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

Genome‐wide association studies (GWAS) have identified APOE2 allele as linked to exceptional longevity,with carriers exhibiting a reduced risk of Alzheimer's disease (AD). Apolipoprotein E (APOE),a glycoprotein involved in lipid transport,has three major alleles. However,alterations in lipid metabolism alone do not fully explain APOE2's protective effects. In contrast,APOE4 is the strongest genetic risk factor for AD. To investigate how APOE2 promotes neuronal longevity and confers neuroprotection,we generated human isogenic APOE iPSC‐derived models of both inhibitory GABAergic and excitatory neurons. In GABAergic neurons,APOE alleles differentially influenced endogenous DNA damage,DNA repair,and neuronal motility. Single‐cell RNA sequencing revealed APOE4‐specific gene expression signatures associated with AD,whereas APOE2 GABAergic neurons were enriched for DNA repair and signaling pathways. Consistent with this,APOE2 neurons exhibited significantly lower levels of DNA damage. APOE4 GABAergic neurons exhibit increased expression of repetitive ribosomal RNA,which is associated with DNA damage and cellular senescence. To determine whether the effects extended to excitatory neurons,we used a separate human model of Ngn2‐induced glutamatergic neurons,and found that APOE2 excitatory neurons were more resistant to cellular senescence and DNA damage than isogenic APOE3 and APOE4 neurons. Similarly,we found human APOE2‐targeted replacement mice exhibited less nucleolar enlargement and increased nuclear Lamin A/C,Hmgb1,and H3K9me3 compared to APOE4 counterparts. Together,our findings identify DNA repair and suppression of senescence‐associated processes as key mechanisms by which APOE2 is associated with neuronal resilience,providing mechanistic insight into its association with exceptional longevity and protection against AD. Neurons expressing APOE2 were more resistant to endogenous DNA damage,activated transcriptional signaling pathways associated with DNA repair,and were resilient to stress‐induced DNA damage and cellular senescence. In contrast,APOE4 neurons exhibited elevated expression of rRNA repetitive elements and were prone to becoming senescent. View Publication

High expression of the Breast Cancer Resistance Protein (BCRP) gene has been shown to be involved in resistance to chemotherapeutic drugs. Knowledge of the localization of BCRP protein in normal tissues may help unravel the normal function of this protein. Therefore,we characterized the tissue distribution and cellular localization of BCRP in frozen sections of normal human tissues. For this purpose,we used the recently described monoclonal antibody BXP-34 and another independently developed monoclonal antibody directed against BCRP,BXP-21. Both monoclonal antibodies show specific BCRP plasma membrane staining on cytospins obtained from topotecan- or mitoxantrone-selected cell lines,as well as from BCRP-transfected cell lines. Immunoprecipitation experiments using either BXP-21 or BXP-34 yielded a clear M(r) 72,000 BCRP band from BCRP-overexpressing tumor cells. In the topotecan-selected T8 and mitoxantrone-selected MX3 tumor cell lines,BCRP turned out to be differentially glycosylated. In contrast to BXP-34,BXP-21 is able to detect the M(r) 72,000 BCRP protein on immunoblots and is reactive with BCRP in formalin-fixed,paraffin-embedded tissues. Using BXP-21 and BXP-34,prominent staining of BCRP was observed in placental syncytiotrophoblasts,in the epithelium of the small intestine and colon,in the liver canalicular membrane,and in ducts and lobules of the breast. Furthermore,BCRP was present in veinous and capillary endothelium,but not in arterial endothelium in all of the tissues investigated. In the tissues studied,the mRNA levels of BCRP were assessed using reverse transcription-PCR,and these corresponded with the levels of BCRP protein estimated from immunohistochemical staining. The presence of BCRP at the placental syncytiotrophoblasts is consistent with the hypothesis of a protective role of BCRP for the fetus. The apical localization in the epithelium of the small intestine and colon indicates a possible role of BCRP in the regulation of the uptake of p.o. administered BCRP substrates by back-transport of substrate drugs entering from the gut lumen. Therefore,it may be useful to attempt to modulate the uptake of p.o. delivered BCRP substrates,e.g.,topotecan or irinotecan,by using a BCRP inhibitor. Clinical trials testing this hypothesis have been initiated in our institute. View Publication

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here,we provide a detailed characterization of RG108,a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly,RG108 caused demethylation and reactivation of tumor suppressor genes,but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation. View Publication

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV),another member of the same subfamily. hMPV causes respiratory tract illnesses that,similar to human RSV,occur predominantly during the winter months and have symptoms that range from mild to severe cough,bronchiolitis,and pneumonia. Like RSV,the hMPV virus can be subdivided into two genetic subgroups,A and B. With RSV,a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups,this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV. View Publication

BACKGROUND Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC),particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies,and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct,the authors sought to determine whether differences in the number of CSCs could account for this clinical observation. METHODS CSC populations in HPV16-positive and HPV-negative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH),an in vitro tumorsphere formation assay,and an in vivo limiting cell dilution in nonobese diabetic/severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1,a CSC marker,to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC. RESULTS HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining,further extending the association between HPV16 and CSCs. CONCLUSIONS The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore,the reliance on the CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression. View Publication

