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

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells,of which basal cells are the major type in human airways. In this study,helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery,and direct bronchoscopic instillation,respectively. Vector transduction was assessed by immunostaining of lung tissue sections,which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition,efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore,we successfully delivered the human CFTR gene to airway basal cells from CF patients,and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases. View Publication

Despite the success of chimeric antigen receptor T (CART) cell therapy in hematological malignancies,durable remissions remain low. Here,we report CART senescence as a potential resistance mechanism in 41BB-costimulated CART cell therapy. To mimic cancer relapse,we utilized an in vitro model with repeated CART cell activation cycles followed by rest periods. Using CD19-targeted CART cells with costimulation via 4-1BB-CD3ζ (BBζ) or CD28-CD3ζ (28ζ),we showed that CART cells undergo functional,phenotypical,and transcriptomic changes of senescence,which is more prominent in BBζ. We then utilized two additional independent strategies to induce senescence through MYC activation and irradiation. Induction of senescence impaired BBζ activity but improved 28ζ activity in preclinical studies. These findings were supported by analyses of independent patient data sets; senescence signatures in CART cell products were associated with non-response to BBζ but with improved clinical outcomes in 28ζ treatment. In summary,our study identifies senescence as a potential mechanism of failure predominantly in 41BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1. SignificanceWe identified senescence as a cause of failure in CART cell therapy,predominantly in 4-1BB-costimulated CART cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-025-02371-1. View Publication

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However,current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility,limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here,we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow,and reconstitute human haematopoiesis in immunocompromised mice. Importantly,nonhuman primate autologous gene-modified CD34+ cell products are capable of stable,polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy,there is enormous potential for this approach to treat patients on a global scale. View Publication

Glioblastoma is the most common form of primary malignant brain tumour. These tumours are highly proliferative and infiltrative resulting in a median patient survival of only 14 months from diagnosis. The current treatment regimens are ineffective against the small population of cancer stem cells residing in the tumourigenic niche; however,a new therapeutic approach could involve the removal of these cells from the microenvironment that maintains the cancer stem cell phenotype. We have isolated multipotent sphere-forming cells from human high grade glioma (glioma sphere-forming cells (GSCs)) to investigate the adhesive and migratory properties of these cells in vitro. We have focused on the role of two closely related metalloproteinases ADAM10 and ADAM17 due to their high expression in glioblastoma and GSCs and their ability to activate cytokines and growth factors. Here,we report that ADAM10 and ADAM17 inhibition selectively increases GSC,but not neural stem cell,migration and that the migrated GSCs exhibit a differentiated phenotype. We also observed a correlation between nestin,a stem/progenitor marker,and fibronectin,an extracellular matrix protein,expression in high grade glioma tissues. GSCs adherence on fibronectin is mediated by α5β1 integrin,where fibronectin further promotes GSC migration and is an effective candidate for in vivo cancer stem cell migration out of the tumourigenic niche. Our results suggest that therapies against ADAM10 and ADAM17 may promote cancer stem cell migration away from the tumourigenic niche resulting in a differentiated phenotype that is more susceptible to treatment. 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

We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells,but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions. View Publication

Cryopreservation is an essential technique to preserve stem cells,semipermanently sustaining their potentials. There are two main approaches of cryopreservation for human pluripotent stem cells (hPSCs). The first is the vitrification,which involves instantaneous freeze and thaw of hPSCs. The second is the conventional slow-cooling method and a rapid thaw. Both cryopreservation protocols have been standardized and optimized to yield high survivability of hPSCs. View Publication

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However,it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here,we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days,at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis,thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional,morphological and functional signatures of differentiated neurons,with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons,suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. View Publication

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells,yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes,but how much epigenomes differ remains unclear. Here,we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts,we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks,which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally,we observe novel,context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment. textcopyright 2010 Elsevier Inc. View Publication

BACKGROUND: Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications,including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have quantitatively compared promoter activities of five commonly used constitutive promoters,including the human β-actin promoter (ACTB),cytomegalovirus (CMV),elongation factor-1α,(EF1α),phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. CONCLUSION/SIGNIFICANCE: The ACTB,EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation,promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells,it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages,suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs. View Publication