搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time,cost and high inter-species variability. Here,we developed a human-relevant in vitro model,which recapitulated two cellular events characteristic of embryogenesis,to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation,epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human. View Publication

The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha,beta and gamma. Since RAR gamma is predominantly expressed in adult skin,specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay,we have identified three compounds with high RAR gamma selectivity. View Publication

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here,we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells,as predicted from a numerical model of cell migration,and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further,reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications. View Publication

Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus,many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study,we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells,which are shown by multiple marker or phenotypes of CSCs. Thus,these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus,further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer. View Publication

Autophagy is characterized by the sequestration of bulk cytoplasm,including damaged proteins and organelles,and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity,the mechanism is not yet clear. Here we report that cytosolic FoxO1,a forkhead O family protein,is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation,but this process was independent of the transcriptional activity of FoxO1. In response to stress,FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2),a NAD(+)-dependent histone deacetylase,and the acetylated FoxO1 bound to Atg7,an E1-like protein,to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy. View Publication

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease,the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study,a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus,preventing it from infecting cells in an in vitro cell-based assay,including multiple viral variants that are currently circulating in the human population. In addition,we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease. View Publication

Diabetic keratopathy (DK),a significant complication of diabetes,often leads to corneal damage and vision impairment. Effective models are essential for studying DK pathogenesis and evaluating potential therapeutic interventions. This study developed a novel biomimetic full-thickness corneal model for the first time,incorporating corneal epithelial cells,stromal cells,endothelial cells,and nerves to simulate DK conditions in vitro. By exposing the model to a high-glucose (HG) environment,the pathological characteristics of DK,including nerve bundle disintegration,compromised barrier integrity,increased inflammation,and oxidative stress,were successfully replicated. Transcriptomic analysis revealed that HG downregulated genes associated with axon and synapse formation while upregulating immune response and oxidative stress pathways,with C-C Motif Chemokine Ligand 5 (CCL5) identified as a key hub gene in DK pathogenesis. The therapeutic effects of Lycium barbarum glycopeptide (LBGP) were evaluated using this model and validated in db/db diabetic mice. LBGP promoted nerve regeneration,alleviated inflammation and oxidative stress in both in vitro and in vivo models. Notably,LBGP suppressed the expression of CCL5,highlighting its potential mechanism of action. This study establishes a robust biomimetic platform for investigating DK and other corneal diseases,and identifies LBGP as a promising therapeutic candidate for DK. These findings provide valuable insights into corneal disease mechanisms and pave the way for future translational research and clinical applications. Graphical abstractImage 1 Highlights•A full-thickness biomimetic corneal model containing corneal epithelium,nerves,stroma,and endothelium was constructed.•Using this model,the pathological characteristics of diabetic keratopathy were successfully replicated in vitro.•Lycium barbarum glycopeptide (LBGP) alleviated high-glucose-induced damage in vitro and in vivo models.•CCL5 plays an important role in the pathogenesis of diabetic keratopathy. View Publication

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases,including those with genetic origins. Currently,primary ECs are the main source for disease modelling in this field. However,they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level,we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor,known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level,which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly,the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability,while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary,we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-024-09925-9. View Publication

The development of immunocompetent skin models marks a significant advancement in in vitro methods for detecting skin sensitizers while adhering to the 3R principles,which aim to reduce,refine,and replace animal testing. This study introduces for the first time an advanced immunocompetent skin model constructed entirely from induced pluripotent stem cell (iPSC)-derived cell types,including fibroblasts (iPSC-FB),keratinocytes (iPSC-KC),and fully integrated dendritic cells (iPSC-DC). To evaluate the skin model’s capacity,the model was treated topically with a range of well-characterized skin sensitizers varying in potency. The results indicate that the iPSC-derived immunocompetent skin model successfully replicates the physiological responses of human skin,offering a robust and reliable alternative to animal models for skin sensitization testing,allowing detection of extreme and even weak sensitizers. By addressing critical aspects of immune activation and cytokine signaling,this model provides an ethical,comprehensive tool for regulatory toxicology and dermatological research. View Publication

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer,an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo–electron microscopy at near-atomic resolution and implemented novel,cost-effective,high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease,exemplifying the potential of our approach. View Publication

Endometrial receptivity is a critical determinant of embryo implantation and early pregnancy success; however,current methods for assessing endometrial receptivity remain poorly validated and insufficiently reliable for clinical application. Here,we establish a patient-derived vascularised endometrium-on-a-chip (EoC),successfully replicating the dynamic microenvironment and both temporal and spatial architecture of native endometrial tissue. Using our EoC,we develop a clinically relevant endometrial receptivity scoring system,ERS2,which integrates molecular profiling of established receptivity markers with quantitative analyses of angiogenesis. The ERS2 enables personalised assessment of endometrial health and implantation potential,addressing inter-patient variability often overlooked by conventional techniques. By leveraging our EoC to therapeutic monitoring,we observe progressive restoration of the endometrial microenvironment following platelet-rich-plasma treatments,highlighting the translational utility of our model. This study represents the innovative application of a patient-derived EoC and scoring system to assess receptivity,offering personalised infertility management and advancing targeted therapies in reproductive medicine. Accurate assessment of endometrial receptivity remains a challenge in infertility care. Here,authors present a patient-derived vascularised endometrium-on-a-chip and a scoring system for receptivity evaluation. View Publication

Purpose: Current lung cancer organoid models often fail to replicate the complex tumor immune microenvironment,reducing their predictive value for immunotherapy and radiotherapy. Therefore,it is crucial to establish an optimized lung cancer organoid model which could recapitulate the tumor immune microenvironment,enabling more accurate evaluation of therapeutic responses. Methods: We developed an optimized air-liquid interface (ALI) culture method to generate patient-derived lung cancer organoids (ALI-LUOs) from 19 lung cancer samples. The tumor microenvironment,including immune and stromal components,was characterized using immunofluorescence,flow cytometry,and single-cell RNA sequencing. The organoids were further used to assess responses to αPD-1 therapy and radiotherapy. Results: The optimized method significantly improved organoid formation efficiency while preserving immune cell viability for up to 30 days. Immune and fibroblast populations were confirmed by immunofluorescence and flow cytometry. Single-cell RNA sequencing demonstrated that ALI-LUOs accurately replicate the tumor immune landscape. Key tumor immunity pathways such as cGAS-STING could be captured by ALI-LUOs. Importantly,ALI-LUOs modeled clinical responses to immune checkpoint inhibitors and radiotherapy with high fidelity. Conclusions: The ALI-LUOs,developed through an optimized culture method,faithfully capture the key characteristics of lung cancer,including its immunosuppressive tumor microenvironment. Our findings highlight this modified ALI-LUOs as a valuable preclinical platform for evaluating antitumor immunity and refining lung cancer treatments. View Publication