BACKGROUND:Circulating tumour cells (CTCs) are mediators of cancer dissemination and the formation of metastasis, which is the leading cause of cancer-related deaths. Experimental models derived from CTCs contribute to understanding the biology of CTCs, their role in dissemination, and the discovery of potential drugs targeting CTCs. METHODS:A xenograft was derived from CTCs isolated from a patient diagnosed with metastatic invasive ductal carcinoma of the breast. The characterisation of the CTCs-derived xenograft (CDX) was conducted through in vivo experimental metastatic assays, RNA-Seq, spectral flow cytometry, and drug sensitivity tests. RESULTS:The CTCs-enriched fraction formed a CDX within 6 months, and its metastatic potential was confirmed. CDX cells were propagated in vitro, where the enrichment of CD44+/CD24- breast cancer stem cells was confirmed. An RNA-Seq-based comparison of CDX with the primary tumour from the same patient unravelled substantial changes in genes related to cell growth, metabolism, and extracellular signalling. CDX and in vitro cell culture showed sensitivity to carboplatin. A partial response was also observed for vandetanib, which was selected through in silico analysis of transcriptomic data. CONCLUSIONS:We present and characterise a novel model derived from CTCs for understanding the plasticity and behaviour of CTCs and advanced breast cancer. CDX_IBP_01 was established from the CTC-enriched fraction obtained from the patient with progressing breast cancer. Once stably re-transplanted and growing in vivo, the transcriptomes of CDX and archived primary BCa1 samples were compared. 2D and 3D in vitro cell cultures were established from sorted human cancer cells from an in vivo xenograft. Phenotypes of established models and their stability were characterised using spectral flow cytometry. The metastatic potential of CDX was evaluated in an in vivo assay. Finally, the applicability of the established model for in vivo and in vitro drug screening was evaluated. Created in https://BioRender.com .

Decellularized lung scaffolds represent a promising platform for respiratory tissue engineering, yet it remains unclear how decellularization remodels the proteome, which extracellular matrix (ECM) components are depleted or retained, and whether residual non-matrisome proteins persist despite DNA-based clearance benchmarks. Moreover, systematic cross-species comparison remains unaddressed. We applied a single detergent-based protocol to mouse, pig, and human lung tissue and characterized the resulting scaffolds using histology, ultrastructural imaging, immunofluorescence, mass spectrometry-based proteomics with matrisome annotation, and STRING-based protein–protein interaction (PPI) network analysis. Decellularization achieved consistent cellular clearance while preserving alveolar ECM architecture. Proteomic profiling identified a 96-protein matrisome core shared across all three species, dominated by structural collagens, basement membrane glycoproteins, and elastic fibre components, whereas matrisome-associated proteins showed substantially lower cross-species conservation. PPI network analysis mapped this core onto three modules of known and predicted associations. Beyond the matrisome, species-specific residual cellular protein signatures persisted despite scaffolds meeting DNA-based clearance criteria. As an initial demonstration of cytocompatibility, human expandable lung epithelium progenitor cells on pig scaffolds remained attached and proliferative over 28 days. These findings reframe scaffold assessment beyond cellular clearance toward molecular evaluation of ECM composition and network architecture, arguing for proteomic criteria alongside anatomical considerations in lung bioengineering.

