BACKGROUND:Satellite cells are the primary drivers of skeletal muscle growth and regeneration and are a major cell source in both muscle tissue engineering and cultivated meat. However, current bovine satellite cell isolation can be technically demanding or yield heterogeneous cell populations. METHODS:This study presents a workflow optimized for simplicity and cost-efficiency by integrating rapid muscle processing using electric mincing with magnetic-activated cell sorting targeting Integrin-α7 to reliably enrich satellite cells. The isolated cells were characterized by Pax7 immunostaining and myogenic differentiation assays. RESULTS:ITGA7-based magnetic sorting yielded an enriched satellite cell population corresponding to 49.39 ± 14.70 × 10³ cells per gram of minced tissue. The isolated cells exhibited high Pax7 expression (98 ± 1.34% Pax7-positive cells) and strong myogenic differentiation capacity (differentiation index: 94.39 ± 3.58%), confirming their identity and functional potential. CONCLUSION:By minimizing hands-on effort and avoiding specialized equipment, this workflow lowers technical and financial barriers while enabling the acquisition of highly enriched, early-passage satellite cell populations. This workflow provides an adaptable foundation for advancing cultivated meat bioprocess development and accelerating research in skeletal muscle biology.
Gut homeostasis relies on the tightly controlled balance between intestinal stem cell self-renewal and differentiation. The clathrin adaptor complex AP-1B plays a pivotal role in establishing the polarity of enterocytes as well as in the asymmetric distribution of membrane proteins, including the brush border transporters that govern intestinal absorption. Additionally, AP-1B has been involved in the control of intestinal cell proliferation, which suggests that it may regulate various aspects of gut functional organisation. In this study, we investigated the consequences of conditional mutations of the gene encoding the AP-1B subunit μ1B (Ap1m2) in mouse enteroids. We first showed in this model that Ap1m2 mutations also induce strong polarity defects at the subcellular level in the absorptive enterocytes. Next, we unveiled that AP-1B regulates intestinal cell differentiation at the tissular level, through the commitment of stem/progenitor cells towards the secretory lineage and cell positioning along the crypt-villus axis. Furthermore, we showed that AP-1B inhibition also induces hyperproliferation in enteroids. Notably, we unravelled that, complementary to the Wnt/β-catenin-mediated proliferation described in null mice, AP-1B downregulation triggers a tissue-autonomous, mTOR/YAP-dependent, proliferative pathway. Overall, these results enlighten the pleiotropic roles played by AP-1B in the homeostasis of the gut epithelium.
BACKGROUND:Gallbladder cancer (GBC) is a highly lethal malignancy with limited experimental models to study disease biology or evaluate therapeutic responses. Although canonical Wnt activation is commonly used for patient-derived organoid (PDO) development and expansion, gallbladder PDOs have also been generated under Wnt-inhibitory conditions. No comparative assessments have determined how Wnt pathway modulation influences gallbladder PDO development, phenotype, or drug response. METHODS:This study systematically compared the impact of canonical Wnt activation (WNTAct medium containing CHIR99021) versus inhibition (WNTInh medium containing DKK1) on the establishment, propagation, molecular features, and therapeutic responses of PDOs generated from malignant or non-malignant gallbladder tissues derived from the same patient. RESULTS:Both media supported successful PDO generation with comparable efficiency, preserving biliary epithelial functions and marker expression. Transcriptomic profiling confirmed selective enrichment of canonical Wnt target genes in PDOs generated in WNTAct cultures. WNTAct conditions enabled markedly superior long-term propagation, whereas WNTInh cultures more consistently retained the dysplastic features in malignant samples. Gemcitabine response assays demonstrated significantly greater drug sensitivity in PDOs grown in WNTAct medium, a phenotype reversible upon media switching but requiring extended adaptation, indicating a dynamic and context-dependent influence of Wnt-signaling on chemotherapeutic vulnerability. CONCLUSION:Collectively, the findings reveal a trade-off between long-term propagation and histological fidelity in gallbladder PDOs and show that Wnt-signaling modulates gemcitabine sensitivity in a reversible manner. This comparative framework provides practical guidance for selecting culture conditions for gallbladder PDO-based disease modeling, and precision oncology applications.
