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

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study,we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg,HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV,thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection,we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients,we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. View Publication

Cancer remains one of the leading causes of mortality worldwide,leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade,CD103 + T cells have been associated with better clinical prognosis in patients with cancer. However,the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here,we show an unexpected and transient CD61 expression,which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling,improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically,the presence of CD61 + tumor-infiltrating T lymphocytes is associated with improved clinical outcomes,mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion,this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells,which potentiates a new target for immune-based cellular therapies. Subject terms: T cells,Tumour immunology,Lymphocyte activation View Publication

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD,even before symptom onset. Because these early disease phenotypes remain incompletely understood,we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients,presymptomatic C9ORF72-HRE (C9-HRE) carriers,and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition,single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids,including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further,molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly,organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology,whereas the extent to which more downstream cellular defects,as found in C9-ALS/FTD models,were detected varied for the different presymptomatic C9-HRE cases. Together,these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies. The online version contains supplementary material available at 10.1186/s40478-024-01857-1. View Publication

Gastrointestinal (GI) dysfunction,characterized by severe diarrhea and dehydration,is a central contributor to morbidity and mortality in filovirus disease in patients,yet the role of the epithelium in this clinical outcome remains poorly defined. Here,we employ induced pluripotent stem cell (iPSC)-derived human intestinal (HIOs) and colonic organoids (HCOs) to model Ebola virus (EBOV) and Marburg virus (MARV) infection. These organoids are permissive to filovirus infection and support viral replication. Bulk RNA sequencing revealed distinct intestinal and colonic epithelial responses,including apical and junctional disruption and a delayed virus-specific induction of interferon-stimulated genes. Moreover,infection impaired adenylate cyclase signaling and CFTR-mediated ion transport,providing mechanistic insight into virus-induced secretory diarrhea. This platform recapitulates key features of human GI pathology in filoviral disease and serves as a powerful system to dissect host-pathogen interactions and identify therapeutic targets. Author summaryEbola virus (EBOV) and Marburg virus (MARV) are among the most lethal viruses known. Infection with these viruses leads to severe disease and death. One of their most harmful effects is damage to the gastrointestinal tract,causing intense diarrhea and life-threatening dehydration. Yet,how these viruses affect the gut remains poorly understood. In this study,we used human mini-guts—small,three-dimensional tissues grown from stem cells that mimic the human intestinal and colonic epithelium—to investigate how these viruses interact with gut epithelial cells. We found that both EBOV and MARV infect and replicate in these tissues,disrupt key barrier structures,and interfere with the cells’ ability to regulate fluid secretion. These effects mirror the severe symptoms seen in patients. Our study provides new insight into how EBOV and MARV damage the gut and identifies specific cellular pathways that may be targeted for treatment. This research not only improves our understanding of EBOV and MARV infections but also offers new infection platforms for testing therapies aimed at protecting the gastrointestinal system during filovirus outbreaks. View Publication

AbstractIntroductionNovel strategies are needed to address the rising burden of osteoporosis and fragility fractures. High-intensity resistance and impact (HiRIT) exercise has shown benefit in improving bone density in postmenopausal women with osteoporosis/osteopenia. Whether HiRIT can enhance the therapeutic effects of osteoporosis pharmacotherapy has not been established. ROLEX-DUO is a randomised controlled trial designed to assess the efficacy of romosozumab on various bone and muscle outcomes in combination with different exercise interventions in women with postmenopausal osteoporosis/osteopenia.Methods and analysisROLEX-DUO is an 8-month randomised placebo-controlled trial conducted at two tertiary referral centres for patients with osteoporosis/osteopenia in Sydney,New South Wales,Australia. The study is implementing the combination of romosozumab or placebo with different forms of exercise in postmenopausal women with osteoporosis/osteopenia without recent fragility fracture (n=102). Eligible women will be randomised 1:1:1 into one of three groups: (1) romosozumab with supervised HiRIT,(2) romosozumab with unsupervised low-intensity exercise or (3) placebo with unsupervised low-intensity exercise. Co-primary outcomes are the mean percentage change in lumbar spine bone mineral density (BMD),and mean change in five times sit-to-stand test performance (seconds) at 8 months. Secondary/exploratory outcomes include BMD changes at the femoral neck,total hip and distal radius,three-dimensional dual-energy X-ray absorptiometry (DXA) hip outcomes,DXA-derived lean and fat mass,serum markers of bone turnover (procollagen type 1 peptide,C-telopeptide of type 1 collagen) and bone biomarkers (dickkopf-1),serum extracellular vesicle analyses,36-Item Short Form Survey (SF-36) quality-of-life scores,Menopause-Specific Quality Of Life (MENQOL) Questionnaire menopause symptom burden scores,number of falls and fractures. Mixed-effects models will be performed to compare longitudinal outcome results between groups using intention-to-treat analysis.Ethics and disseminationThe trial was approved by the Northern Sydney Local Health District Human Research Ethics Committee (2022/ETH01794,protocol V.8,dated 03 July 2024). Participants will provide written informed consent prior to inclusion. Findings will be disseminated via peer-reviewed journals,scientific conferences and summary reports to funding bodies.Trial registration numberACTRN12623000867695. View Publication

