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

SummaryA strong interest in drugs targeting the tumor microenvironment (TME) necessitates new experimental systems that incorporate key TME components. Compared to traditional 2D cell lines,3D ex vivo spheroids from patient-derived xenograft (PDX) materials may better capture patient tumor characteristics. We developed and validated a 3D tumor spheroid model from non-small cell lung cancer (NSCLC) PDXs to enable T cell infiltration. Histologic and transcriptomic analysis suggested that tumor spheroids closely recapitulate the source PDX tumor tissues. Consistent T cell infiltration into tumor spheroids was achieved using a well-established magnetic nanoparticle technology,which maintained T cell function and tumor-killing activity. Drug treatment studies with immunotherapy agents also demonstrated the potential scalability of 3D tumor-T cell spheroids in assessing drug activity,including tumor viability and cytokine secretion. This platform provides a useful tool for evaluating drug candidates that can be translated to patient tumor responses related to both tumor intrinsic and TME factors. Graphical abstract Highlights•We developed a 3D tumor spheroid model from lung cancer patient-derived xenografts•The model enabled robust T cell infiltration and preserved T cell cytotoxic functions•Histology and RNA-seq showed that tumor spheroids closely resembled source tumors•Proof-of-concept experiments showed this platform’s utility in preclinical drug testing Biological sciences; Biotechnology; Natural sciences; Tissue Engineering View Publication

In the present study,we combined stem cell technology with a non-absorbable biomaterial for the reconstruction of the ruptured ACL. Towards this purpose,multipotential stromal cells derived either from subcutaneous human adipose tissue (hAT-MSCs) or from induced pluripotent stem cells (iPSCs) generated from human foreskin fibroblasts (hiPSC-MSCs) were cultured on the biomaterial for 21days in vitro to generate a 3D bioartifical ACL graft. Stem cell differentiation towards bone and ligament at the ends and central part of the biomaterial was selectively induced using either BMP-2/FGF-2 or TGF-β/FGF-2 combinations,respectively. The bioartificial ACL graft was subsequently implanted in a swine ACL rupture model in place of the surgically removed normal ACL. Four months post-implantation,the tissue engineered ACL graft generated an ACL-like tissue exhibiting morphological and biochemical characteristics resembling those of normal ACL. View Publication

Tunneling nanotubes (TNTs) are ultrafine,filamentous actin-based cytoplasmic extensions which form spontaneously to connect cells at short and long-range distances. We have previously described long-range intercellular communication via TNTs connecting mesothelioma cells in vitro and demonstrated TNTs in intact tumors from patients with mesothelioma. Here,we investigate the ability of TNTs to mediate a viral thymidine kinase based bystander effect after oncolytic viral infection and administration of the nucleoside analog ganciclovir. Using confocal microscopy we assessed the ability of TNTs to propagate enhanced green fluorescent protein (eGFP),which is encoded by the herpes simplex virus NV1066,from infected to uninfected recipient cells. Using time-lapse imaging,we observed eGFP expressed in infected cells being transferred via TNTs to noninfected cells; additionally,increasing fluorescent activity in recipient cells indicated cell-to-cell transmission of the eGFP-expressing NV1066 virus had also occurred. TNTs mediated cell death as a form of direct cell-to-cell transfer following viral thymidine kinase mediated activation of ganciclovir,inducing a unique long-range form of the bystander effect through transmission of activated ganciclovir to nonvirus-infected cells. Thus,we provide proof-of-principle demonstration of a previously unknown and alternative mechanism for inducing apoptosis in noninfected recipient cells. The conceptual advance of this work is that TNTs can be harnessed for delivery of oncolytic viruses and of viral thymidine kinase activated drugs to amplify the bystander effect between cancer cells over long distances in stroma-rich tumor microenvironments. View Publication

UNLABELLED Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within central nervous system (CNS) glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved,infection of NG2 glia was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs),we examined here whether their infection by neurovirulent (FrCasE) or nonneurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Here,we demonstrate that OPCs,but not OLs,are major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture did not affect survival,proliferation,or OPC progenitor marker expression but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that,while MLVs did not affect cellular engraftment or survival,they did inhibit OL differentiation,irrespective of MLV neurovirulence. In addition,in chimeric brains,where FrCasE-infected NPC transplants caused neurodegeneration,the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation but only FrCasE induces overt neurodegeneration,restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitates disease. IMPORTANCE A variety of human and animal retroviruses are capable of causing central nervous system (CNS) neurodegeneration manifested as motor and cognitive deficits. These retroviruses infect a variety of CNS cell types; however,the specific role each cell type plays in neuropathogenesis remains to be established. The NG2 glia,whose CNS functions are only now emerging,are a newly appreciated viral target in murine leukemia virus (MLV)-induced neurodegeneration. Since one role of NG2 glia is that of oligodendrocyte progenitor cells (OPCs),we investigated here whether their infection by the neurovirulent MLV FrCasE contributed to neurodegeneration by affecting OPC viability and/or development. Our results show that both neurovirulent and nonneurovirulent MLVs interfere with oligodendrocyte differentiation. Thus,NG2 glial infection could contribute to neurodegeneration by preventing myelin formation and/or repair and by suspending OPCs in a state of persistent susceptibility to excitotoxic insult mediated by neurovirulent virus effects on other glial subtypes. View Publication

Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised,thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently,the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator,phosphoinositide-dependent protein kinase-1 (PDK1). By using a multidisciplinary approach including molecular modelling,classical biochemical assays,and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM),a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor) and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore,these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs. View Publication

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement),two SOPs were assessed,by using two cell culture media (Test A,containing GM-CSF; and Test B,containing G-CSF,GM-CSF,IL-3,IL-6 and SCF),and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer),the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance),dedicated to a training set of six anticancer drugs (adriamycin,flavopindol,morpholino-doxorubicin,pyrazoloacridine,taxol and topotecan),a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions,and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs,respectively,that were selected as prototype xenobiotics. As expected,both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B,without any loss of performance or predictive accuracy. On the basis of these results,we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential. View Publication

The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1,ANXA11,CST4,PRDX5,PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1,ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. 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

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

The human pre-T-cell receptor alpha (TCRalpha; pTalpha) gene encodes a polypeptide which associates with the TCRbeta chain and CD3 molecules to form the pre-TCR complex. The surface expression of the pre-TCR is pTalpha dependent,and signaling through this complex triggers an early alphabeta T-cell developmental checkpoint inside the thymus,known as beta-selection. E2A transcription factors,which are involved at multiple stages of T-cell development,regulate the transcription of the pTalpha gene. Here we show that the regulatory protein Tax of the human T-cell leukemia virus type 1 (HTLV-1) efficiently suppresses the E47-mediated activation of the pTalpha promoter. Furthermore,we report that in Tax lentivirally transduced human MOLT-4 T cells,which constitutively express the pTalpha gene,the amount of pTalpha transcripts decreases. Such a decrease is not observed in MOLT-4 cells transduced by a vector encoding the Tax mutant K88A,which is unable to interact with p300. These data underline that Tax inhibits pTalpha transcription by recruiting this coactivator. Finally,we show that the expression of Tax in human immature thymocytes results in a decrease of pTalpha gene transcription but does not modify the level of E47 transcripts. These observations indicate that Tax,by silencing E proteins,down-regulates pTalpha gene transcription during early thymocyte development. They further provide evidence that Tax can interfere with an important checkpoint during T-cell differentiation in the thymus. 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