Introduction:The safe and effective application of human pluripotent stem cells (hPSCs) in research and regenerative medicine requires precise control over pluripotency and cell fate. Pluripotency is characterized by high histone acetylation and aerobic glycolysis, while differentiation involves metabolic remodeling and reduced acetylation. Pyruvate dehydrogenase (PDH) links these processes by converting glycolytic pyruvate into acetyl coenzyme A (Ac-CoA), the key substrate for histone acetylation. Methods:We investigated how PDH activity regulates histone acetylation and pluripotency maintenance under physiologically relevant oxygen levels (5% and 21% O₂). PDH contribution to histone acetylation was assessed using a specific PDH inhibitor, followed by rescue experiments with acetyl-CoA precursors. hPSCs were exposed to variations in FGF2 signaling and reactive oxygen species (ROS) using H₂O₂ treatment to evaluate redox-dependent modulation of PDH and downstream effects on pluripotency factors. Protein levels and post-translational modifications were analyzed by Western blotting and quantitative PCR, relative metabolite concentrations by LC-MS, and ROS levels by fluorescence microscopy. Results:Active PDH promoted global histone H3 acetylation and upregulated the expression of the pluripotency factor NANOG, specifically under 5% O₂. Mechanistic analysis revealed a novel FGF2-MEK1/2-ERK1/2-ROS signaling axis that regulates PDH activity through redox-sensitive mechanisms. This regulatory pathway was oxygen-dependent and absent under atmospheric oxygen levels (21% O₂). Discussion:These findings identify PDH as a redox-sensitive metabolic switch connecting cellular metabolism with the epigenetic control of pluripotency by modulating Ac-CoA availability. Conclusion:Our study highlights the importance of oxygen tension, ROS homeostasis, and growth factor signaling in shaping the metabolic-epigenetic landscape of hPSCs, with implications for optimizing stem cell culture and differentiation protocols.

Lipopolysaccharide (LPS)-induced inflammation of lung tissues triggers irreversible alterations in the lung parenchyma, leading to fibrosis and pulmonary dysfunction. While the molecular and cellular responses of immune and connective tissue cells in the lungs are well characterized, the specific epithelial response remains unclear due to the lack of representative cell models. Recently, we introduced human embryonic stem cell-derived expandable lung epithelial (ELEP) cells as a novel model for studying lung injury and regeneration. ELEPs were derived from the CCTL 14 human embryonic stem cell line through activin A-mediated endoderm specification, followed by further induction toward pulmonary epithelium using FGF2 and EGF. ELEPs exhibit a high proliferation rate and express key structural and molecular markers of alveolar progenitors, such as NKX2-1. The effects of Escherichia coli LPS serotype O55:B5 on the phenotype and molecular signaling of ELEPs were analyzed using viability and migration assays, mRNA and protein levels were determined by qRT-PCR, western blotting, and immunofluorescent microscopy. We demonstrated that purified LPS induces features of a hybrid epithelial-to-mesenchymal transition in pluripotent stem cell-derived ELEPs, triggers the unfolded protein response, and upregulates intracellular β-catenin level through retention of E-cadherin within the endoplasmic reticulum. Human embryonic stem cell-derived ELEPs provide a biologically relevant, non-cancerous lung cell model to investigate molecular responses to inflammatory stimuli and address epithelial plasticity. This approach offers novel insights into the fine molecular processes underlying lung injury and repair.

3D bioprinting is transforming tissue engineering by enabling spatial arrangement of cells and cell aggregates within supportive hydrogels. Among available materials, alginate remains widely used for its biocompatibility, printability, and cost-effectiveness. However, its bioinert nature and lack of adhesive moieties restrict its capacity to support essential processes like cell adhesion, migration, and proliferation. In this study, we propose a comprehensive approach to enhance alginate hydrogels focusing on stem, stromal, and endothelial cell types to support extended growth and vascular network formation. Key innovations include the incorporation of the TYRAY peptide in 3D alginate hydrogels─its first application in this context─to promote cell adhesion and migration, accompanied by Ca(OH)2-modified surfaces for stable hydrogel anchoring and an ultrasonic mist cross-linking to preserve 3D structure fidelity. Functionalization with the TYRAY peptide significantly enhanced cell proliferation, promoted multicellular spheroid fusion, and supported endothelial network development in comparative culture setting. Together, these findings establish this defined, xeno-free alginate system as a versatile bioink material suitable for 3D culture and bioprinting applications.

Interstitial lung diseases (ILDs) is a large and heterogeneous group of disorders with a variable degree of lung inflammation and lung fibrosis. In some ILDs, we can observe a progressive-fibrosing phenotype—PF-ILD (e.g., idiopathic pulmonary fibrosis, fibrotic phenotype of hypersensitivity pneumonitis, familial lung fibrosis, etc.). Lung fibrosis is characterized by overgrowth, stiffening, and scarring of tissues due to excess deposition of extracellular matrix. In some patients suffering from PF-ILD, progression and fatal outcomes occur despite treatment. Therefore, there is a great need for the development of lung fibrosis models that will help to understand and recapitulate the etiopathogenesis of the disease and may thus serve as tools for unraveling its underlying profibrotic mechanisms and potential therapeutic targets. In this review, we summarize ILD etiopathogenesis, current and novel therapeutic options, and discuss in vivo, ex vivo, and in vitro near-to-native lung fibrosis models, which help to elucidate specific processes within ILD pathophysiology.