BACKGROUND:Oxidative stress has a significant role in the origination and progression of oral squamous cell carcinoma (OSCC) by interrupting cellular redox homeostasis and gene regulation. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is a major regulator of antioxidant defense, activating cytoprotective genes under stress conditions. AIM:The study highlight the dual role of the Kelch-like ECH-associated protein-1 (Keap1)/Nrf2 signaling pathway in the development, progression, and therapeutic resistance of OSCC. METHODOLOGY:Literature was conducted to evaluate the role of oxidative stress and Keap1/Nrf2 signaling in OSCC. RESULTS:Nrf2 protects normal cells from oxidative damage, its persistent activation in cancer cells promotes tumor progression, metabolic reprogramming, and resistance to chemotherapy and radiotherapy. Dysregulation of the Keap1/Nrf2 axis enhances cancer cell survival and contributes to radioresistance in OSCC. However, Nrf2 displays a dual function as both a tumor suppressor and promoter. CONCLUSION:The Keap1/Nrf2 signaling pathway plays a key role in OSCC pathogenesis and treatment resistance. Targeting the Keap1/Nrf2 signaling pathway may provide a promising therapeutic strategy to overcome treatment resistance in OSCC.
Colorectal cancer (CRC) develops through the stepwise accumulation of genetic alterations, including mutations in APC, KRAS, and TP53, which drive tumor initiation and progression. Although advances in genomic profiling have significantly improved our understanding of CRC, translating these alterations into functional outcomes and therapeutic responses remains a major challenge. This limitation arises from tumor heterogeneity, context-dependent signaling, and the dynamic nature of tumor evolution. Recent advances in patient-derived organoids (PDOs) have provided a platform that preserves tumor-specific architecture and genetic features, enabling functional interrogation of drug responses. However, PDOs lack critical components of the tumor microenvironment, such as stromal, immune, and mechanical cues. Organ-on-a-chip (OoC) technologies, particularly organoid-on-a-chip systems in which PDOs are integrated into microfluidic devices, further address these limitations by recapitulating physiological conditions and multicellular interactions. In this review, we discuss the genetic evolution of CRC and highlight how emerging functional models, including PDOs and organoid-on-a-chip platforms, bridge the gap between genomic alterations and tumor behavior. We propose that integrating these platforms offers a promising framework for advancing functional precision medicine and improving our understanding of CRC biology.
AIM:Pancreatic islet δ-cells produce somatostatin, a paracrine regulator of insulin and glucagon secretion within islets. Although adaptive changes in α- and β-cell populations during pregnancy have been described in both animals and humans, data on δ-cell plasticity are sparse and entirely lacking in human pregnancy. We aimed to determine whether pancreatic islet δ-cell mass undergoes morphological adaptation during human pregnancy. METHODS AND RESULTS:Formalin-fixed paraffin-embedded pancreatic tissue from pregnant (n = 7) and non-pregnant (n = 7) donors was analysed. Sections were immunolabelled for somatostatin to identify δ cells, and whole-slide quantitative analysis was performed using an unbiased automated imaging pipeline. δ-cell area was measured across the entire pancreatic sections and compared between groups. In contrast to previously reported expansion of α- and β-cell populations in pregnancy, δ-cell area was not significantly different between pregnant and non-pregnant donors. No quantitative architectural alterations in δ-cell distribution within islets were observed. CONCLUSION:Pancreatic δ-cell area does not increase during human pregnancy. These findings demonstrate that endocrine cell plasticity within the maternal pancreas is selective and does not uniformly involve all islet cell subtypes.
