In Vitro Cellular & Developmental Biology. Animal最新文献

Immunoglobulin A (IgA) nephropathy (IgAN) is characterized by the deposition of IgA1 in the glomerular mesangium, which induces secondary glomerular and tubulointerstitial inflammation and subsequently leads to podocyte apoptosis and fibrosis. This condition often progresses to end-stage renal disease and lacks effective targeted treatment. Our study aimed to explore the role of M2 macrophage-mediated Ubiquitin C-terminal hydrolase L1 (UCHL1) expression in podocytes and its potential impact on the progression of IgAN. This study established an IgAN cellular model by exposing podocytes to aggregated IgA1 (aIgA1)-treated glomerular mesangial cells supernatants and assessed the impact of M2 macrophage polarization on UCHL1 expression and podocyte apoptosis. Additionally, we utilized siRNA technology and overexpression constructs to investigate the direct effects of UCHL1 modulation on podocyte apoptosis. The supernatant from aIgA1-treated glomerular mesangial cells significantly induced apoptosis in podocytes. Based on this, M2 macrophage polarization was induced using interleukin (IL)-4. The results showed that M2 macrophages (CD163⁺) effectively alleviated podocyte apoptosis by reducing the secretion of inflammatory cytokines IL-6, tumor necrosis factor (TNF)-α, and IL-1β, as well as downregulating the expression of apoptosis-related proteins. Notably, M2 macrophages (CD163⁺) inhibited the expression of UCHL1 in podocytes. Blockade of UCHL1 promoted podocyte proliferation, reduced apoptosis, and downregulated the protein expression of the fibrotic markers vascular endothelial growth factor and collagen type IV. Overexpression of UCHL1 reversed the protective effects of M2 macrophages on podocyte apoptosis. M2 macrophage (CD163⁺)-mediated UCHL1 downregulation in podocytes presents a potential therapeutic approach for IgAN by alleviating apoptosis.

Culturing stem cells in species-specific serum ensures physiological relevance, reduces variability, and supports safer clinical use, highlighting the urgent need to develop reliable, species-matched systems for research and therapy. To address the concept of species specificity in cell culture, we investigated the effects of goat serum (GS) on the derivation of goat muscle stem cells (MuSCs). First, MuSCs were isolated from goat muscle tissue and cultured in media supplemented with either 10% goat GS or fetal bovine serum (FBS). Next, the isolated cells underwent characterization and differentiation. Finally, the effects of varying concentrations of GS and FBS on cell proliferation were evaluated. The results demonstrated that goat MuSCs grew in a GS-containing medium and were positively immunostained for CD29 and Pax7. Gene expression analysis revealed no significant differences in the expression of Pax7, MyoD, and MyoG genes between goat MuSCs grown in GS- or FBS-containing medium. Cells grown in GS-containing medium showed more efficient differentiation toward myogenic and adipogenic lineages than those grown in FBS. Supplementing the culture medium with 10% GS resulted in the greatest enhancement of goat MuSC proliferation, as evidenced by the MTT assay, increased Ki67 expression, and a higher number of colony-forming units. This study demonstrated that GS supplementation is notably beneficial for the proliferation of goat MuSCs.

ARP2/3 is a seven-subunit protein complex involved in the formation of actin filament branching, which is essential for the formation of membrane protrusions, cell migration, and establishment of cell polarity. Among these functions, ARP2/3 has been implicated in myoblast fusion. Since myogenesis is a complex multistep process, here we decided to explore deeper the distribution patterns and functions of ARP2/3 during the initial steps of embryonic chicken skeletal myogenesis. The chosen biological experimental model was the cell culture of pectoralis muscle obtained from 11-d-old chick embryos, which is composed of myoblasts, multinucleated myotubes, and muscle fibroblasts. Our results show that ARP2/3 was found in myoblasts, myotubes, and muscle fibroblasts in four main distributions: the perinuclear region, in small puncta in the cytoplasm, along F-actin structures in the cytoplasm, and in circular structures in myotubes. Inhibition of ARP2/3 function by CK-666 led to a significant reduction in several parameters of skeletal myogenesis, including the area of muscle cells (desmin-positive cells), myotube thickness, the number of myoblasts, the number of nuclei within myotubes, the number of fibroblasts, the total number of nuclei (including nuclei in myoblasts, myotubes, and fibroblasts), and the myoblast fusion index. Interestingly, CK-666 reduced myotube formation and induced the formation of spindle-shaped myoblasts. Live cell video microscopy showed that inhibition of ARP2/3 induced a decrease in myoblast cell migration and the formation of blebs in the membranes of cells. The collection of our results shows that ARP2/3 is essential for the initial steps of embryonic chick skeletal myogenesis, and its inhibition leads to a major reduction in myoblast proliferation, migration, fusion, and muscle fiber formation.

Skeletal muscle tissue consists of not only myofibers, i.e., muscle cells, but also intramuscular adipocytes. Our previous study demonstrated that adipocytes produce secretory factors during differentiation, leading us to hypothesize that soluble factors derived from adipocytes regulate gene expression and cellular function in muscle cells. Yet the mechanism by which coexisting adipocytes influence muscle cells remains unclear. Here, microarray analysis was used to examine transcriptional changes in muscle cells under two co-culture conditions: myoblasts co-cultured with differentiated adipocytes and myotubes co-cultured with preadipocytes. Gene Ontology terms related to cell adhesion, extracellular matrix (ECM) organization, and metabolic processes were significantly enriched in both conditions. We also assessed the influence of adipocyte co-culture on myogenic differentiation and fiber type-specific gene expression. In myoblasts, co-culture with differentiated adipocytes had no significant effect on the expression of myogenic regulatory factors, whereas Myh2 and Myh4 expression was markedly increased in myotubes co-cultured with preadipocytes. These results indicate that adipocyte-derived soluble factors alter the transcriptional landscape of muscle cells, especially genes involved in ECM remodeling and metabolic regulation. This intercellular communication likely contributes to structural and metabolic adaptations in skeletal muscle tissue in vivo.

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.