Lead nanoparticles (PbNPs) in air pollution pose a significant threat to human health, especially due to their neurotoxic effects. In this study, we exposed mice to lead(II) oxide nanoparticles (PbONPs) in inhalation chambers to mimic real-life exposure and assess their impact on the brain. PbONPs caused the formation of Hirano bodies and pathological changes related to neurodegenerative disorders through cytoskeletal disruptions without the induction of inflammation. Damage to astrocytic endfeet and capillary endothelial cells indicated a compromised blood-brain barrier (BBB), allowing PbONPs to enter the brain. Additionally, NPs were detected along the olfactory pathway, including fila olfactoria, suggesting that at least a proportion of PbNPs enter the brain directly by passing through the olfactory epithelium. PbNP inhalation severely damaged the apical parts of olfactory epithelial cells, including the loss of microtubules in their ciliary distal segments. Inhalation of PbONPs led to the rapid accumulation of lead in the brain, while more soluble lead(II) nitrate NPs did not accumulate significantly until 11 weeks of exposure. PbNPs induced disruption of the BBB at multiple levels, ranging from ultrastructural changes to functional impairments of the barrier; however, they did not induce systemic inflammation in the brain. The clearance ability of the brain to remove Pb was very low for both types of NPs, with significant pathological effects persisting even after a long clearance period. Cation-binding proteins (ZBTB20 and calbindin1) were distributed unevenly in the brain, with the strongest signal located in the hippocampus, which exhibited the greatest defects in nuclear architecture, indicating that this area is the most sensitive structure for PbNP exposure. PbNP exposure also altered the PI3K/Akt/mTOR signaling pathway, and tau phosphorylation in the hippocampus and inhibition of tau phosphorylation by GSK-3 inhibitor rescued the negative effect of PbONPs on the intracellular calcium level in trigeminal ganglion cultures. In zebrafish larvae, PbONPs affected locomotor activity and reduced calcium levels in the medium enhanced negative effect of PbONP on animal mobility, even increasing lethality. These findings suggest that cytoskeletal disruption and calcium dysregulation are key factors in PbNP-induced neurotoxicity, providing potential targets for therapeutic intervention to prevent neurodegenerative changes following PbNP exposure.

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease marked by alveolar type 2 (AT2) stem cell dysfunction and excessive matrix deposition, with no effective treatments. Recent advances have recognized that AT2 cells act as stem cells, in addition to their role in the production of pulmonary surfactants in the distal alveolar space. We and others have reported a failure of AT2 regeneration and a loss of AT2 cells in IPF. We recently further reported that there is a defect in lipid metabolism in IPF AT2 cells and we discovered a selective loss of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in AT2 cells from IPF, as well as in AT2 cells from bleomycin-injured mice. Pharmacological and genetic experiments confirm that LPCAT1 is required for AT2 cell renewal in 3D organoid assays. AT2 cell-specific Lpcat1 deletion resulted in reduced AT2 renewal, spontaneous lung fibrosis, and heightened susceptibility to bleomycin-induced fibrosis in mice in vivo. Expression-based high-content drug screening with an LPCAT1 knock-in cell line identified several drug families that upregulated LPCAT1 expression. We further confirmed that anti-malarial artesunate and PLA2 inhibitor ONO-RS-082 increased LPCAT1 mRNA expression, promoted AT2 renewal, and attenuated bleomycin-induced lung fibrosis in mice in vivo. Our findings establish LPCAT1 as a critical regulator of AT2 renewal and lipid metabolism in IPF, suggesting that reactivation of LPCAT1 could offer a novel therapeutic strategy for restoring alveolar progenitor function and mitigating lung fibrosis.