The complement activation product,C5a,is a pivotal member of the innate immune response; however,a diverse number of nonimmune functions are now being ascribed to C5a signaling,including roles during embryonic development. Here,we identify the expression of the C5a precursor protein,C5,as well as the C5a receptors,C5aR and C5L2,in both human embryonic stem cells and human-induced pluripotent stem cells. We show that administration of a physiologically relevant dose of purified human C5a (1 nM) stimulates activation of ERK1/2 and AKT signaling pathways,and is able to promote maintenance of the pluripotent state in the absence of FGF2. C5a also reduced cell loss following dissociation of human pluripotent stem cells. Our results reveal that complement C5a signaling supports human stem cell pluripotency and survival,and thus may play a key role in shaping early human embryonic development. Stem Cells 2014;32:3278-3284. View Publication

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation,but they often clump in culture,which has always represented a challenge for neurodifferentiation. In this study,we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO2,which doubled the expression of the NESTIN,PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore,an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the neurosphederm". The large neural tube-type rosette (NTTR) structure formed from the neurosphederm� View Publication

RATIONALE: Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized,the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes. OBJECTIVE: Our aim was to develop a human embryonic stem cell-based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis. METHODS AND RESULTS: Approximately 550 known pathway modulators were screened in a high-content screening assay,with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell,which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell-derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably,several other Wnt inhibitors were very efficient in inducing cardiogenesis,including a molecule that prevents Wnts from being secreted by the cell,which confirmed that Wnt inhibition was the relevant biological activity. CONCLUSIONS: Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications. View Publication

Copy number variation (CNV) is a common chromosomal alteration that can occur during in vitro cultivation of human cells and can be accompanied by the accumulation of mutations in coding region sequences. We describe here a systematic application of current molecular technologies to provide a detailed understanding of genomic and sequence profiles of human embryonic stem cell (hESC) lines that were derived under GMP-compliant conditions. We first examined the overall chromosomal integrity using cytogenetic techniques to determine chromosome count,and to detect the presence of cytogenetically aberrant cells in the culture (mosaicism). Assays of copy number variation,using both microarray and sequence-based analyses,provide a detailed view genomic variation in these lines and shows that in early passage cultures of these lines,the size range and distribution of CNVs are entirely consistent with those seen in the genomes of normal individuals. Similarly,genome sequencing shows variation within these lines that is completely within the range seen in normal genomes. Important gene classes,such as tumor suppressors and genetic disease genes,do not display overtly disruptive mutations that could affect the overall safety of cell-based therapeutics. Complete sequence also allows the analysis of important transplantation antigens,such as ABO and HLA types. The combined application of cytogenetic and molecular technologies provides a detailed understanding of genomic and sequence profiles of GMP produced ES lines for potential use as therapeutic agents. View Publication

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437),originally identified as a retinoic acid receptor gamma-selective retinoid,was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study,we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis,a process that is accompanied by rapid induction of c-Jun,nur77,and p21(WAF1/CIP1). In addition,we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein,a dominant-negative inhibitor of c-Jun,in A549 cells,nur77 expression and apoptosis induction by AHPN/CD437 were impaired,whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore,overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus,expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together,our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer. View Publication

Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively,an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with β5i and β6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells,and to a lesser extent in activated human T cells. Reversible,β5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells. View Publication

BACKGROUND AIMS microRNAs (miRNAs) are small non-coding RNAs that bind to 3'UTR regions of mRNAs to promote their degradation or block their translation. Mice with disruption of the trefoil factor 1 gene (Tff1) develop gastric neoplasms. We studied these mice to identify conserved miRNA networks involved in gastric carcinogenesis. METHODS We performed next-generation miRNA sequencing analysis of normal gastric tissues (based on histology) from subjects without evidence of gastric neoplasm from patients (n=64) and TFF1-knockout mice (n=22). We validated our findings using 270 normal gastric tissues (including 61 samples from patients without evidence of neoplastic lesions) and 234 gastric tumor tissues from 3 separate cohorts of patients and from mice. We performed molecular and functional assays using cell lines (MKN28,MKN45,STKM2,and AGS cells),gastric organoids,and mice with xenograft tumors. RESULTS We identified 117 miRNAs that were significantly deregulated in mouse and human gastric tumor tissues,compared with non-tumor tissues. We validated changes in levels of 6 miRNAs by quantitative real-time PCR analyses of neoplastic gastric tissues from mice (n=39) and 3 independent cohorts patients (332 patients total). We found levels of MIR135B-5p,MIR196B-5p,and MIR92A-5p to be increased in tumor tissues whereas levels of MIR143-3p,MIR204-5p,and MIR133-3p were decreased in tumor tissues. Levels of MIR143-3p were reduced not only in gastric cancer tissues,but also in normal tissues adjacent to tumors in humans and low-grade dysplasia in mice. Transgenic expression of MIR143-3p in gastric cancer cell lines reduced their proliferation and restored their sensitivity to cisplatin. AGS cells with stable transgenic expression of MIR143-3p grew more slowly as xenograft tumors in mice than control AGS cells; tumor growth from AGS cells that expressed MIR143-3p,but not control cells,was sensitive to cisplatin. We identified and validated bromodomain containing 2 (BRD2) as a direct target of MIR143-3p; increased levels of BRD2 in gastric tumors associated with shorter survival times of patients. CONCLUSIONS In an analysis of miRNA profiles of gastric tumors from mice and human patients,we identified a conserved signature associated with early stages of gastric tumorigenesis. Strategies to restore MIR143-3p or inhibit BRD2 might be developed for treatment of gastric cancer. View Publication