. ITM2A+ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.
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Curación de Fractura , Proteínas de la Membrana , Periostio , Animales , Periostio/metabolismo , Periostio/citología , Ratones , Curación de Fractura/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Células Madre/metabolismo , Células Madre/citología , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Fracturas Óseas/patología , Fracturas Óseas/metabolismo , Fracturas Óseas/terapia , Fracturas Óseas/genética , Osteoblastos/metabolismo , Osteoblastos/citología , Diferenciación Celular , Condrocitos/metabolismo , Condrocitos/citología , Masculino , Linaje de la CélulaRESUMEN
Diabetic foot ulcers (DFUs) are chronic wounds and one of the most common complications of diabetes, imposing significant physical and mental burdens on patients due to their poor prognosis and treatment efficacy. Adipose-derived stem cells (ADSCs) have been proven to promote wound healing, with studies increasingly attributing these beneficial effects to their paracrine actions. Consequently, research on ADSC secretome as a novel and promising alternative for DFU treatment has been extensively conducted. This article provides a comprehensive review of the mechanisms underlying refractory DFU wounds, the secretome of ADSCs, and its role in promoting wound healing in diabetes foot ulcers. And the review aims to provide reliable evidence for the clinical application of ADSC secretome in the treatment of refractory DFU wounds.
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Tejido Adiposo , Pie Diabético , Secretoma , Cicatrización de Heridas , Humanos , Pie Diabético/terapia , Pie Diabético/metabolismo , Pie Diabético/patología , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Secretoma/metabolismo , Células Madre/metabolismo , Células Madre/citología , AnimalesRESUMEN
Low back pain significantly impacts individuals' quality of life, with intervertebral disc degeneration (IDD) being a primary contributor to this condition. Currently, IDD treatment primarily focuses on symptom management and does not achieve a definitive cure. The cartilage endplate (CEP), a crucial nutrient-supplying tissue of the intervertebral disc, plays a pivotal role in disc degeneration. This review examines the mechanisms underlying CEP degeneration, summarizing recent advancements in understanding the structure and function of CEP, the involvement of various signaling pathways, and the roles of cartilage endplate stem cells (CESCs) and exosomes (Exos) in this process. The aim of this review is to provide a comprehensive reference for future research on CEP. Despite progress in understanding the role of CEP in IDD, the mechanisms underlying CEP degeneration remain incompletely elucidated. Future research poses significant challenges, necessitating further investigations to elucidate the complexities of CEP.
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Cartílago , Degeneración del Disco Intervertebral , Degeneración del Disco Intervertebral/patología , Degeneración del Disco Intervertebral/metabolismo , Humanos , Cartílago/metabolismo , Cartílago/patología , Animales , Disco Intervertebral/patología , Disco Intervertebral/metabolismo , Exosomas/metabolismo , Células Madre/metabolismo , Células Madre/citología , Células Madre/patología , Transducción de SeñalRESUMEN
A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-ß1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.
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Colágeno Tipo I , Fibrina , Células Madre , Tendones , Andamios del Tejido , Factor de Crecimiento Transformador beta1 , Humanos , Tendones/citología , Tendones/metabolismo , Andamios del Tejido/química , Células Madre/metabolismo , Células Madre/citología , Fibrina/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tendinopatía/metabolismo , Tendinopatía/patología , Células Cultivadas , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Persona de Mediana Edad , Masculino , Supervivencia Celular/efectos de los fármacos , Ingeniería de Tejidos/métodos , Proteínas de la MembranaRESUMEN
BACKGROUND: Hepatic progenitor cells serve not only as the origin of combined hepatocellular cholangiocarcinoma (cHCC-CCA) but are also responsible for malignancy recurrence after surgical resection. Nucleophosmin 1 (NPM1) has been implicated in cancer metastasis and poor prognosis. This study aimed to determine the expression of NPM1 by hepatic progenitor cells in cHCC-CCA and the effects of targeting NPM1 on hepatic progenitor cells and BEL-7402 cells with characteristics of both progenitor cells and cHCC-CCA. METHODS: First, NPM1 was detected by RTâPCR, western blotting, and double-immunofluorescence staining in cHCC-CCA tissues. NPM1 expression was subsequently analysed in rat hepatic progenitor cells cultured in vitro and in interleukin 6 (IL6)-treated cells. The effects and mechanism of NPM1 on hepatic progenitor cells were determined by knocking down NPM1 and performing RNA sequencing analysis. Finally, NSC348884, a small-molecule inhibitor that