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

Brain Microphysiological Systems,including neural organoids derived from human induced pluripotent stem cells,offer a unique lens to study the intricate workings of the human brain. This paper investigates the foundational elements of learning and memory in neural organoids by quantifying immediate early gene expression in response to chemical modulation,input-specific short- and long-term synaptic plasticity,neuronal network dynamics,connectivity,and criticality to demonstrate the utility of these organoids in basic science research. Neural organoids showed synapse formation,glutamatergic and GABAergic receptor expression,immediate early gene expression basally and evoked,functional connectivity,criticality,and synaptic plasticity in response to theta-burst stimulation. In addition,pharmacological interventions on GABAergic and glutamatergic receptors and input-specific theta-burst stimulation further shed light on the capacity of neural organoids to mirror synaptic modulation,specifically short- and long-term potentiation and depression,demonstrating their potential as tools for studying neurophysiological and neurological processes and informing therapeutic strategies for diseases. Neural organoids exhibit key aspects of learning and memory,including input-specific synaptic plasticity,basal and evoked immediate early gene expression,and critical network dynamics,highlighting their value in modeling human neurophysiology. View Publication

Suspension bath bioprinting,whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing,has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here,we report a systematic investigation of suspension bath properties that support the printing,fusion,and culture of spheroid-based bioinks without added stabilisation. Specifically,agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150 µ m in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however,the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing,fusion,and culture of spheroid-based inks,either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture. View Publication

Aurora kinases play an important role in chromosome alignment,segregation,and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases,and ZM447439,a potent inhibitor of Aurora kinases,effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152,a highly selective inhibitor of Aurora B kinase,on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60,NB4,MOLM13),ALL line (PALL-2),biphenotypic leukemia (MV4-11),acute eosinophilic leukemia (EOL-1),and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM,as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis,as measured by cell-cycle analysis and annexin V staining,respectively. Of note,AZD1152 synergistically enhanced the antiproliferative activity of vincristine,a tubulin depolymerizing agent,and daunorubicin,a topoisomerase II inhibitor,against the MOLM13 and PALL-2 cells in vitro. Furthermore,AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together,AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation. 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

There is increasing evidence that many cancers,including breast cancer,contain populations of cells that display stem-cell properties. These breast cancer stem cells,by virtue of their relative resistance to radiation and cytotoxic chemotherapy,may contribute to treatment resistance and relapse. The elucidation of pathways that regulate these cells has led to the identification of potential therapeutic targets. A number of agents capable of targeting breast cancer stem cells in preclinical models are currently entering clinical trials. Assessment of the efficacy of the agents will require development of innovative clinical trial designs with appropriate biologic and clinical end points. The effective targeting of breast cancer stem cells has the potential to significantly improve outcome for women with both early-stage and advanced breast cancer. View Publication

The self-renewing epithelium of the small intestine is ordered into stem/progenitor crypt compartments and differentiated villus compartments. Recent evidence indicates that the Wnt cascade is the dominant force in controlling cell fate along the crypt-villus axis. Here we show a rapid,massive conversion of proliferative crypt cells into post-mitotic goblet cells after conditional removal of the common Notch pathway transcription factor CSL/RBP-J. We obtained a similar phenotype by blocking the Notch cascade with a gamma-secretase inhibitor. The inhibitor also induced goblet cell differentiation in adenomas in mice carrying a mutation of the Apc tumour suppressor gene. Thus,maintenance of undifferentiated,proliferative cells in crypts and adenomas requires the concerted activation of the Notch and Wnt cascades. Our data indicate that gamma-secretase inhibitors,developed for Alzheimer's disease,might be of therapeutic benefit in colorectal neoplastic disease. View Publication

Lysophosphatidic acid (LPA) is enriched in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests cancer-associated myofibroblasts play a pivotal role in tumorigenesis through secreting stromal cell-derived factor-1 (SDF-1). In the present study,we demonstrate that LPA induces expression of alpha-smooth muscle actin (alpha-SMA),a marker for myofibroblasts,in human adipose tissue-derived mesenchymal stem cells (hADSCs). The LPA-induced expression of alpha-SMA was completely abrogated by pretreatment of the cells with Ki16425,an antagonist of LPA receptors,or by silencing LPA(1) or LPA(2) isoform expression with small interference RNA (siRNA). LPA elicited phosphorylation of Smad2/3,and siRNA-mediated depletion of endogenous Smad2/3 or adenoviral expression of Smad7,an inhibitory Smad,abrogated the LPA induced expression of alpha-SMA and phosphorylation of Smad2/3. LPA-induced secretion of transforming growth factor (TGF)-beta1 in hADSCs,and pretreatment of the cells with SB431542,a TGF-beta type I receptor kinase inhibitor,or anti-TGF-beta1 neutralizing antibody inhibited the LPA-induced expression of alpha-SMA and phosphorylation of Smad2. Furthermore,ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of alpha-SMA and phosphorylation of Smad2,and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of alpha-SMA and phosphorylation of Smad2. In addition,LPA increased the expression of SDF-1 in hADSCs,and pretreatment of the cells with Ki16425 or SB431562 attenuated the LPA-stimulated expression of SDF-1. These results suggest that cancer-derived LPA stimulates differentiation of hADSCs to myofibroblast-like cells and increases SDF-1 expression through activating autocrine TGF-beta1-Smad signaling pathway. View Publication

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of $$4$$7 and CCR9 by Peyer's patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid-dependent manner. This paradigm,however,cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration,we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly,we found that lung DCs,like CD103(+) MLN DCs,up-regulate the gut-homing integrin $$4$$7 in vitro and in vivo,and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses,lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs,which has the potential to inform the design of novel vaccines against mucosal pathogens. View Publication

The incidence of cardiovascular disease represents a significant and growing health-care challenge to the developed and developing world. The ability of native heart muscle to regenerate in response to myocardial infarct is minimal. Tissue engineering and regenerative medicine approaches represent one promising response to this difficulty. Here,we present methods for the construction of a cell-seeded cardiac patch with the potential to promote regenerative outcomes in heart muscle with damage secondary to myocardial infarct. This method leverages iPS cells and a fibrin-based scaffold to create a simple and commercially viable tissue-engineered cardiac patch. Human-induced pluripotent stem cells (hiPSCs) can,in principle,be differentiated into cells of any lineage. However,most of the protocols used to generate hiPSC-derived endothelial cells (ECs) and cardiomyocytes (CMs) are unsatisfactory because the yield and phenotypic stability of the hiPSC-ECs are low,and the hiPSC-CMs are often purified via selection for expression of a promoter-reporter construct. In this chapter,we describe an hiPSC-EC differentiation protocol that generates large numbers of stable ECs and an hiPSC-CM differentiation protocol that does not require genetic manipulation,single-cell selection,or sorting with fluorescent dyes or other reagents. We also provide a simple but effective method that can be used to combine hiPSC-ECs and hiPSC-CMs with hiPSC-derived smooth muscle cells to engineer a contracting patch of cardiac cells. View Publication

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication