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

Melanoma,a potentially lethal skin cancer,is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses,limited knowledge exists on the role of mature B cells. We describe an approach,including a cell-based ELISA,to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (Ptextless0.0001). Interestingly,we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (Ptextless0.0001). Overall,28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly,a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients,which is reduced with disease progression,adding to previous reports of tumor-reactive antibodies in patient sera,and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. View Publication

Realizing the potential of human pluripotent stem cell (hPSC)-based therapy requires the development of defined scalable culture systems with efficient expansion,differentiation and isolation protocols. We report an engineered 3D microfiber system that efficiently supports long-term hPSCs self-renewal under chemically defined conditions. The unique feature of this system lies in the application of a 3D ECM-like environment in which cells are embedded,that affords: (i) uniform high cell loading density in individual cell-laden constructs (∼10 7 cells/ml); (ii) quick recovery of encapsulated cells (textless10min at 37°C) with excellent preservation of cell viability and 3D multicellular structure; (iii) direct cryopreservation of the encapsulated cells in situ in the microfibers with textgreater17-fold higher cell viability compared to those cultured on Matrigel surface; (iv) long-term hPSC propagation under chemically defined conditions. Four hPSC lines propagated in the microfibrous scaffold for 10 consecutive passages were capable of maintaining an undifferentiated phenotype as demonstrated by the expression of stem cell markers and stable karyotype invitro and the ability to form derivatives of the three germ layers both invitro and invivo. Our 3D microfibrous system has the potential for large-scale cultivation of transplantable hESCs and derivatives for clinical applications. textcopyright 2011 Elsevier Ltd. View Publication

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

Long-term management for leukemia is challenging due to the painful and invasive procedure of bone marrow (BM) biopsy. At present,non-invasive liquid (blood) biopsy is not utilized for leukemia,due to lower counts of leukemia blast cells in the blood. Here,we described a robust system for the simultaneous detection and enrichment of rare blast cells. Enrichment of blast cells was achieved from blood with a one-step microfluidic blast cell biochip (BCB) sorting system,without specific targeting of proteins by antibodies. Non-target cells encountered a differential net force as compared to stiffer blast cells and were removed. The efficiency of the BCB promotes high detection sensitivity (1 in 106 cells) even from patients with minimal residual disease. The procedure was validated using actual blast cells from patients with various types of leukemia. Outcomes were compared to current evaluation standards,such as flow cytometry,using BM aspirates. Blast cell detection efficiency was higher in 55.6{\%} of the patients using the BCB as compared to flow cytometry,despite the lower concentrations of blast cells in liquid biopsy. These studies promote early-stage detection and routine monitoring for minimal residual disease in patients. View Publication

Molecular- and cellular-based therapies have the potential to reduce obesity-associated disease. In response to cold,beige adipocytes form in subcutaneous white adipose tissue and convert energy stored in metabolic substrates to heat,making them an attractive therapeutic target. We developed a robust method to generate a renewable source of human beige adipocytes from induced pluripotent stem cells (iPSCs). Developmentally,these cells are derived from FOXF1+ mesoderm and progress through an expandable mural-like mesenchymal stem cell (MSC) to form mature beige adipocytes that display a thermogenically active profile. This includes expression of uncoupling protein 1 (UCP1) concomitant with increased uncoupled respiration. With this method,dysfunctional adipogenic precursors can be reprogrammed and differentiated into beige adipocytes with increased thermogenic function and anti-diabetic secretion potential. This resource can be used to (1) elucidate mechanisms that underlie the control of beige adipogenesis and (2) generate material for cellular-based therapies that target metabolic syndrome in humans. View Publication

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data,while reducing turnover time and costs,and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay,a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes,we sought to introduce standardization steps to the protocol,improving the overall robustness of the PluriLum assay,as well as a shortening of the assay protocol. First,we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm,robust differentiation can be anticipated. In terms of reproducibility,exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data,compared to more reliable concentration–response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol,resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid,both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally,we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter,which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion,we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing. View Publication

Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality,however,continuous expansion of pluripotent cells does not occur in vivo,and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency,the transcription factor Oct-4,in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059,which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2,results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation. View Publication