Background Therapeutic resistance is a major cause of treatment failure in glioblastoma (GBM), highlighting the need for physiologically relevant models to identify actionable resistance mechanisms. While two-dimensional (2D) cultures are widely used for target discovery, they poorly represent the tumor microenvironment. In contrast, three-dimensional (3D) spheroid cultures better recapitulate spatial heterogeneity, hypoxic gradients, and stress-adaptive signaling observed in tumors.Methods We applied an integrated 2D-3D quantitative proteomic approach to identify microenvironment-dependent regulators of chemoresistance in GBM. Proteomic profiling was performed in U87MG and U251MG cells grown as 2D monolayers or 3D spheroids. Differentially expressed proteins were validated by quantitative RT-PCR, and functional studies were conducted using genetic depletion followed by assessment of temozolomide (TMZ) sensitivity.Results Comparative analysis identified 13 proteins consistently differentially expressed between 2D and 3D cultures: NDUFB5, RNGTT, MLK4, SYN1, DDX5, EIF2AK2, ITGA1, ZNF33B, ZNF343, WDR19, JPH3, CCT8L2, and FNDC3A. Among these, Mixed Lineage Kinase 4 (MLK4) showed strong and reproducible upregulation in 3D spheroids in both GBM cell lines. Genetic depletion of MLK4 significantly increased TMZ sensitivity without affecting basal cell viability, suggesting a specific role in therapy response. Notably, MLK4 expression was induced only under 3D conditions.Conclusion MLK4 functions as a microenvironment-responsive regulator of chemoresistance in GBM. These findings demonstrate that 3D culture systems reveal clinically relevant resistance pathways not detectable in conventional 2D models and highlight 3D proteomic profiling as a powerful strategy for identifying therapeutically actionable targets.
BACKGROUND:Two isoforms of the 90-kDa heat shock protein (Hsp90), stress-inducible Hsp90α and constitutively expressed Hsp90β, function in mammalian cells as molecular chaperones that promote the folding of specific client proteins involved in essential cellular processes and regulatory pathways. A number of Hsp90 client proteins take part in cancer progression, and the inhibition of Hsp90 induces the degradation of oncogenic client proteins and cancer cell death. Hsp90 inhibitors specific for individual Hsp90 isoforms have a significant potential for the development of anticancer therapeutics due to reduced toxicity. Cells with knocked-out genes encoding Hsp90 isoforms represent excellent cellular models to investigate the rearrangement of the cell chaperone machinery in response to the suppression/loss of the Hsp90 isoforms. RESULTS:Recently, we have shown that the knockout of the HSP90AA1 gene encoding Hsp90α in human fibrosarcoma HT1080 cells does not affect basic cellular processes in normal and stressful conditions, which suggests an adaptation of the cell chaperone machinery to the loss of Hsp90α. Here, we demonstrated that the lack of Hsp90α in HT1080 cells leads to an up-regulation of the constitutively expressed Hsp90β and several important Hsp90 co-chaperones (Aha1, Hop, and others). The expression of the major chaperones of the Hsp70 machinery (Hsp70-1, Hsp70-2, Hsc70) was also significantly induced. The components of the prefoldin-chaperonin folding arm and PFDL, R2TP, and R2SP complexes, as well as the major mitochondrial chaperones, were also largely up-regulated in Hsp90α-KO cells, while the expression of ER-resident chaperones/co-chaperones was either repressed or did not change. CONCLUSIONS AND SIGNIFICANCE:We demonstrated here for the first time an adaptation of the cell chaperone machinery to the loss of the Hsp90α chaperone, which may be important for understanding the molecular mechanisms of action of Hsp90α-specific inhibitors and elaborating new therapy strategies in combating cancer, including the combination of Hsp90α-targeted therapy.
