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

Teratogens,substances that may cause fetal abnormalities during development,are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here,we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity,leading to better prediction of teratogenicity. In particular,our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition,this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity,where it correctly predicted the teratogenicity for seven of the eight drugs. Thus,our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways. ?? 2010 Elsevier Inc. View Publication

Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible,cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05[x02013]2x105 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo,but after isolation can be expanded considerably,generating clinically useful numbers of cells. In this study,we demonstrate the use of a defined embryonic stem cell expansion medium,mTeSR (Stem Cell Technologies),for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates,demonstrate tri-lineage differentiation potential,and show similar surface marker profiles (CD29(+),CD44(+),CD49a(+),CD73(+),CD90(+),CD105(+),CD146(+),CD166(+),CD34(-),and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However,expression of early differentiation transcription factors runt-related transcription factor 2,sex-determining region Y box 9,and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated,whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS,adipogenic differentiation was significantly decreased in mTeSR-expanded cells,both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further,this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs,such as bone repair and cartilage generation. View Publication

BackgroundHigh densities of tertiary lymphoid structures (TLSs) are associated with improved clinical outcomes in various malignancies,including human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). However,the role of TLSs in shaping antitumor immunity in HPV-induced cervical cancer (CESC) remains unclear. Therefore,we analyzed the density,composition,and prognostic impact of TLSs in patients with CESC as well as patients with HNSCC.MethodsMultiplex immunofluorescence,immunohistochemistry,and spatial transcriptomics were used to analyze TLS density and composition in HNSCC and CESC tissue sections with respect to patient prognosis. The spatial approach was supplemented by flow cytometry-based analysis of the polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) phenotype in freshly resected primary tumor tissues.ResultsAlthough both indications were associated with HPV infection,we confirmed a positive correlation between TLS density and improved overall survival only in patients with HNSCC. The TLS composition differed markedly between HNSCC and CESC samples,with a shift toward high regulatory T cell (Treg) and PMN-MDSC abundance in CESC samples. The highest Treg and PMN-MDSC levels were observed in patients with CESC who died of the disease. CESC-infiltrating PMN-MDSCs showed high arginase 1 expression,which correlated with diminished T-cell receptor (TCR)ζ chain expression in CESC-infiltrating T cells. Additionally,the high number of PMN-MDSCs in TLSs was associated with the absence of HPV-specific T cells in CESC.ConclusionsUnlike in HNSCC,the composition of TLSs,rather than their quantity,was associated with the overall survival of patients with CESC. High numbers of Tregs and PMN-MDSCs infiltrating immature TLSs prevail in patients with CESC who succumbed to the disease and seem to affect tumor-specific immune responses. View Publication

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach,often involving the use of viral vectors. Electroporation using square-wave generating devices,like Lonza's Nucleofector,is a widely used option,but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work,we show that our in-house developed buffers,termed Chicabuffers,can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device,we electroporated 14 different cell lines and also primary cells,like mesenchymal stem cells and cord blood CD34+,providing optimized protocols for each of them. Moreover,when combined with sleeping beauty-based transposon system,long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells,facilitating the widespread adoption of this technology. View Publication

Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced,AML remains a therapeutic challenge with insufficient cure rates. In the last years,immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced,which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However,the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here we report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression,unimpeded proliferation,and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore,CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects. View Publication

A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene,which was readily identified because of its high degree of homology to the gibbon sequence,also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells,proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3. View Publication

The present study describes and compares functional properties of Nuli-1 cells and primary human nasal epithelial cells (HNEC) including TLR expression and function. Differences in gene expression were identified for non-TLR genes that play a role in TLR response pathways. However,experiments comparing TLR gene expression for both Nuli-1 cells and HNECs indicated conserved expression in both cell types. Stimulation of the two cell types resulted in a conserved response to TLR3 agonists,but in differences in response to agonists for TLR5 and TLR6/2. HNECs were much more susceptible to infection with Staphylococcus aureus than NuLi-1 cells. Furthermore,when cultured at air-liquid interface (ALI),NuLi-1 cells possessed much lower trans-epithelial resistance than primary HNEC and did not exhibit maintenance of cell morphology or mucous production which was observed in HNECs. Nor did they produce the characteristic interconnecting pattern of tight junction complexes at the apicolateral margin of adjacent cells. Caution should therefore be exercised when selecting cell lines for immunological studies and a thorough screen of properties relevant to the study should always be carried out prior to commencement. View Publication

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation,two hESCs lines were cultured on mixed feeder cells (MFCs,MEFs: HFFs = 1:1) and HFFs feeder,respectively,and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry,quantitative fluorescent real-time PCR,transmission and scanning electron microscopy,and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However,compared to hESCs line on MFCs feeder,hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2,PITX3,NURR1,and TH genes. In addition,the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion,HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons,but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore,feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines,but also electrophysiological properties of hESCs-derived DA neurons. View Publication

Biallelic mutations in PIGK cause GPI biosynthesis defect 22 (GPIBD22),characterized with developmental delay,hypotonia,and cerebellar atrophy. The understanding of the underlying causes is limited due to the lack of suitable disease models. To address this gap,we generated a mouse model with PIGK deficits,specifically in Purkinje cells (Pcp2-cko) and an induced pluripotent stem cell (iPSC) model using the c.87dupT mutant (KI) found in GPIBD22 patients. Pcp2-cko mice demonstrated cerebellar atrophy,ataxia and progressive Purkinje cells loss which were accompanied by increased apoptosis and neuroinflammation. Similarly,KI iPSCs exhibited increased apoptosis and accelerated neural rosette formation,indicating that PIGK defects could impact early neural differentiation that confirmed by the RNA-Seq results of neural progenitor cells (NPCs). The increased apoptosis and accelerated NPC differentiation in KI iPSCs are associated with excessive unfolded protein response (UPR) pathway activation,and can be rescued by UPR pathway inhibitor. Our study reveals potential pathogenic mechanism of GPIBD22 and providing new insights into the therapeutic strategy for GPIBD. View Publication