Inhaled cadmium oxide nanoparticles (CdONPs) represent an underrecognized environmental and occupational hazard because of their potential for systemic bioaccumulation and organ-specific toxicity. In this study, mice were exposed to subchronic inhalation of CdONPs, and cadmium distribution, clearance, and tissue responses were assessed over a 21-day recovery period using atomic absorption spectrometry, laser ablation inductively coupled plasma mass spectrometry, histopathology, and gene expression analysis. Cadmium accumulated predominantly in the lungs, where clearance was slow and accompanied by persistent inflammation and foam cell formation. The intestines exhibited efficient cadmium reduction, likely due to high epithelial turnover, while the liver showed minimal accumulation and no overt damage. By contrast, the kidneys retained cadmium primarily in the cortex, with partial clearance and ultrastructural changes, including mitochondrial disorganization and lipid accumulation. Bone tissues demonstrated differential retention: jaw bones effectively cleared cadmium, whereas femurs showed sustained or increased levels, suggesting redistribution from other organs. Gene expression analysis revealed moderate but consistent upregulation of Abca1, Apoe, and Ptch1 in the kidneys of clearance groups, indicating adaptations in lipid metabolism and membrane transport. These findings highlight organ-specific clearance kinetics and molecular responses to inhaled CdONPs, underscoring the need for tissue-targeted risk assessment frameworks in nanoparticle toxicology.

The limiting factor in the current state-of-the-art bioengineering of human tissues/organoids in vitro is the lack of functional vasculature that would support growth, metabolic waste removal, and oxygen supply of the formed constructs. The three-dimensional (3D) tissues can be formed by utilization of specific cell types, different hydrogels and scaffolds that provide a physical support, in addition to specific medium composition. Establishment of chemically defined xeno-free conditions is of great interest when keeping in mind the transition from basic research to therapeutic applications. In this study, we present the formation of vascular network of human umbilical vein endothelial cells co-cultured with stromal cells (SCs), human dental pulp SCs or human adipose SCs, in natural hydrogels (Matrigel, GelMA) as well as xeno-free VitroGel hydrogels. As medium composition plays a pivotal role in the successful generation of biological constructs in vitro, we evaluated the potency of specific growth factors (GFs) to induce vascular network formation in 3D hydrogels. We confirmed that vascular endothelial growth factor (VEGF), the most used pro-angiogenic factor, is not mandatory in culture medium for vascular network formation. Rather the endothelial cell (EC) network in co-culture of ECs and SCs can be formed through stimulation of SCs with GFs such as insulin-like growth factor 1 (IGF1), basic fibroblast growth factor (FGF2), and epidermal growth factor (EGF) regardless of serum presence in the medium. Also, we tested completely chemically defined medium that, when used together with xeno-free hydrogel, presents a step forward to application of such tissue constructs in translational regenerative medicine.

Lately, the need for three-dimensional (3D) cell culture has been recognized in order to closely mimic the organization of native tissues. Thus, 3D scaffolds started to be employed to facilitate the 3D cell organization and enable the artificial tissue formation for the emerging tissue engineering applications. 3D scaffolds can be prepared by various techniques, each with certain advantages and disadvantages. Decellularization is an easy method based on removal of cells from native tissue sample, yielding extracellular matrix (ECM) scaffold with preserved architecture and bioactivity. This chapter provides a detailed protocol for decellularization of pig lung and also some basic assays for evaluation of its effectivity, such as determination of DNA content and histological verification of the selected ECM components. Such decellularized scaffold can subsequently be used for various tissue engineering applications, for example, for recellularization with cells of interest, for natural ECM hydrogel preparation, or as a bioink for 3D bioprinting.

The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface in vitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.

