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

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach,we discover that measured BMP signaling history correlates strongly with fate in individual cells. We find that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However,both the level and duration of signaling activity control cell fate choices only by changing the time integral. Therefore,signaling duration and level are interchangeable in this context. In a stem cell model for patterning of the human embryo,we show that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Our data suggest that mechanistically,BMP signaling is integrated by SOX2. The interpretation of the key developmental signal BMP remains poorly understood. Here,the authors show that the total time-integrated signaling controls differentiation in a stem cell embryo model and provide a possible mechanism. View Publication

Background/Objectives: The limited translatability of preclinical experimental findings to patients remains an obstacle for successful treatment of brain diseases. Relevant models to elucidate mechanisms behind brain pathogenesis,including cell-specific contributions and cell-cell interactions,and support successful targeting and prediction of drug responses in humans are urgently needed,given the species differences in brain and blood-brain barrier (BBB) functions. Human microphysiological systems (MPS),such as Organ-Chips,are emerging as a promising approach to address these challenges. Here,we examined and advanced a Brain-Chip that recapitulates aspects of the human cortical parenchyma and the BBB in one model. Methods: We utilized human primary astrocytes and pericytes,human induced pluripotent stem cell (hiPSC)-derived cortical neurons,and hiPSC-derived brain microvascular endothelial-like cells and included for the first time on-chip hiPSC-derived microglia. Results: Using Tumor necrosis factor alpha (TNF?) to emulate neuroinflammation,we demonstrate that our model recapitulates in vivo-relevant responses. Importantly,we show microglia-derived responses,highlighting the Brain-Chip’s sensitivity to capture cell-specific contributions in human disease-associated pathology. We then tested BBB crossing of human transferrin receptor antibodies and conjugated adeno-associated viruses. We demonstrate successful in vitro/in vivo correlation in identifying crossing differences,underscoring the model’s capacity as a screening platform for BBB crossing therapeutic strategies and ability to predict in vivo responses. Conclusions: These findings highlight the potential of the Brain-Chip as a reliable and time-efficient model to support therapeutic development and provide mechanistic insights into brain diseases,adding to the growing evidence supporting the value of MPS in translational research and drug discovery. View Publication

Brain organoids offer unprecedented insights into brain development and disease modeling and hold promise for drug screening. Significant hindrances,however,are morphological and cellular heterogeneity,inter-organoid size differences,cellular stress,and poor reproducibility. Here,we describe a method that reproducibly generates thousands of organoids across multiple hiPSC lines. These High Quantity brain organoids (Hi-Q brain organoids) exhibit reproducible cytoarchitecture,cell diversity,and functionality,are free from ectopically active cellular stress pathways,and allow cryopreservation and re-culturing. Patient-derived Hi-Q brain organoids recapitulate distinct forms of developmental defects: primary microcephaly due to a mutation in CDK5RAP2 and progeria-associated defects of Cockayne syndrome. Hi-Q brain organoids displayed a reproducible invasion pattern for a given patient-derived glioma cell line. This enabled a medium-throughput drug screen to identify Selumetinib and Fulvestrant,as inhibitors of glioma invasion in vivo. Thus,the Hi-Q approach can easily be adapted to reliably harness brain organoids’ application for personalized neurogenetic disease modeling and drug discovery. Human brain organoids are plagued by heterogeneity and poor reproducibility,critical parameters for reliable disease modeling and drug testing. Here,the authors report on Hi-Q organoids which solve these limitations and can be cryopreserved in large quantities. View Publication

Genetic deletion of Dusp1 eliminates CSF3R-induced leukemia. Inhibition of Dusp1 induces the expression of Bim and p53 in oncogenic context,resulting in selective demise of leukemic cells. View Publication

R2 elements,a class of non-long terminal repeat (non-LTR) retrotransposons,have the potential to be harnessed for transgene insertion. However,efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here,we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3′ UTR to initiate target-primed reverse transcription. Guided by these insights,we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system’s size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5′ end of the donor RNA and optimizing delivery,creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools. Subject terms: Transferases,Protein design,Genetic engineering View Publication

Viral transduction is a critical step in the manufacturing of genetically modified T cells for immunotherapies,yet conventional transduction methods suffer from low to medium efficiency,high vector consumption,and limited scalability. To address these challenges,we introduce the Transduction Boosting Device (TransB),an innovative,automated,and closed-system platform designed to enable efficient and scalable gene delivery and overcome the limitations of conventional transduction methods. TransB improves cell-virus interactions by facilitating proximity between target cells and viral vectors. TransB demonstrated up to 1-fold decrease in processing time,3-fold reduction in viral vector consumption,and 0.7-fold increase in transduction efficiency compared to 24—well plate method for donor T cell transduction in studies evaluating its impact on transduction process. Comparison studies transducing T cells from three different donors with Lenti-GFP vectors showed that TransB achieved an average 0.5-fold improvement in transduction efficiencies while maintaining comparable post-transduction cell recovery,viability,growth,and phenotype compared to 24—well plate. Furthermore,TransB delivered consistent performance across two different input cell numbers demonstrating scalability of the process. These findings suggest that TransB could significantly shorten the transduction time,reduce the transduction cost and improve the transduction efficiency for manufacturing genetically modified T cell therapies. It shows strong potential as a robust,efficient,and scalable platform to enhance T cell therapy manufacturing and help overcome current manufacturing challenges in the field. The online version contains supplementary material available at 10.1186/s12967-025-06836-1. View Publication

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However,it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells,designated tumor-evoked Bregs (tBreg),phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-$\beta$-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs,4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications,as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis. View Publication

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here,we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits,both purified naive CD8+ T cells and purified naive CD4+ T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period,most of the cells died,but some survived in a quiescent state for a month. The survivors had small round cell bodies,expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia,if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats. 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

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

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