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

Development and lineage choice are driven by interconnected transcriptional,epigenetic and metabolic changes. Specific metabolites,such as α-ketoglutarate (αKG),function as signalling molecules affecting the activity of chromatin-modifying enzymes. However,how metabolism coordinates cell-state changes,especially in human pre-implantation development,remains unclear. Here we uncover that inducing naive human embryonic stem cells towards the trophectoderm lineage results in considerable metabolic rewiring,characterized by αKG accumulation. Elevated αKG levels potentiate the capacity of naive embryonic stem cells to specify towards the trophectoderm lineage. Moreover,increased αKG levels promote blastoid polarization and trophectoderm maturation. αKG supplementation does not affect global histone methylation levels; rather,it decreases acetyl-CoA availability,reduces histone acetyltransferase activity and weakens the pluripotency network. We propose that metabolism functions as a positive feedback loop aiding in trophectoderm fate induction and maturation,highlighting that global metabolic rewiring can promote specificity in cell fate decisions through intricate regulation of signalling and chromatin. Subject terms: Embryonic stem cells,Embryology View Publication

Anti-interleukin (IL)-4Rα monoclonal antibodies (mAb) improve lung function and decrease the number of exacerbations in patients with COPD type (T)2 inflammation. However,the involvement of early innate immune responses underlying these treatment effects is not well known. We sought to understand the effect and mechanisms of IL-4Rα mAb treatment on bronchial epithelial cells (BECs) from COPD patients under T2 inflammatory conditions with and without rhinoviral infection. Primary BECs from healthy and COPD patients were grown at an air–liquid interface and stimulated with IL-4 or IL-13 cytokines in the presence of IL-4Rα mAb. Cells were infected with human rhinovirus 1B and collected 24 h after infection. Antiviral mediators ( i.e.,interferons (IFNs) and pattern recognition receptors (PRRs)),as well as chemokine and alarmin expression,were measured by reverse transcriptase quantitative PCR and ELISA. Treatment with IL-4Rα mAb (100 nM) inhibited the eotaxin-3 (CCL26) gene after IL-4/IL-13 induction (p<0.05) in COPD BECs. However,no significant changes in rhinovirus-induced IFN-β,PRRs or thymic stromal lymphopoietin gene responses were observed with IL-4/IL-13 stimulation and IL-4Rα mAb treatment. A significant increase in mucin 5AC gene expression was observed with both IL-4 and IL-13 stimulation,but it was not reduced with IL-4Rα treatment in BECs. Inhibition of IL-4Rα reduced CCL26 levels without affecting antiviral immune responses in BECs from COPD patients. Inhibition of IL-4Rα reduced IL-4/IL-13 signalling without broadly suppressing the immune system,which might suggest that inhibition of the IL-4Rα pathways may prevent COPD exacerbations through reduction of eosinophil chemotaxis. View Publication

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the uncontrolled proliferation of white blood cells. Tyrosine kinase inhibitors (TKIs) are the standard treatment; however,resistance to BCR::ABL1 mutations remains challenging. WEE1,a checkpoint kinase involved in mitosis and DNA repair,is a potential therapeutic target for CML treatment. Ponatinib-resistant CML cells were screened to identify candidates for overcoming drug resistance. The efficacy of the ABL TKI asciminib and the WEE1 inhibitor MK-1775 was evaluated using proliferation and colony formation assays. Public database analysis ( GSE100026 ) assessed WEE1/PKMYT1 expression in CML. In vitro screening identified MK-1775 as a promising therapeutic candidate. WEE1/PKMYT1 expression was elevated in CML cells compared to healthy cells. Both asciminib and MK-1775 inhibited CML cell proliferation after 72 h,with enhanced cytotoxicity when combined. Co-treatment reduced colony formation and induced G2/M arrest,whereas an increase in the sub-G1 cell population indicated apoptosis. Furthermore,the combination treatment disrupted the mitochondrial membrane potential. The combination of asciminib and WEE1 inhibition demonstrated greater efficacy than either drug alone,suggesting a novel therapeutic strategy for treating CML. These findings provide insights into overcoming TKI resistance and highlight a promising approach for future clinical applications. The online version contains supplementary material available at 10.1007/s12672-025-03036-7. View Publication

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation,high levels of enucleation (80-90%) and low levels of cell death (textless10%) are achieved. We compared the yield of erythroblasts obtained from PBMC,CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available. View Publication