Intravenous thrombolysis with a recombinant tissue plasminogen activator (rt-PA) is the first-line treatment of acute ischemic stroke. However, successful recanalization is relatively low and the underlying processes are not completely understood. The goal was to provide insights into clinically important factors potentially limiting rt-PA efficacy such as clot size, rt-PA concentration, clot age and also rt-PA in combination with heparin anticoagulant. We established a static in vitro thrombolytic model based on red blood cell (RBC) dominant clots prepared using spontaneous clotting from the blood of healthy donors. Thrombolysis was determined by clot mass loss and by RBC release. The rt-PA became increasingly less efficient for clots larger than 50 μl at a clinically relevant concentration of 1.3 mg/l. A tenfold decrease or increase in concentration induced only a 2-fold decrease or increase in clot degradation. Clot age did not affect rt-PA-induced thrombolysis but 2-hours-old clots were degraded more readily due to higher activity of spontaneous thrombolysis, as compared to 5-hours-old clots. Finally, heparin (50 and 100 IU/ml) did not influence the rt-PA-induced thrombolysis. Our study provided in vitro evidence for a clot size threshold: clots larger than 50 μl are hard to degrade by rt-PA. Increasing rt-PA concentration provided limited thrombolytic efficacy improvement, whereas heparin addition had no effect. However, the higher susceptibility of younger clots to thrombolysis may prompt a shortened time from the onset of stroke to rt-PA treatment.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.

The effect of silica (SiO2) addition (0 wt%-20 wt%) on the microstructural and mechanical properties, as well as the in vitro response of calcium phosphate scaffolds for potential application in bone tissue engineering (BTE) was investigated in this research. Scaffolds characterized by high porosity (77%-88 %) and interconnected spherical pores with a broad range of pore sizes (5-600 mu m) were fabricated using in-situ foaming method. Incorporated silica affected the phase transformation of hydroxyapatite (HA) to beta-tricalcium phosphate (beta-TCP) and led to the development of new crystalline silica-rich phases like silicocarnotite and wollastonite. The reinforcement of silica became apparent during the tests of mechanical properties. Scaffolds with 5 wt% of SiO2 exhibited compressive strength (1.13 MPa) higher than pure HA scaffolds (0.93 MPa). Bone bonding potential of the materials was tested in simulated body fluid (SBF), demonstrating this potential in silica-doped samples. Additionally, degradation experiments showed gradual material degradation, making it suitable for BTE applications. Furthermore, cell culture studies using human mesenchymal stromal cells (MSC) confirmed the scaffold's non- toxicity and provided insights into how the silica content influences cell viability, morphology, and osteogenic potential. The findings of this study offer valuable insights into the design and development of advanced scaffolds with tailored properties for effective BTE applications.

Human embryonic stem cells (hESCs) have unique abilities that enable their use in cell therapy, disease modeling, and drug development. Their derivation is usually performed using a feeder layer, which is undefined and can potentially cause a contamination by xeno components, therefore there is a tendency to replace feeders with xeno-free defined substrates in recent years. Three hESC lines were successfully derived on the vitronectin with a truncated N-terminus (VTN-N) in combination with E-cadherin in xeno-free conditions for the first time, and their undifferentiated state, hESC morphology, and standard karyotypes together with their potential to differentiate into three germ layers were confirmed. These results support the conclusion that the VTN-N/E-cadherin is a suitable substrate for the xeno-free derivation of hESCs and can be used for the derivation of hESCs according to good manufacturing practices.

Figure S1. In vitro profiling of SCH900776 and its selected analogs. Figure S2. Analogs of MU380 with the N-trifluoromethylpyrazol pharmacophore. Figure S3. Dot plots of flow cytometric analysis using γH2AX and Annexin V staining related to Figure 2D. Figure S4. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentrations in combination with HU in the concentration range shown on the x-axis (related to Figure 3A). Figure S5. Dynamics of cytotoxic effects of CHK1 inhibitors alone and in combination with HU. Figure S6. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with GEM in concentration range shown on the x-axis (related to Figure 3B). Figure S7. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with irradiation in the dose range shown on the x-axis. Figure S8. Western blot analysis of total CHK1 and p-CHK1 (S296) levels in a panel of selected cell lines after indicated treatments. Figure S9. Drug retention analysis. Figure S10. MU380 induces CHK1 pS345 in mice hair follicles. Figure S11. Bioluminescent signal of SHO mice bearing MiaPaCa2 luc xenografts treated with GEM, SCH900776, or MU380 alone or in indicated combinations. Weight of MiaPaCa2 luc tumor-bearing animals. Representative images of γH2AX staining in MiaPaCa2 luc tumor sections.