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

Introduction To complement islet transplantation for type1 diabetic patients,cell-based therapy using pluripotent stem cells such as ES cells and iPS cells is promising. Many papers have already reported the induction of pancreatic $\beta$ cells from these cell types,but a suspension culture system has not usually been employed. The aim of this study is to establish a suspension culture method for inducing functional islet-like cells from human iPS cells. Methods We used 30 ml spinner type culture vessels for human iPS cells throughout the differentiation process. Differentiated cells were analyzed by immunostaining and C-peptide secretion. Cell transplantation experiments were performed with STZ-induced diabetic NOD/SCID mice. Blood human C-peptide and glucagon levels were measured serially in mice,and grafts were analyzed histologically. Results We obtained spherical pancreatic beta-like cells from human iPS cells and detected verifiable amounts of C-peptide secretion in vitro. We demonstrated reversal of hyperglycemia in diabetic model mice after transplantation of these cells,maintaining non-fasting blood glucose levels along with the human glycemic set point. We confirmed the secretion of human insulin and glucagon dependent on the blood glucose level in vivo. Immunohistological analysis revealed that grafted cells became $\alpha$,$\beta$ and $\delta$ cells in vivo. Conclusions These results suggest that differentiated cells derived from human iPS cells grown in suspension culture mature and function like pancreatic islets in vivo. View Publication

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue,but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus,fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies,but that of BOECs was lower. In terms of invasiveness of biopsy sampling,biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs,but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative. View Publication

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment,tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors,whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms),suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However,CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically,the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer,tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly,in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion,the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress. View Publication

Small non‐coding microRNAs (miRNAs) play essential roles in Alzheimer's disease (AD) pathogenesis. Repressor element 1‐silencing transcription factor (REST) is involved in AD,though its regulation remains unclear. We performed real‐time quantitative polymerase chain reaction (qPCR) in autopsied brain tissues to determine miR‐153‐3p and AD associations. A reporter‐based assay measured the activity of REST mRNA 3′‐untranslated region (3′‐UTR). Induced pluripotent stem cells (iPSC)‐derived neurons and human cell lines were applied to determine miR‐153‐3p regulation of endogenous proteins. Elevation of miR‐153‐3p is associated with a reduced probability of AD,while elevated REST is associated with a greater probability of AD. The 3′‐UTR functional assay pinpointed the miR‐153‐3p binding sites. miR‐153‐3p treatment reduced REST,amyloid precursor protein (APP),and α‐synuclein (SNCA) 3′‐UTR activities and protein levels. miR‐153‐3p treatment altered REST and neuronal differentiation in iPSC‐derived neuronal stem cells. RNA‐sequencing and proteomics revealed miR‐153‐3p‐associated networks. miR‐153‐3p reduces REST,APP,and SNCA expression,pointing toward its therapeutic and biomarker potential in neurodegenerative diseases. With the increased emphasis on comorbidities of Alzheimer's disease (AD) and other neurodegenerative diseases,we identified that miR‐153‐3p,as a master regulator,reduced a group of neurodegeneration related proteins: REST,amyloid precursor protein (APP) and α‐synuclein (SNCA) levels. The elevation of miR‐153‐3p levels is associated with reduced probability of AD in posterior cingulate cortex (PCC),while REST,by contrast,is associated with a greater probability of AD. miR‐153‐3p treatment alters REST protein levels and neuronal differentiation in induced pluripotent stem cells (iPSC) derived neuronal cells. RNA sequencing proteomics and interactome analysis revealed the role of miR‐153‐3p in axonal guidance. View Publication

5-Ethynyl-2′-deoxyuridine (EdU) has revolutionized DNA replication and cell cycle analyses through fast,efficient click chemistry detection. However,commercial EdU kits suffer from high costs,proprietary formulations,limited antibody multiplexing capabilities,and difficulties with larger biological specimens. Here,we present OpenEMMU (Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics),an optimized,affordable,and user-friendly click chemistry platform utilizing off-the-shelf reagents. OpenEMMU enhances efficiency,brightness,and multiplexing capabilities of EdU staining with both non-conjugated and conjugated antibodies across diverse cell types,including T cell activation and proliferation assays. We validated its effectiveness for the fluorescent imaging of nascent DNA synthesis in developing embryos and organs,including embryonic heart,forelimbs,and 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled the deep-tissue 3D imaging of DNA synthesis in zebrafish larvae and under replication stress in embryos at high spatial resolution. This approach opens new avenues for understanding organismal development,cell proliferation,and DNA replication dynamics with unprecedented precision and flexibility. Subject areas: Biochemistry,Cell biology,Developmental biology,Computational bioinformatics View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication

BACKGROUND Adoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors,but its clinical application has been stalled because of toxicity. Here,we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors. METHODS We generated a cell membrane-anchored IL-12 (aIL12),a tumor-targeted IL-12 (ttIL12),and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells,chimeric antigen receptor-T cells,and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells. RESULTS attIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically,attIL12-T cells targeted tumor cells expressing cell-surface vimentin,enriching effector T cell and interferon $\gamma$ production in tumors,which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects. CONCLUSIONS This novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors. View Publication

Highlights•Preformation of aggregates tuned by cell density enable cultivation of hiPSCs in scale-down shear environments.•Scale-down systems utilizing preformation protocols achieve comparable fold expansion with commercial systems.•Expression of pluripotency markers and functional differentiation capacity is maintained following passage in scale-down culture.•Successful application of hiPSC protocols at < 20 mL scales enable rapid and cost-effective research into cell phenotype under dynamic conditions. Human induced pluripotent stem cell (hiPSC) derived therapeutics require clinically relevant quantities of high-quality cell populations for applications in regenerative medicine. The lack of efficacy exhibited across clinical trials suggests deeper understanding of the networks governing phenotype is needed. Further,costs limit study throughput in characterizing the artificial niche relative to outcomes. We present herein an optimized strategy to enable high-throughput hiPSC expansion at <20 mL research scale. We assessed viability of single cell inoculation and aggregate preformation to facilitate proliferation. We modeled aggregate characteristics against agitation rate. Our results demonstrate tunable control with fold expansion comparable to commercial systems. Marker quantification and teratoma assay confirm functional pluripotency. This approach constitutes a scalable protocol to accelerate hiPSC research,and a significant step in advancing the rate of progress in elucidating links to derivative functionality. This work will enable statistically rigorous studies targeting hiPSC and downstream phenotype for clinical manufacturing. Graphical abstractImplementation of adapted protocols enable scale-down systems as a tool for high-throughput iPSC biomanufacturing research,in platforms conducive to scale-up for clinical manufacturing.Image,graphical abstract View Publication

Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity,their large size often leads to necrotic core formation,limiting reproducibility and challenging the integration of microglia. Here,we present a detailed,reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons,astrocytes,and optionally microglia,all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells,generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling),microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications,including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories,demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes. View Publication

Using a high-throughput chemical screen,we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action,we discovered an essential role of E-cadherin signaling for ESC survival. Specifically,we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling,which then leads to a fatal perturbation of integrin signaling. Furthermore,we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally,we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology,growth factor requirement,and sensitivity to enzymatic cell dissociation. View Publication