In Vitro Cellular & Developmental Biology. Animal最新文献

Rapid growth of the aquaculture industry is hampered by infectious diseases in marine invertebrates, causing economic losses. Marine invertebrate cell cultures offer tools to evaluate biological properties and cellular responses in different conditions. Long-term culture aims to isolate tissue-specific cells and identify bioactive compounds from stem cells. Echinometra mathaei, known as Persian Gulf sea urchin, has lots of benefits in various fields including aquaculture, embryology, and evolutionary biology. However, its cell culture faces challenges due to poorly characterized microenvironmental and specific cultivation requirements. This study aims to establish and optimize a long-term cell culture for coelomocyte derived from E. mathaei, focusing on the characterization of microenvironment conditions to overcome the limitations of current marine invertebrate cell culture. After the collection of E. mathaei from Lark Island, Persian Gulf, Iran, and their acclimatization in artificial seawater, coelomocytes were isolated from different sources including the coelomic fluid, the coelomic epithelium, and the axial organ. Various cell dissociation methods, culture media, growth supplements, culture dishes, and physical conditions were tested to determine optimal conditions for coelomocyte in vitro culture. Moreover, coelomocytes were differentiated to pigment-producing cells, and naphthoquinone pigments were extracted and identified using spectrophotometry. Light microscopy identified several coelomocyte types, including petaloid, filopodial, vibratile cells, and spherulocytes. The HCCM medium supplemented with coelomic fluid proved most effective for cell growth and viability. Moreover, coelomic fluid is the best culture media for differentiation of coelomocyte into the cell producing naphthoquinone pigments. These findings contribute to developing in vitro cell culture methods for sea urchin, providing a foundation for further research on sea urchin immunology, cell biology, and cellular responses to pathogens and other biological stress.

Coho salmon (Oncorhynchus kisutch) is an important salmonid species differing from other salmonids in its tolerance and response to pathogens endemic to the aquaculture industry, such as infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV). Consequently, coho salmon has become a subject of increased scientific interest to investigate the underlying genetic mechanisms behind these and other host-pathogen interactions. Currently, most research studying coho salmon has been conducted using live animal models as there have been few in vitro tools readily available. Here, we present the first cell line from an adult coho salmon, Coho Salmon Fibroblast-Like 1 Norway-Canada (CSFL-1NC) and its preliminary characterisation. CSFL-1NC is a homogenous, spontaneously immortalised cell line from the pectoral fin of a wild adult coho salmon, with a consistent and stable fibroblastic morphology. The cell line has a relatively stable transcriptome across several passages, with high expression of key fibroblastic marker genes, displays rapid migration, and can be genetically manipulated both by transfection and transduction with varying efficiency using plasmids, lentivirus, and/or CRISPR methodology. Virus challenges show clear susceptibility to IPNV as evidenced by cytopathic effects and efficient viral replication, yet it shows little to no response when exposed to ISAV (HPRD).

Mesenchymal stem cells (MSCs) hold promise for treating inflammatory and immune-related diseases; however, their clinical application is limited by poor survival and function post-transplantation. Collagen hydrogels may support MSC viability and function by mimicking the extracellular matrix. This study aimed to evaluate how cell density and collagen concentration within three-dimensional (3D) collagen matrices affect the immunomodulatory behavior of MSCs under inflammatory conditions. MSCs were embedded in collagen hydrogels of varying stiffness and seeded at different densities. Constructs were stimulated with proinflammatory cytokines (tumor necrosis factor-α and interferon-γ), and changes in Gene expression, hydrogel contraction, and cell viability were analyzed. Lower collagen concentrations and higher seeding densities enhanced MSC immunomodulatory Gene expression and matrix contraction. High cell density increased contraction but reduced cell viability in softer gels. Mechanical properties of the matrix, such as stiffness and viscoelasticity, influenced cell behavior via mechanotransduction pathways. Both physical and biological cues within 3D collagen hydrogels significantly regulated MSC immunomodulatory responses. Optimizing collagen concentration and seeding density may improve the therapeutic potential of MSC-based treatments.

Rheumatoid arthritis (RA) is a chronic autoimmune condition that leads to joint damage. Mesenchymal stem cells (MSCs) are being recognized as a promising treatment option because of their capacity to modulate immune responses. Their therapeutic effects are mediated by released extracellular vesicles (EVs) which contain microRNAs known to influence inflammatory processes. This research focused on the impact of bone marrow MSC (BM-MSC)-derived EVs overexpressing miR-10a on cytokine production in a mouse model of collagen-induced arthritis (CIA). miR-10a was overexpressed in MSCs derived from bone marrow using Transfectamin. EVs were then isolated from the culture media of both miR-control and miR-10a-modified MSCs. Immunizing mice established the CIA model with type II collagen, after which they received either miR-control or miR-10a-enriched MSC-EVs. The severity of arthritis was evaluated through joint swelling measurements, and the concentrations of pro-inflammatory cytokines (such as interleukin (IL)-17a, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α) alongside anti-inflammatory cytokines (including transforming growth factor (TGF)-β, IL-10, and IL-4) in the joints and serum were assessed using real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Our results indicated that treatment with miR-10a MSC-EVs led to a notable decrease in arthritis severity and joint damage in CIA mice. Furthermore, these EVs were found to lower levels of pro-inflammatory cytokines while enhancing anti-inflammatory cytokines compared to those treated with miR-control MSC-EVs. This study highlights how enhancing miR-10a expression can improve the therapeutic efficacy of MSC-EVs by altering the cytokine environment in CIA models.

