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

Facile and sensitive detection and isolation of circulating tumor cells (CTCs) was achieved using the aptamer-targeted magnetic nanoparticles (Apt-MNPs) in conjugation with a microfluidic device. Apt-MNPs were developed by the covalent attachment of anti-MUC1 aptamer to the silica-coated magnetic nanoparticles via the glutaraldehyde linkers. Apt-MNPs displayed high stability and functionality after 6 months of storage at 4 °C. The specific microfluidic device consisting of mixing,sorting and separation modules was fabricated through conventional photo- and soft-lithography by using polydimethylsiloxane. The capture efficiency of Apt-MNPs was first studied in vitro on MCF-7 and MDA-MB-231 cancer cell lines in the bulk and microfluidic platforms. The cell capture yields of more than 91% were obtained at the optimum condition after 60 minutes of exposure to 50 $\mu$g mL-1 Apt-MNPs with 10 to 106 cancer cells in different media. CTCs were also isolated efficiently from the blood samples of breast cancer patients and successfully propagated in vitro. The isolated CTCs were further characterized using immunofluorescence staining. The overall results indicated the high potential of the present method for the detection and capture of CTCs. View Publication

BackgroundSETBP1 Haploinsufficiency Disorder (SETBP1-HD) is characterised by mild to moderate intellectual disability,speech and language impairment,mild motor developmental delay,behavioural issues,hypotonia,mild facial dysmorphisms,and vision impairment. Despite a clear link between SETBP1 mutations and neurodevelopmental disorders the precise role of SETBP1 in neural development remains elusive. We investigate the functional effects of three SETBP1 genetic variants including two pathogenic mutations p.Glu545Ter and SETBP1 p.Tyr1066Ter,resulting in removal of SKI and/or SET domains,and a point mutation p.Thr1387Met in the SET domain.MethodsGenetic variants were introduced into induced pluripotent stem cells (iPSCs) and subsequently differentiated into neurons to model the disease. We measured changes in cellular differentiation,SETBP1 protein localisation,and gene expression changes.ResultsThe data indicated a change in the WNT pathway,RNA polymerase II pathway and identified GATA2 as a central transcription factor in disease perturbation. In addition,the genetic variants altered the expression of gene sets related to neural forebrain development matching characteristics typical of the SETBP1-HD phenotype.LimitationsThe study investigates changes in cellular function in differentiation of iPSC to neural progenitor cells as a human model of SETBP1 HD disorder. Future studies may provide additional information relevant to disease on further neural cell specification,to derive mature neurons,neural forebrain cells,or brain organoids.ConclusionsWe developed a human SETBP1-HD model and identified perturbations to the WNT and POL2RA pathway,genes regulated by GATA2. Strikingly neural cells for both the SETBP1 truncation mutations and the single nucleotide variant displayed a SETBP1-HD-like phenotype.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13229-024-00625-1. View Publication

R428 (BGB324) is an anti-cancer drug candidate under clinical investigation. It inhibits the receptor tyrosine kinase Axl and induces apoptosis of many types of cancer cells,but the relationship between the two has not been well established. We investigated the molecular mechanisms of the R428-induced apoptosis and found that R428 induced extensive cytoplasmic vacuolization and caspase activation,independent of its inhibitory effects on Axl. Further analyses revealed that R428 blocked lysosomal acidification and recycling,accumulated autophagosomes and lysosomes,and induced cell apoptosis. Inhibition of autophagy by autophagy inhibitors or autophagic gene-knockout alleviated the R428-induced vacuoles formation and cell apoptosis. Our study uncovered a novel function and mechanism of R428 in addition to its ability to inhibit Axl. These data will help to better direct the application of R428 as an anti-cancer reagent. It also adds new knowledge to understand the regulation of autophagy and apoptosis. View Publication

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation,and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI,and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I),we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI,we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation,and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together,our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation,and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. View Publication

Although modulation of protein levels is an important tool for study of protein function,it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes,a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach,which confers instability to the tagged protein in the absence of the compound Shield-1,has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies,partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles,this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles,we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent,and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein,and that destabilization of DD-TCOF1 resulted in impaired cell growth,as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells. View Publication

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection,characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure,the RosetteSep-Applied Imaging Rare Event (RARE) detection method,which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample,thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity,by analyzing a 5-10 fold larger volume of blood,compared with a direct immunocytochemical (ICC) technique,with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods,both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had textgreater or = 1 tumor cell detected by the RARE procedure,compared with 5.2% after direct ICC analysis,analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB,as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However,the easy access of peripheral blood,and the increased sensitivity obtained by increasing the sample volume with the RARE procedure,suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed. View Publication

Natural killer (NK) cells are thought to develop from common lymphoid progenitors in the bone marrow. However,immature thymocytes also retain NK potential. Currently,the contribution of the thymus-dependent pathway in normal steady-state NK-cell development is unknown. Here,we show that TCRgamma genes are rearranged in approximately 5% of neonatal and 1% of adult mouse splenic NK cells,and similar levels are detected in NK cells from TCRbeta,delta double-knockout mice,excluding the possibility of T-cell contamination. NK-cell TCRgamma gene rearrangement is thymus dependent because this rearrangement is undetectable in nude mouse NK cells. These results change the current view of NK-cell development and show that a subset of NK cells develops from immature thymocytes that have rearranged TCRgamma genes. View Publication

Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied,in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines,but the role of glycoprotein 130 activation has not yet been investigated. In this study,CD133(+) cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6-sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry,2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction,3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay,4) analyze cell-cycle status,and 5) perform long-term culture-initiating cell assays. The level of mIL-6R(-) cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs,independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133(+) 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor-alpha protease inhibitor-1,an inhibitor of a disintegrin and metalloprotease proteases,suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion,expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment. View Publication

Disruption of pathways leading to programmed cell death plays a major role in most malignancies,including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL,preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study,we show that ABT-737 is cytotoxic to MM cell lines,including those resistant to conventional therapies,and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally,several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6,vascular endothelial growth factor or insulin-like growth factor,suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM. View Publication

There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study,we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation,the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry,immunoblotting,and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore,the cells were tested for functions associated with mature hepatocytes,including glycogen storage and indocyanine green uptake and release,and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate,numerous epithelial cells resembling hepatocytes were observed,and approximately 74% of the cells expressed the hepatic marker,albumin,after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers,and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development,and could provide a source of hepatocytes for transplantation therapy and drug screening. View Publication

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice,myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6,accompanied by substantial expression of IL-2,TNF-α,and CCL2. In contrast,NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo,IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation,enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study,we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases. View Publication

PURPOSE: To investigate the distribution of CD44(+)/CD24(-) cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy. METHODS: CD44(+)/CD24(-) tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays,respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays,and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST). RESULTS: In 27/78 cases,complete remission (CR) was identified using RECIST. However,18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy,24 cases involved cancer cells that were confined to the tumor outline,and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition,there were 6 patients who were insensitive to chemotherapy,and in these cases,both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy. CONCLUSION: CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion. View Publication