Recent Articles

3D Cell CultureBelow are the titles and abstracts from the most recent articles published in In Vitro Cellular and Developmental Biology – Animal. Click on the title to view the full article at Springerlink.


  • Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform–dependent manner
    on May 17, 2024 at 12:00 am

    Abstract Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin–proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 […]

  • Cardiac differentiation of chimpanzee induced pluripotent stem cell lines with different subspecies backgrounds
    on May 16, 2024 at 12:00 am

    Abstract The comparative analysis between humans and non-human primates is an instrumental approach for elucidating the evolutional traits and disease propensity of humans. However, in primates, cross-species analyses of their developmental events have encountered constraints because of the ethical and technical limitations in available sample collection, sequential monitoring, and manipulations. In an endeavor to surmount these challenges, in recent years, induced pluripotent stem cells (iPSCs) have garnered escalating interest as an in vitro tool for cross-species analyses between humans and non-human primates. Meanwhile, compared to humans, there is less information on in vitro differentiation of non-human primate iPSCs, and their genetic diversity including subspecies may cause different eligibility to in vitro differentiation methods. Therefore, antecedent to embarking on a comparative analysis to humans, it is a prerequisite to develop the efficacious […]

  • Bringing cell biology into classroom: tips to culture and observe skeletal muscle cells in high school and college
    on May 14, 2024 at 12:00 am

    Abstract Watching living cells through a microscope is much more exciting than seeing pictures of cells in high school and college textbooks. However, bringing cell cultures into the classroom is challenging for biology teachers since culturing cells requires sophisticated and expensive instruments such as a CO2 incubator and an inverted phase-contrast microscope. Here, we describe easy and affordable methods to culture and observe skeletal muscle cells using the L-15 culture medium, tissue culture flask, standard dry incubator, standard upright microscope, and modified Smartphone microscope. Watching natural living cells in a “Do-It-Yourself (DIY)” way may inspire more students’ interest in cell biology.

  • Evaluating osteogenic potential of a 3D-printed bioceramic-based scaffold for critical-sized defect treatment: an in vivo and in vitro investigation
    on May 14, 2024 at 12:00 am

    Abstract The integration of precision medicine principles into bone tissue engineering has ignited a wave of research focused on customizing intricate scaffolds through advanced 3D printing techniques. Bioceramics, known for their exceptional biocompatibility and osteoconductivity, have emerged as a promising material in this field. This article aims to evaluate the regenerative capabilities of a composite scaffold composed of 3D-printed gelatin combined with hydroxyapatite/tricalcium phosphate bioceramics (G/HA/TCP), incorporating human dental pulp–derived stem cells (hDPSCs). Using 3D powder printing, we created cross-shaped biphasic calcium phosphate scaffolds with a gelatin layer. The bone-regenerating potential of these scaffolds, along with hDPSCs, was assessed through in vitro analyses and in vivo studies with 60 rats and critical-sized calvarial defects. The assessment included analyzing cellular proliferation, differentiation, and alkaline phosphatase […]

  • The role of semaphorin 3A on chondrogenic differentiation
    on May 10, 2024 at 12:00 am

    Abstract Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100 ng/mL) was examined using real-time reverse transcription polymerase […]

  • Characteristics of nuclear architectural abnormalities of myotubes differentiated from LmnaH222P/H222P skeletal muscle cells
    on May 9, 2024 at 12:00 am

    Abstract The presence of nuclear architectural abnormalities is a hallmark of the nuclear envelopathies, which are a group of diseases caused by mutations in genes encoding nuclear envelope proteins. Mutations in the lamin A/C gene cause several diseases, named laminopathies, including muscular dystrophies, progeria syndromes, and lipodystrophy. A mouse model carrying with the LmnaH222P/H222P mutation (H222P) was shown to develop severe cardiomyopathy but only mild skeletal myopathy, although abnormal nuclei were observed in their striated muscle. In this report, we analyzed the abnormal-shaped nuclei in myoblasts and myotubes isolated from skeletal muscle of H222P mice, and evaluated the expression of nuclear envelope proteins in these abnormal myonuclei. Primary skeletal muscle cells from H222P mice proliferated and efficiently differentiated into myotubes in vitro, similarly to those from wild-type mice. During cell proliferation, few abnormal-shaped nuclei were […]

  • Potential treatment of squamous cell carcinoma by targeting heparin-binding protein 17/fibroblast growth factor-binding protein 1 with vitamin D3 or eldecalcitol
    on May 7, 2024 at 12:00 am

    Abstract Heparin-binding protein 17 (HBp17), first purified in 1991 from the conditioned medium of the human A431 squamous cell carcinoma (SCC) cell line, was later renamed fibroblast growth factor-binding protein 1 (FGFBP-1). HBp17/FGFBP-1 is specifically expressed and secreted by epithelial cells, and it reversibly binds to fibroblast growth factor (FGF)-1 and FGF-2, as well as FGFs-7, -10, and -22, indicating a crucial involvement in the transportation and function of these FGFs. Our laboratory has investigated and reported several studies to elucidate the function of HBp17/FGFBP-1 in SCC cells and its potential as a molecular therapeutic target. HBp17/FGFBP-1 transgene exoression in A431-4 cells, a clonal subline of A431 that lacks tumorigenicity and does not express HBp17/FGFBP-1, demonstrated a significantly enhanced proliferation in vitro compared with A431-4 cells, and it acquired tumorigenicity in the subcutis of nude mice. Knockout (KO) of the HBp17/FGFBP-1 […]

  • Wnt signaling in cardiac development and heart diseases
    on May 6, 2024 at 12:00 am

    Abstract The Wnt signaling pathway is a fundamental cellular communication system with extensive implications in various organs including the heart. In cardiac homeostasis, it governs essential processes like cellular proliferation, differentiation, and apoptosis, ensuring the heart’s structural and functional integrity from embryonic stages and throughout life. Both canonical and non-canonical Wnt signaling pathways play a critical role during embryonic heart development in a region- and stage-specific manner. Canonical Wnt signaling also plays a significant role in heart diseases such as myocardial infarction and heart failure. However, the role of non-canonical Wnt signaling in heart diseases has not been fully elucidated. Wnt5a is a major ligand that activates non-canonical Wnt pathway, and recent studies start to clarify the role of the Wnt5a signaling axis in cardiac health and disease. In this review, we will briefly summarize the previous findings on the […]

  • Differentiation of bone marrow mesenchymal stem cells into Leydig-like cells with testicular extract liquid in vitro
    on May 2, 2024 at 12:00 am

    Abstract Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3β-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell […]

  • BMSC-derived exosomes regulate the Treg/Th17 balance through the miR-21-5p/TLR4/MyD88/NF-κB pathway to alleviate dry eye symptoms in mice
    on April 29, 2024 at 12:00 am

    Abstract Bone marrow mesenchymal stem cell (BMSC)–derived exosomes (BMSC-Exos) have a variety of biological functions and are extensively involved in the regulation of inflammatory diseases, as well as tissue repair and regeneration. However, the mechanism of action of these compounds in dry eye disease (DED) in mice is still unclear. This study demonstrated that the Treg/Th17 ratio was strongly imbalanced in DED clinical samples. BMSC-Exos can modulate the Treg/Th17 balance, improve the integrity of the corneal epithelial layer, and ameliorate DED progression in mice. Mechanistically, BMSC-Exos dramatically decreased the levels of IL-17 and IL-22; increased the levels of IL-4, IL-10, and TGF-β1; and increased tear secretion and the number of goblet cells in the conjunctiva in mice, thus alleviating the progression of DED. This effect is achieved by BMSC-Exos through the delivery of miR-21-5p to target and restrain TLR4, thereby restraining the MyD88/NF-κB […]

  • PRRX1 upregulates PD-L1 in human mesenchymal stem cells
    on April 25, 2024 at 12:00 am

    Abstract Mesenchymal stem cells (MSCs) have been demonstrated to be efficacious in clinical applications for the amelioration of immune disorders, including graft-versus-host disease (GvHD) and Crohn's disease. The immunosuppressive role of Programmed death-ligand 1 (PD-L1) in MSCs is pivotal, yet the regulatory mechanisms governing its expression remain to be fully elucidated. In this study, we explored the influence of paired-related homeobox (PRRX1), a determinant of multipotency and self-renewal in MSCs, on the expression of various surface antigens, notably PD-L1. Multiple isoforms of PRRX1 were found to augment the mRNA levels of MSC markers, such as CD26 and CD317, with all isoforms elevating PD-L1 expression at both mRNA and protein levels. This study reveals that PRRX1 may act as a potential immunomodulatory factor in MSCs by regulating the PD-L1 pathway.

  • Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes
    on April 25, 2024 at 12:00 am

    Abstract The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida–free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the […]

  • The migration pattern of cells during the mesoderm and endoderm differentiation from human pluripotent stem cells
    on April 24, 2024 at 12:00 am

    Abstract Gastrulation is the first major differentiation process in animal embryos. However, the dynamics of human gastrulation remain mostly unknown owing to the ethical limitations. We studied the dynamics of the mesoderm and endoderm cell differentiation from human pluripotent stem cells for insight into the cellular dynamics of human gastrulation. Human pluripotent stem cells have properties similar to those of the epiblast, which gives rise to the three germ layers. The mesoderm and endoderm were induced with more than 75% purity from human induced pluripotent stem cells. Single-cell dynamics of pluripotent stem cell–derived mesoderm and endoderm cells were traced using time-lapse imaging. Both mesoderm and endoderm cells migrate randomly, accompanied by short-term directional persistence. No substantial differences were detected between mesoderm and endoderm migration. Computer simulations created using the measured parameters revealed that random movement […]

  • Adult hair follicle stem cells differentiate into neuronal cells in explanted rat intestinal tissue
    on April 24, 2024 at 12:00 am

    Abstract Hair follicle stem cells (HFSCs) are adult stem cells located in the outer root sheath of the follicle bulge with high neural plasticity, which promise a potential for the stem cell therapy for neurological diseases. Hirschsprung’s disease (HD) is characterized by the absence of ganglia in the distant bowel. In this study, we elucidated the capacity of HFSCs to differentiate into neuronal cells in the aganglionic colon from embryonic rat. HFSCs were isolated from adult Sprague–Dawley (SD) rats and formed spheres that could be passaged. The cultured HFSCs expressed neural crest stem cells (NCSCs) markers such as SOX10, CD34, and nestin, which indicated their neural crest lineage. Subsequent differentiation assays demonstrated that these cells could give rise to neural progeny that expressed neuronal or glial markers. The aganglionic colon from the embryonic intestine was applied as in vitro explant to test the capacity of proliferation and differentiation […]

  • Tumor endothelial cell-derived Sfrp1 supports the maintenance of cancer stem cells via Wnt signaling
    on April 16, 2024 at 12:00 am

    Abstract Cancer stem cells (CSCs), which are critical targets for cancer therapy as they are involved in drug resistance to anticancer drugs, and metastasis, are maintained by angiocrine factors produced by particular niches that form within tumor tissue. Secreted frizzled-related protein 1 (Sfrp1) is an extracellular protein that modulates Wnt signaling. However, the cells that produce Sfrp1 in the tumor environment and its function remain unclear. We aimed to elucidate angiocrine factors related to CSC maintenance, focusing on Sfrp1. Although Sfrp1 is a Wnt pathway-related factor, its impact on tumor tissues remains unknown. We investigated the localization of Sfrp1 in tumors and found that it is expressed in some tumor vessels. Analysis of mice lacking Sfrp1 showed that tumor growth was suppressed in Sfrp1-deficient tumor tissues. Flow cytometry analysis indicated that CSCs were maintained in the early tumor growth phase in the Sfrp1 knockout (KO) mouse model of […]

  • Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells
    on April 15, 2024 at 12:00 am

    Summary Skeletal muscle's regenerative ability is vital for maintaining muscle function, but chronic diseases like Duchenne muscular dystrophy can deplete this capacity. Muscle satellite cells, quiescent in normal situations, are activated during muscle injury, expressing myogenic regulatory factors, and producing myogenic progenitor cells. It was reported that muscle stem cells in primary culture and reserve cells in C2C12 cells express anti-apoptotic protein Bcl-2. Although the role of Bcl-2 expressed in myogenic cells has been thought to be to enhance cell viability, we hypothesized that Bcl-2 may promote the formation of reserve cells. The expression pattern analysis showed the expression of Bcl-2 in undifferentiated mononucleated cells, emphasizing its usefulness as a reserve cell marker and reminding us that cells expressing Bcl-2 have low proliferative potential. Silencing of Bcl-2 by transfection with siRNA decreased cell viability and the number of reserve […]