disrupts NPM1 dimer formation, was used to confirm the function of NPM1 in BEL-7402 cells. RESULTS: Both human hepatic progenitor cells in cHCC-CCA tissues and rat in vitro cultured hepatic progenitor cells highly expressed NPM1. IL6, a cytokine involved in the malignant transformation of hepatic progenitor cells, dose-dependently increased NPM1 and PCNA expression. Knocking down NPM1 reduced IL6R transcription (P < 0.0001) and inhibited the proliferation (P = 0.0065) of hepatic progenitor cells by suppressing the mTOR signalling pathway and activating the apoptosis pathway. Furthermore, knocking down NPM1 in hepatic progenitor cells resulted in more apoptotic cells (7.33 ± 0.09% vs. 3.76 ± 0.13%, P < 0.0001) but fewer apoptotic cells in the presence of NSC348884 (47.57 ± 0.49% vs. 63.40 ± 0.05%, P = 0.0008) than in the control cells, suggesting that low-NPM1-expressing cells are more resistant to NSC348884. In addition, NSC348884 induced the apoptosis of BEL-7402 cells with an IC50 of 2.77 µmol/L via the downregulation of the IL-6R and mTOR signalling pathways and inhibited the growth of BEL-7402 cells in a subcutaneous xenograft tumour model (P = 0.0457). CONCLUSIONS: Targeting NPM1 inhibits proliferation and induces apoptosis in hepatic progenitor cells and BEL-7402 cells, thus serving as a potential therapy for cHCC-CCA.
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Apoptosis , Proliferación Celular , Proteínas Nucleares , Nucleofosmina , Transducción de Señal , Células Madre , Serina-Treonina Quinasas TOR , Humanos , Apoptosis/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Proliferación Celular/efectos de los fármacos , Animales , Transducción de Señal/efectos de los fármacos , Ratas , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Células Madre/metabolismo , Células Madre/citología , Masculino , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Línea Celular Tumoral , RatonesRESUMEN
BACKGROUND: Understanding the role of cytokines in tooth development is critical for advancing dental tissue engineering. Fibroblast growth factor 9 (FGF9) is the only FGF consistently expressed throughout dental epithelial tissue, from the initiation of tooth bud formation to tooth maturation. However, mice lacking Fgf9 (Fgf9-/-) surprisingly show no obvious abnormalities in tooth development, suggesting potential compensation by other FGFs. Here we report findings from an Fgf9S99N mutation mouse model, a loss-of-function mutation with a dominant negative effect. Our study reveals that Fgf9 is crucial for dental epithelial stem cell (DESC) survival and enamel formation. METHODS: To dissect the role of Fgf9 in tooth development, we performed the micro-CT, histomorphological analysis and gene expression assay in mice and embryos with S99N mutation. In addition, we assessed the effect of FGF9 on the DESC survival and dental epithelial differentiation by DESC sphere formation assay and tooth explant culture. Cell/tissue culture methods, gene expression analysis, specific inhibitors, and antibody blockage analysis were employed to explore how Fgf9 regulates enamel differentiation and DESC survival through both direct and indirect mechanisms. RESULTS: The Fgf9S99N mutation in mice led to reduced ameloblasts, impaired enamel formation, and increased apoptosis in the cervical loop (CL). DESC sphere culture experiments revealed that FGF9 facilitated DESC survival via activating ERK/CREB signaling, without affecting cell proliferation. Furthermore, in vitro tissue culture experiments demonstrated that FGF9 promoted enamel formation in a manner dependent on the presence of mesenchyme. Interestingly, FGF9 stimulation inhibited enamel formation in isolated enamel epithelia and DESC spheres. Further investigation revealed that FGF9 supports DESC survival and promotes amelogenesis by stimulating the secretion of FGF3 and FGF10 in dental mesenchymal cells via the MAPK/ERK signaling pathway. CONCLUSIONS: Our study demonstrates that Fgf9 is essential for DESC survival and enamel formation. Fgf9 performs as a dual-directional regulator of the dental enamel epithelium, not only inhibiting DESC differentiation into ameloblasts to preserve the stemness of DESC, but also promoting ameloblast differentiation through epithelial-mesenchymal interactions.
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Esmalte Dental , Células Epiteliales , Factor 9 de Crecimiento de Fibroblastos , Células Madre , Animales , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Factor 9 de Crecimiento de Fibroblastos/genética , Ratones , Esmalte Dental/metabolismo , Células Madre/metabolismo , Células Madre/citología , Células Epiteliales/metabolismo , Incisivo/metabolismo , Supervivencia Celular , Diferenciación CelularRESUMEN
BACKGROUND: The formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known. METHODS: Primary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air-liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential. RESULTS: Herein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis. CONCLUSION: Cx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.