Mitochondria exhibit a complex spatially organized distribution within muscle, tailored to the energy requirements of ATPases and contractile filaments, which exhibit precise intracellular positioning. Mitochondrial distribution varies across longitudinal and transverse axes as well as based on fiber composition within the muscle. The differential mitochondrial capacity can be localized in muscle by succinate dehydrogenase (SDH) activity. Given the distinct energy requirements of the fore-limb and hind-limb muscles, this study aimed to investigate the distribution of mitochondrial activity within individual fibers and their composition within fascicles across different tetrapod taxa. We analyzed pectoralis and gastrocnemius from toad, garden lizard, duck, pigeon, quail, chicken, rat, rabbit, goat, and buffalo. The study revealed unique patterns of mitochondrial activity distribution within the same muscle across various tetrapods. Toad and lizard muscles showed mostly fibers with intermediate SDH-activity (SDHInt) in both muscles. The muscles only from birds and mammals exhibited fibers with negligible SDH-activity termed SDHLow, which might indicate that such fibers are evolutionarily more recent. Interestingly, avian pectoralis showed a very unique fiber composition compared to mammals, which displayed a mosaic pattern of different fibers. Among mammals, slow-grazers (buffaloes, goats) had higher percentages of SDHHigh and SDHInt fibers, whereas sprint-runners (rats, rabbits) possessed a high abundance of SDHLow fibers. These findings provide evidence for localized mitochondrial enrichment as an adaptation strategy to create muscle group heterogeneity.Summary Statement This study characterizes the spatial distribution of mitochondrial activity in skeletal muscle across tetrapods, from the single-fiber scale to the fascicular level.
Xenopus laevis survives seasonal droughts by entering a hypometabolic state known as aestivation. One of the mechanisms employed by X. laevis to mitigate aestivation-induced tissue atrophy is gene regulation of pro-survival proteins. We further expand on the role of anti-apoptotic signaling in X. laevis by investigating the effect of signal transducer and activator of transcription (STAT) signaling on downstream anti-apoptotic genes in control and dehydrated liver and skeletal muscle of X. laevis. Herein, we found that STAT signaling is differentially regulated between tissues. STAT3 signaling in the liver and STAT5 signaling in skeletal muscle lead to the selective upregulation of downstream anti-apoptotic proteins. Additionally, pro-apoptotic STAT1 signaling is found to be attenuated in both tissues during dehydration stress. Overall, our results indicate an important role for anti-apoptotic proteins during dehydration stress and their contribution in mitigating aestivation-induced atrophy.
Super-resolution microscopy has become an indispensable tool for investigating molecular architectures in their native cellular environment. However, most super-resolution techniques face limitations that prevent rapid, deep imaging of live samples. Random Illumination Microscopy (RIM), based on natural laser speckle illumination, is a method of choice to overcome these challenges. RIM combines laser speckle illumination at the optical resolution with an algorithm that exploits the statistical invariance of speckle patterns.In this approach, a stack of hundreds of random speckle images is acquired using a random diffusive element and then processed to reconstruct the super-resolved optical section. The invariant statistical properties of speckle patterns, which persist even as they diffuse through biological samples, enable deep-tissue imaging. Additionally, the wide-field configuration of both illumination and detection ensures high acquisition speeds and minimal sample photodamage.Here, we present the implementation of our RIM prototype within a microscopy core facility. We describe the system setup, characterization, and optimization, identifying the key elements required for its reliable operation. As a proof of concept, we also provide biological examples demonstrating the prototype's performance in resolving subcellular structures.
BACKGROUND:Patient-derived neural organoids (NOs) have emerged as powerful tools for modeling human neurodevelopmental disorders, especially when animal models are unavailable or fail to recapitulate human-specific cortical development. However, significant variability in differentiation potential, even among healthy donor lines, and especially in fragile patient-derived induced pluripotent stem cells (iPSCs), poses major methodological challenges. Protocols that succeed in one line may fail in others, leading to poor organoid formation, reduced growth, impaired neuroepithelial patterning, or complete failure to generate neural tissues. METHODS:By systematically comparing multiple published protocols, we identified key sources of variability and ultimately developed optimized protocols for generating both whole-brain (WB) and cortical organoids (CO) from human iPSCs (hiPSCs), including lines from progeroid Cockayne syndrome patients. RESULTS:Through iterative refinement of critical parameters, including cell seeding density, Matrigel incorporation, and timing and type of pathway inhibition, we achieved consistent organoid growth and structural organization across all tested lines. The resulting pipeline is adaptable and can be further tailored for newly derived or particularly challenging hiPSC lines. CONCLUSION:Collectively, this methodological framework enables robust and reproducible generation of NOs from genetically diverse hiPSC sources, providing a reliable platform for studying human neurodevelopment and disease mechanisms in progeroid and other patient-specific contexts.