  • 3,3′-Diindolylmethane inhibits Th17 cell differentiation via impairing IRF-7-mediated plasmacytoid dendritic cell activation in imiquimod-induced psoriasis mice
    on April 11, 2024 at 12:00 am

    Abstract Psoriasis is a paradigmatic condition characterised by a heightened autoimmune response and chronic inflammation. However, the exact nature and the pathological causes behind it are still unknown. Growing evidence suggest dysregulated cytokine network as a result of over-activated T cells and plasmacytoid dendritic cells (pDCs) as the critical drivers in the development of psoriasis. In the present study, we aimed to investigate the therapeutic efficacy of 3,3′-diindolylmethane (DIM) on pDC activation and Th17 cell development in imiquimod (IMQ)–induced psoriasis mice. Our in vitro research investigated the IRF-7 signalling in pDCs that explained the reduced expression of the transcription factor IRF-7 responsible for pDC activation as a result of DIM treatment. Concurrently, DIM treatment decreased the release of Th17 cell polarising cytokines (IFN-α, IL-23, and IL-6) by pDCs which validated a reduction in differentiated pathogenic Th17 cell population […]

  • Role of the Ror family receptors in Wnt5a signaling
    on April 8, 2024 at 12:00 am

    Abstract Ror-family receptors, Ror1 and Ror2, are type I transmembrane proteins that possess an extracellular cysteine-rich domain, which is conserved throughout the Frizzled-family receptors and is a binding site for Wnt ligands. Both Ror1 and Ror2 function primarily as receptors or co-receptors for Wnt5a to activate the β-catenin-independent, non-canonical Wnt signaling, thereby regulating cell polarity, migration, proliferation, and differentiation depending on the context. Ror1 and Ror2 are expressed highly in many tissues during embryogenesis but minimally or scarcely in adult tissues, with some exceptions. In contrast, Ror1 and Ror2 are expressed in many types of cancers, and their high expression often contributes to the progression of the disease. Therefore, Ror1 and Ror2 have been proposed as potential targets for the treatment of the malignancies. In this review, we provide an overview of the regulatory mechanisms of Ror1/Ror2 expression and discuss […]

  • Inhibition of circ_0073932 attenuates myocardial ischemia‒reperfusion injury via miR-493-3p/FAF1/JNK
    on April 5, 2024 at 12:00 am

    Abstract Oxidative stress and apoptosis play crucial roles in myocardial ischemia‒reperfusion injury (MIRI). In this study, we investigated the role of circ_0073932 in MIRI as well as its molecular mechanism. A hypoxia/reoxygenation (H/R) cardiomyocyte model was established with H9C2 cardiomyocytes, and RT–qPCR was used to measure gene expression. We observed that circ_0073932 expression was abnormally increased in the H/R cardiomyocyte model and in blood samples from MIRI patients. Inhibition of circ_0073932 suppressed H/R-induced cell apoptosis, oxidative stress (ROS, LDH and MDA), and p-JNK expression. Dual luciferase reporter assays showed that circ_0073932 targeted the downregulation of miR-493-3p, and miR-493-3p targeted the downregulation of FAF1. Furthermore, si-circ_0073932, an miR-493-3p inhibitor, oe-FAF1, or si-FAF1 were transfected into H9C2 cardiomyocytes to investigate the roles of these factors in MIRI. Our results showed that compared with the […]

  • Eco-friendly green synthesis of N‑pyrazole amino chitosan using PEG-400 as an anticancer agent against gastric cancer cells via inhibiting EGFR
    on April 1, 2024 at 12:00 am

    Abstract The present study was conducted to develop a green process that provides access to the development of Schiff base derivatives of chitosan with the heterocyclic moiety as a novel class of anti-gastric cancer agent. In the present study, we have synthesized these derivatives by reacting various pyrazoles with chitosan using CAN in PEG400. The compounds were synthesized in 20 min in excellent yield by using CAN at 5% in PEG400 at 80°C in the shortest reaction time of 20 min. The PEG400 could be efficiently recycled for the three consecutive runs. The developed compounds were tested for EGFR-TK inhibition using a Kinase-Glo Plus luminescence kinase assay kit where they exhibited significant activity revealing compound 2d as the most potent analog, while other compounds showed mild to moderate inhibitory activity. MTT assay was conducted to determine the effect of the three most potent EGFR inhibitors (2b, 2c, and 2d) on the proliferation of gastric cancer […]

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