BACKGROUND: 1,25-Dihydroxyvitamin D(3) inhibits growth of several types of human cancer cells in vitro,but its therapeutic use is hampered because it causes hypercalcemia. 19-nor-1,25-Dihydroxyvitamin D(2) (paricalcitol) is a noncalcemic vitamin D analog that is approved by the Food and Drug Administration for the treatment of secondary hyperparathyroidism. We investigated the antitumor activity and mechanism of action of paricalcitol in vitro and in vivo. METHODS: Effects of paricalcitol on proliferation,the cell cycle,differentiation,and apoptosis were examined in cancer cell lines. Effects on tumor growth were examined with colon cancer cell xenografts in nude mice (five in the experimental group and five in the control group). The interaction of paricalcitol with the vitamin D receptor (VDR) in mononuclear spleen cells and myeloid stem cells from wild-type and VDR knockout mice was examined. All statistical tests were two-sided. RESULTS: Paricalcitol inhibited the proliferation of myeloid leukemia cell lines HL-60,NB-4,and THP-1 cells at an effective dose that inhibited growth 50% (ED(50)) of 2.4-5.8 x 10(-9) M by inducing cell cycle arrest and differentiation. Paricalcitol inhibited the proliferation of NCI-H929 myeloma cells at an ED(50) of 2.0 x 10(-10) M by inducing cell cycle arrest and apoptosis. Paricalcitol also inhibited the proliferation of colon cancer cell lines HT-29 (ED(50) = 1.7 x 10(-8) M) and SW837 (ED(50) = 3.2 x 10(-8) M). HT-29 colon cancer xenografts in paricalcitol-treated nude mice were smaller (1044 mm(3) and 1752 mm(3),difference = 708 mm(3),95% confidence interval = 311 to 1104 mm(3); P =.03) and weighed less (1487 mg and 4162 mg,difference = 2675 mg,95% confidence interval = 2103 to 3248 mg; Ptextless.001) than those in vehicle-treated mice. Paricalcitol induced committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate as macrophages. CONCLUSION: Paricalcitol has anticancer activity against myeloid leukemia,myeloma,and colon cancer cells that may be mediated through the VDR. Because it has been approved by the Food and Drug Administration,clinical trials of this agent in certain cancers are reasonable. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors,transformed cell lines,and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves,as has been seen in a number of human breast cancers,or through mutation of intermediates in the Wnt pathway,such as adenomatous polyposis coli or beta-catenin,as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development,but overexpression of certain Wnts,such as Wnt-1,leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG,morphological changes and increased proliferation are accompanied by increased levels of CK2,as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins,which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin,Dvl-2,and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells,abrogates phosphorylation of beta-catenin,and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. View Publication

The development of dengue antivirals and vaccine has been hampered by the incomplete understanding of molecular mechanisms of dengue virus (DENV) infection and pathology,partly due to the limited suitable cell culture or animal models that can capture the comprehensive cellular changes induced by DENV. In this study,we differentiated human pluripotent stem cells (hPSCs) into hepatocytes,one of the target cells of DENV,to investigate various aspects of DENV-hepatocyte interaction. hPSC-derived hepatocyte-like cells (HLCs) supported persistent and productive DENV infection. The activation of interferon pathways by DENV protected bystander cells from infection and protected the infected cells from massive apoptosis. Furthermore,DENV infection activated the NF-$$B pathway,which led to production of proinflammatory cytokines and downregulated many liver-specific genes such as albumin and coagulation factor V. Our study demonstrates the utility of hPSC-derived hepatocytes as an in vitro model for DENV infection and reveals important aspects of DENV-host interactions. View Publication

A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects,we first established a liver organoid using human induced pluripotent stem cells (iPSCs),mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids,resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after in vivo organoid transplantation. To assess the paracrine contributions,we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form,their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs,resulting in the expression of bile salt export pump. In conclusion,the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids,but organoid self-organization requires cell-to-cell surface contact. Our in vitro model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes. View Publication

In the central nervous system,neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner,suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist,we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice,GFP expression was restricted to undifferentiated cells in the VZ,which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture,indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development. View Publication

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells,capable of germline transmission,from mouse somatic cells by transduction of four defined transcription factors. Here,we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4,and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology,proliferation,surface antigens,gene expression,epigenetic status of pluripotent cell-specific genes,and telomerase activity. Furthermore,these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. View Publication