Gastric cancer is a significant global health concern due to its high morbidity and mortality. Gastric cancer-associated mesenchymal stem cells (GC-MSCs) significantly contribute to its progression, with circ_0024107 being notably elevated in these cells and essential for their tumor-promoting activities. However, the expression and function of this circRNA in gastric cancer cells as well as its upstream regulators remain unclear. qPCR was used to assess circ_0024107 expression levels. Gain- and loss-of-function experiments evaluated its roles. Transwell assays measured cell migration and invasion. KHDRBS3 was predicted and validated through database analysis and qPCR, and its effects on circ_0024107 were analyzed using qPCR and transwell assays. The expression and clinical implications of KHDRBS3 in gastric cancer were evaluated using the TCGA-STAD database. circ_0024107 expression was elevated in gastric cancer cells, where it promotes migration and invasion. GC-MSCs further enhanced these capabilities by upregulating circ_0024107. KHDRBS3 was identified and validated as a regulator of circ_0024107 expression in both gastric cancer cells and GC-MSCs. Knocking down KHDRBS3 significantly reduced circ_0024107 levels, hindering gastric cancer cell migration and invasion, and weakening the influence of GC-MSCs on tumor cells. KHDRBS3 was abnormally elevated in gastric cancer tissues and correlated with patients' poor prognosis. KHDRBS3-mediated upregulation of circ_0024107 in gastric cancer cells and GC-MSCs synergistically enhances gastric cancer progression. This elucidates novel molecular interactions between GC-MSCs and gastric cancer cells, thereby presenting a promising therapeutic target for effectively mitigating gastric cancer metastasis.

As a persistent osteoarticular degenerative condition, intervertebral disc deterioration (IDD) has been established as a principal causative element in lumbar spine discomfort development. The present investigation seeks to assess the protective effects of casticin against IDD progression and elucidate associated molecular pathways. The CCK8 kit was used to assess the cytotoxicity of casticin on rat nucleus pulposus cells (NPCs). Western blot assay, qRT-PCR, enzyme-linked immunosorbent assay, reactive oxygen species assay, and immunofluorescence were used to detect the expression levels of inflammatory mediators and ROS production between different groups. The nuclear translocation of NF-κB p65 and expression of Nrf2/HO-1 signal pathway in lipopolysaccharide (LPS)-induced NPCs were detected by confocal microscopy. Moreover, histological analysis was used to evaluate the degree of disc degeneration in rats. Casticin treatment inhibited the production of oxygen free radicals and inflammatory mediators induced by LPS, such as ROS, TNF-α, IL-1β, and PGE2. Not only that, we also found that casticin retained the content of type II collagen and aggrecan in NPCs and inhibited the expression of MMP-13 and ADAMTS-5. Moreover, casticin treatment activated the Nrf2/HO-1 signal axis and inhibited nuclear translocation of NF-κB p65 in LPS-exposed NPCs. Histological analysis found that the treatment of casticin in rat IDD models prevented the loss of notochordal cells and the disordered arrangement of fiber loops. Casticin inhibits LPS-stimulated oxidative stress, inflammatory response, and ECM degradation by activating the Nrf2/HO-1 signaling axis and indirectly blocking the NF-κB pathway.

Valvular interstitial cells (VICs) are integral to the progression of calcific aortic valve disease (CAVD). Dentin matrix protein-1 (DMP-1), a member of the Sibling family protein, is implicated in the calcification process. This study aims to investigate the role and mechanisms of DMP-1 in the osteogenic differentiation of VICs. Between April 2018 and December 2018, aortic valve tissues were collected from 14 patients undergoing aortic valve replacement or heart transplantation. DMP-1 expression was quantified in calcified valves versus normal controls. An in vitro model of VICs' osteogenic differentiation was established to study the regulatory mechanism of DMP-1 on valvular calcification using immunoblot, immunohistochemistry, immunofluorescence, etc. The expression of DMP-1 was significantly increased in the calcified aortic valves patients (P < 0.01). DMP-1, both short and long arginine-glycine-aspartic acid (RGD) peptides, induced the osteogenic differentiation in VICs, an effect that was inhibited by an integrin αvβ3 antagonist (P < 0.05). Furthermore, the expression levels of RAF, RAS, MEK, and phosphorylated ERK1/2 were significantly elevated in VICs upon stimulation of DMP-1 (P < 0.05). DMP-1 is involved in the progression of valvular calcification and promotes the osteogenic differentiation of VICs via the integrin αvβ3 receptor. The combination of DMP-1 and integrin αvβ3 via its RGD domain activates the MAPK signaling pathway, leading to enhanced osteogenic gene expression in VICs. Clinical trial number: not applicable.