BACKGROUND:Endometrial cancer (EC) posed a great threat to women's health, especially in postmenopausal women. E2F transcription factor 8 (E2F8), recognized as a central regulator of critical cellular processes, is overexpressed in endometrial carcinomas. However, the underlying mechanisms remains elusive and worthy of further investigation. Herein, we investigated whether E2F8 activates the expression of epidermal growth factor-like domain multiple 6 (EGFL6) through transcriptional regulatory mechanisms to regulate the occurrence and development of EC tumors. METHODS:Western blot and quantitative reverse transcription polymerase chain reaction (qPCR) analyses revealed the expression profiles of E2F8 and EGFL6 in tumor tissues and EC cells. The Ishikawa and KLE cell lines were selected as in vitro models of EC and were subsequently transfected with sh-E2F8 or overexpression EGFL6 (oe-EGFL6) plasmids. CCK-8 assay and Transwell assay were performed to evaluate the cell viability, migration, and invasion of EC cells. A dual luciferase assay was conducted to assess the interaction between E2F8 and EGFL6. E2F8-knockdown Ishikawa cells were subcutaneously transplanted to investigate their effect on EC tumor growth. RESULTS:E2F8 was greatly upregulated in EC cells. Silencing E2F8 inhibits the proliferation, migration, and invasion of EC cells, thereby suppressing tumor growth in vivo. Mechanistically, E2F8 transcriptionally activates EGFL6 by binding to its promoter. oe-EGFL6 rescues the impacts of E2F8 silencing on the proliferation, migration, and invasion of EC cells. CONCLUSION:These results indicated that E2F8 promotes proliferation, migration, and invasion of EC cells by transcriptionally activating EGFL6.
Mammary organoids bridge the gap between reductionist 2D systems and in vivo models by recapitulating bilayered epithelial architecture, branching, hormone responsiveness, and, in advanced platforms, functional readouts of lactation. This review synthesizes organoid models across the reproductive cycle, including branching morphogenesis, pregnancy-induced alveologenesis and milk secretion, and involution; it also surveys emerging directions, including embryonic/pluripotent and cross-species systems, as well as co-culture and organ-on-chip platforms that incorporate stromal, adipose, and immune elements. We outline priorities for building more complex, physiologically faithful ex vivo models that will enable mechanistic dissection of mammary development, yield comparative and translational insights, and create scalable platforms for perturbation and screening, advancing lactation research, breast cancer studies, and women's health in general.
During the last decade, non-invasive methods such as liquid biopsy have increasingly replaced invasive techniques for diagnosing and monitoring diseases. Liquid biopsies are easier to obtain, highly sensitive, cost-effective per sample, and can be used for real-time monitoring, such as tracking drug response. To uncover molecular and cellular characteristics today, blood, saliva, urine, or cerebrospinal fluid are typically analyzed. In this methodological proof-of-concept study, we propose that patient profiling can be facilitated by assessing surface markers on extracellular vesicles (EVs) directly in plasma without prior purification. EV composition reflects the physiological or pathological state of their parent cell, making EVs valuable tools for biomarker discovery and understanding disease mechanisms. Plasma and purified EVs from 30 ischemic stroke patients were analyzed for 17 surface markers using an in-house protein microarray method (EV Array). The findings indicate that measurements on EVs directly in plasma and on purified EVs show similar patterns in expression of surface markers, supporting the feasibility of omitting purification in EV profiling. This knowledge can provide faster monitoring of various diseases, prevent delays in patient profiling, and optimize patient care.
