In Vitro Cellular & Developmental Biology. Animal最新文献

Podocyte injury is a pivotal factor in the advancement of diabetic nephropathy (DN). The present study aimed to delineate the influence of disrupted thyroxine signaling on podocyte apoptosis in DN mouse models. We employed bioinformatics analyses, coupled with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment assessments, to identify differentially expressed genes (DEGs) associated with thyroxine signaling in both human and murine DN datasets. Subsequently, we elucidated the function of thyroid hormone receptor α1 (THRA1) and nuclear receptor co-repressor 1 (NCOR1) on glomerular injury and podocytes apoptosis under hypothyroid and hyperglycemic conditions, respectively. Our findings highlight that hypothyroidism significantly alters glomerular gene expression profiles in DN mice leading to increased podocyte apoptosis. This effect occurs through a dual mechanism: on one hand, the upregulation of THRA1 expression induced by DN results in direct glomerular injury, which was further aggravated by hypothyroidism; on the other hand, the downregulation of NCOR1 expression thereby increases THRA1 activity levels. Our data suggests that disturbed thyroxin signals could trigger podocyte apoptosis and glomerular injury in DN mice, offering new insights into DN pathogenesis while laying groundwork for innovative therapeutic strategies.

In many visual disorders, the neural retinal cells are irreversibly damaged. Because of the low proliferation and repair capacity of these cells in humans, the visual acuity can be highly destroyed in patients, and this is while the current therapies can only partially reduce the extent of this disorder. Although retinal progenitor cells (RPCs) derived from human embryonic stem cells (hESCs) are among one of the most considered sources for the treatment of retinal degenerative disorders, their limited proliferation rate is a significant challenge to provide a sufficient number of cells for transplantation therapy. Thus, this study aims to provide an efficient approach for monitoring RPCs to optimize in vitro maintenance conditions in the future. This was designed by introducing a stable line of hESCs expressing EGFP reporter driven by the human RAX promoter (pRAX-EGFP) as a key biomarker of RPCs. Considering that the pRAX-hESC line showed significant pluripotency capacity and differentiation potential towards RPCs, this cell line can provide a valuable biological tool for optimization and introduction of more suitable in vitro culture conditions in order to ensure the long-term maintenance of the derived RPCs. This cell line might also be considered a reliable cellular source for monitoring the differentiation process towards RPCs in preclinical studies in the future.

Beef is primarily made up of skeletal muscle tissue. Therefore, the cultivation of bovine muscle stem cells (MSCs) to provide a consistent supply of muscle cells would enhance the sustainability of the cultured beef industry. Here, we report a high-yield, simple, economic, and convenient protocol for the isolation of active MSCs from bovine skeletal muscle tissue. We optimized the enzymatic tissue dissociation protocol and the composition of the medium used for differential plating (DP) to enhance the purity of active MSCs isolated from primary cells derived from the tissue. In addition, the optimal source of bovine muscle tissue for the isolation of active MSCs was determined. The yield of active MSCs was maximized by incubating round area-derived skeletal muscle tissue for 30 min in 0.2% (w/v) collagenase type II in high-glucose DMEM (HG-DMEM), followed by 1% (w/v) pronase in HG-DMEM for 5 min, and conducting DP of the enzymatically dissociated skeletal muscle tissue-derived primary cells in HG-DMEM supplemented with 10% (v/v) FBS and 5 ng/mL bFGF. In conclusion, we established a simple, convenient, and inexpensive protocol for the high-yield isolation of active MSCs from bovine skeletal muscle tissue. This protocol could overcome the technical challenges that hamper the large-scale production of bovine muscle cells, thereby enabling the commercialization of cultured beef.

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.

Ageing and reduced levels of physical activity are associated with desensitisation of skeletal muscle to the anabolic effects of amino acids. In vitro studies have indicated that many properties of skeletal muscle tissue are retained in human myotubes, including metabolic alterations associated with exercise and disease. However, the interaction between ageing and physical activity on amino acid sensing and growth has not been explored in human myotubes in vitro. Muscle-derived cells were isolated from biopsies taken from eight young (Y: 23.4 ± 1.9 yr), six older (O: 72.5 ± 5.0 yr), and nine older exercise trained (OT: 71.0 ± 4.1 yr, n = 9) men, and myotube cultures were generated and investigated for growth parameters and amino acid induced changes in mTORC1 signalling and protein synthesis. Our results indicated that muscle cell fusion was similar between groups, but myotube diameter was lower in cultures derived from O individuals. Despite this, mTORC1 signalling, as indicated by immunoblots for phosphorylation of mTOR^Ser2448, rpS6^Ser235/236, and 4E-BP1^Thr37/46 increased to a similar extent in response to amino acid availability in Y, O, and OT myotubes. Furthermore, measures of protein synthesis using the SUnSET assay were increased similarly between groups after the addition of amino acids. These data suggest that skeletal muscle desensitisation to amino acids with ageing is not observed in myotubes cultured in vitro, which could be reflective of the healthy individuals tested in our study or point towards the importance of the muscle niche in the impairments in muscle metabolism in ageing.

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.

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.

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.

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.