In Vitro Cellular & Developmental Biology. Animal最新文献

Resistance to radiation therapy (RT) poses a significant challenge in managing non-small cell lung cancer (NSCLC). Despite research into how tumor-sourced exosome (Exo) miRNAs influence tumor RT resistance and macrophage M2 polarization, the process through which Exos with miR-616-3p modulate macrophage polarization to impact NSCLC RT resistance is still not well understood. The objective of this research was to investigate the molecular processes by which RT regulates M2 polarization of macrophages via the Exos miR-616-3p derived from NSCLC cells. Identification of Exos was conducted using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunofluorescence, and ELISA were employed to verify the macrophage phenotype. The expression of miR-616-3p was identified using RT-qPCR, and the targeting relationship between miR-616-3p and PTEN was confirmed through dual-luciferase reporter gene tests and RIP identification. In NSCLC, miR-616-3p showed high expression levels and was linked to RT and M2 polarization in macrophages. Subsequent research indicated that RT prompted the influx of Exos-miR-616-3p from NSCLC cells into macrophages. Both the H1299 lung cancer cell line and the M0 macrophages underwent co-culture. Findings indicated that NSCLC cells induced by RT and Exos elevated the proportion of CD163 + CD206 + positive cells in macrophages via miR-616-3p and augmented Arg1, IL-10, TGF-β1, and VEGF levels, and enhanced M2 polarization in macrophages. Regarding the molecular process, miR-616-3p suppressed PTEN protein expression while concurrently boosting the levels of p-PI3K/PI3K and p-AKT/AKT; either amplifying PTEN or suppressing PI3K could markedly weaken the impact of ionizing radiation (IR), inhibiting the impact of NSCLC cell Exos on macrophages' M2 polarization. This research reveals that in NSCLC cells induced by IR, Exos with miR-616-3p expression reduce PTEN levels and enhance the PI3K/AKT signaling pathway, leading to increased M2 polarization in macrophages and worsening NSCLC progression.

This study aimed to evaluate the effects of conditioned medium derived from murine skeletal muscle (SMCM) on oxidative stress and testicular morphology in vitro. Initially, Wistar rats underwent treadmill familiarization and a maximal incremental test (MIT). Animals were then submitted to a single exercise session at 60% of the maximum speed established by MIT. In Experiment 1, femoral muscles from trained animals were cultured in αMEM supplemented with 1.25 mg/mL BSA to produce SMCM. In Experiment 2, testes from sedentary rats were fragmented and cultured for 24 h in αMEM alone or αMEM added to irisin at 100 ng/mL or αMEM added to SMCM at 25, 50, 75, or 100%. HPLC confirmed the presence of irisin in SMCM. Oxidative stress analyses demonstrated catalase activity was higher in irisin and 75% of SMCM treatments, while glutathione peroxidase (GPX) activity was significantly higher in the irisin when compared to fresh control. It is important to highlight that 25% of SMCM was similar to fresh control in GPX activity and thiol content. Histological assessment revealed structural alterations in cultured testes, although overall tubular organization was preserved. These findings suggest that skeletal muscle SMCM modulates oxidative balance and testicular structure, with the 25% concentration yielding the most favorable antioxidant profile.

Obesity resulting from chronic overnutrition and physical inactivity promotes the development of metabolic disorders by disrupting physiological processes in metabolically active organs, including skeletal muscles. To investigate whether skeletal muscle stem cells (satellite cells, SCs) are affected by systemic metabolic stress, we established primary SC cultures from male mice fed a high-fat diet (HFD) for 8 wk, and from control mice fed a standard chow (CTL). This model allowed us to assess diet-induced obesity (DIO)-related changes in SC-specific molecular and cellular signatures. Although body weight, body fat composition, and adipose tissue-associated macrophages differed significantly between DIO and CTL ex vivo, we observed no differences in the in vitro behaviour of primary SC-derived myoblasts from either group. Parameters such as proliferation and differentiation following serum deprivation were comparable. Expression levels and distribution patterns of myogenic regulatory factors (MRF), SC-specific markers (Pax7, CD56, Itga7), and hallmarks for senescence (GLB1), autophagy (p62, LC3B), and oxidative stress (ALDH1A1, ALDH1A3) remained unchanged. Thus, potential differences in the signatures of SC-derived myoblasts after 8 wk of a high-fat diet cannot be depicted in vitro. However, future experiments should address whether prolonged and metabolically more susceptible diets will exert long-term effects on myogenesis in vitro or not. Overall, we propose that primary SC cultures are better suited for acute in vitro testing regarding the molecular and cellular plasticity in metabolic shifts as induced by pharmacological treatments or genetical modifications, rather than for modeling long-term dietary effects.

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.