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis,biological activity,and further profiling of these compounds are described. This work resulted in the discovery of 17,GDC-0941,which is a potent,selective,orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer. View Publication

Infusion of T cells modified with a chimeric antigen receptor (CAR) targeting CD19 has achieved exceptional responses in patients with non-Hodgkin’s lymphoma (NHL),which led to the approval of CAR targeting CD19 (CART19) (Axi-cel and Liso-cel) as second line of treatment for adult patients with relapsed/refractory NHL. Unfortunately,60% of patients still relapse after CART19 due to either a loss of expression of the target antigen (CD19) in the tumor cell,observed in 27% of relapsed patients,a limited CAR-T persistence,and additional mechanisms,including the suppression of the tumor microenvironment. Clinic strategies to prevent target antigen loss include sequential treatment with CARs directed at CD20 or CD22,which have caused loss of the second antigen,suggesting targeting other antigens less prone to disappear. CD79b,expressed in NHL,is a target in patients treated with antibody-drug conjugates (ADC). However,the limited efficacy of ADC suggests that a CAR therapy targeting CD79b might improve results. We designed three new CARs against CD79b termed CAR for Lymphoma (CARLY)1,2 and 3. We compared their efficacy,phenotype,and inflammatory profiles with CART19 (ARI0001) and CARTBCMA (ARI0002h),which can treat NHL. We also analyzed the target antigen’s expression loss (CD79b,CD19,and B-cell maturation antigen(BCMA)). We found that CARLY2 and CARLY3 had high affinity and specificity towards CD79b on B cells. In vitro,all CAR-T cells had similar anti-NHL efficacy,which was retained in an NHL model of CD19 − relapse. In vivo,CARLY3 showed the highest efficacy. Analysis of the loss of the target antigen demonstrated that CARLY cells induced CD79b and CD19 downregulation on NHL cells with concomitant trogocytosis of these antigens to T cells,being most notorious in CARLY2,which had the highest affinity towards CD79b and CD19,and supporting the selection of CARLY3 to design a new treatment for patients with NHL. Finally,we created a CAR treatment based on dual targeting of CD79b and BCMA to avoid losing the target antigen. This treatment showed the highest efficacy and did not cause loss of the target antigen. Based on specificity,efficacy,and loss of the target antigen,CARLY3 represents a potential novel CAR treatment for NHL. View Publication

Neural stem cells (NSCs) yield both neuronal and glial progeny,but their differentiation potential toward multiple region-specific neuron types remains remarkably poor. In contrast,embryonic stem cell (ESC) progeny readily yield region-specific neuronal fates in response to appropriate developmental signals. Here we demonstrate prospective and clonal isolation of neural rosette cells (termed R-NSCs),a novel NSC type with broad differentiation potential toward CNS and PNS fates and capable of in vivo engraftment. R-NSCs can be derived from human and mouse ESCs or from neural plate stage embryos. While R-NSCs express markers classically associated with NSC fate,we identified a set of genes that specifically mark the R-NSC state. Maintenance of R-NSCs is promoted by activation of SHH and Notch pathways. In the absence of these signals,R-NSCs rapidly lose rosette organization and progress to a more restricted NSC stage. We propose that R-NSCs represent the first characterized NSC stage capable of responding to patterning cues that direct differentiation toward region-specific neuronal fates. In addition,the R-NSC-specific genetic markers presented here offer new tools for harnessing the differentiation potential of human ESCs. View Publication

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation,a common approach to mimic some of the spatial and temporal aspects of in vivo development,are affected by three factors: input hESC composition,input hESC colony size,and EB size. Using a microcontact printing approach,size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently,size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover,upon forming EBs from these MP-hESCs,we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs,higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely,neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size,typical in most differentiation strategies,produces subsets of appropriate conditions for differentiation into specific cell types. Moreover,our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories. View Publication

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high,and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk,yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast,we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing,including E2F transcription factors,chromatin licensing and DNA replication factor 1 (Cdt1),and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone,showing that progesterone influences epithelial cell lineage differentiation. Therefore,we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers. View Publication

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such,this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However,preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets,predominantly in the distal airways. Analyses of in vivo,ex vivo,and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age,reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models. View Publication