Asunto(s)
Conexinas , Regeneración , Humanos , Animales , Ratones , Conexinas/metabolismo , Conexinas/genética , Diferenciación Celular , COVID-19/metabolismo , COVID-19/virología , COVID-19/patología , Uniones Comunicantes/metabolismo , Autorrenovación de las Células , Calcio/metabolismo , Células Cultivadas , SARS-CoV-2/metabolismo , Masculino , Células Madre/metabolismo , Células Madre/citologíaRESUMEN
Honeybees (Apis mellifera) are a keystone species for managed pollination and the production of hive products. Eusociality in honeybees leads to much of the reproduction in a hive driven by the queen. Queen bees have two large active ovaries that can produce large numbers of eggs if conditions are appropriate. These ovaries are also active throughout the long lives of these insects, up to 5â years in some cases. Recent studies have indicated that the germline precursors of the adult honeybee queen ovary are organized into 8-cell clusters, joined together by a polyfusome; a cytoplasmic bridge. To understand the origin of these clusters, and trace the development of the honeybee queen ovary, we examined the cell types and regionalization of the developing larval and pupal queen ovaries. We used established (nanos and castor), and novel (odd skipped) gene expression markers to determine regions of the developing ovary. Primordial germline cells develop in the honeybee embryo and are organized into ovary structures before the embryo hatches. The ovary is regionalized by larval stage 3 into terminal filaments and germaria. At this stage, clusters of germline cells in the germaria are joined by fusomes and are dividing synchronously. The origin of the 8-cell clusters in the adult germarium is therefore during larval stages. On emergence, the queen ovary has terminal filaments and germaria but has not yet developed any vitellaria, which are produced after the queen embarks on a nuptial flight. The lack of germaria, and the storing of germline progenitors as clusters, may be adaptions for queen bees to endure the metabolic demands of a nuptial flight, as well as rapidly lay large numbers of eggs to establish a hive.
Asunto(s)
Células Germinativas , Larva , Ovario , Animales , Abejas/fisiología , Ovario/citología , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Larva/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Células Madre/citología , Células Madre/metabolismoRESUMEN
BACKGROUND: Periodontal ligament stem cells (PDLSCs) are important seed cells in tissue engineering and clinical applications. They are the priority receptor cells for sensing various mechanical stresses. Yes-associated protein (YAP) is a recognized mechanically sensitive transcription factor. However, the role of YAP in regulating the fate of PDLSCs under tension stress (TS) and its underlying mechanism is still unclear. METHODS: The effects of TS on the morphology and fate of PDLSCs were investigated using fluorescence staining, transmission electron microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). Then qRT-PCR, western blotting, immunofluorescence staining and gene knockdown experiments were performed to investigate the expression and distribution of YAP and its correlation with PDLSCs proliferation. The effects of cytoskeleton dynamics on YAP nuclear translocation were subsequently explored by adding cytoskeleton inhibitors. The effect of cytoskeleton dynamics on the expression of the LINC complex was proved through qRT-PCR and western blotting. After destroying the LINC complex by adenovirus, the effects of the LINC complex on YAP nuclear translocation and PDLSCs proliferation were investigated. Mitochondria-related detections were then performed to explore the role of mitochondria in YAP nuclear translocation. Finally, the in vitro results were verified by constructing orthodontic tooth movement models in Sprague-Dawley rats. RESULTS: TS enhanced the polymerization and stretching of F-actin, which upregulated the expression of the LINC complex. This further strengthened the pull on the nuclear envelope, enlarged the nuclear pore, and facilitated YAP's nuclear entry, thus enhancing the expression of proliferation-related genes. In this process, mitochondria were transported to the periphery of the nucleus along the reconstructed microtubules. They generated ATP to aid YAP's nuclear translocation and drove F-actin polymerization to a certain degree. When the LINC complex was destroyed, the nuclear translocation of YAP was inhibited, which limited PDLSCs proliferation, impeded periodontal tissue remodeling, and hindered tooth movement. CONCLUSIONS: Our study confirmed that appropriate TS could promote PDLSCs proliferation and periodontal tissue remodeling through the mechanically driven F-actin/LINC complex/YAP axis, which could provide theoretical guidance for seed cell expansion and for promoting healthy and effective tooth movement in clinical practice.