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

Chromatin structure affects the extent of DNA damage and repair. Thus,it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here,we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC,heterochromatin is confined to distinct regions,while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives,normal human fibroblasts; and one cancer cell line. At the same time,the number of DNA repair foci were not statistically different among these cells. We showed that in hESC,DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells,and to a lesser extent in IMR90 normal fibroblasts,but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC. View Publication

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder resulting from deficiency of iduronate-2-sulphatase activity. The disease manifests almost exclusively in males; only 16 symptomatic heterozygote girls have been reported so far. We describe the results of X-chromosome inactivation analysis in a 5-year-old girl with clinically severe disease and heterozygous mutation p.Arg468Gln in the IDS gene. X inactivation analysed at three X-chromosome loci showed extreme skewing (96/4 to 99/1) in two patient's cell types. This finding correlated with exclusive expression of the mutated allele. Induced pluripotent stem cells (iPSC) generated from the patient's peripheral blood demonstrated characteristic pluripotency markers,deficiency of enzyme activity,and mutation in the IDS gene. These cells were capable of differentiation into other cell types (cardiomyocytes,neurons). In MPS II iPSC clones,the X inactivation ratio remained highly skewed in culture conditions that led to partial X inactivation reset in Fabry disease iPSC clones. Our data,in accordance with the literature,suggest that extremely skewed X inactivation favouring the mutated allele is a crucial condition for manifestation of MPS II in females. This suggests that the X inactivation status and enzyme activity have a prognostic value and should be used to evaluate MPS II in females. For the first time,we show generation of iPSC from a symptomatic MPS II female patient that can serve as a cellular model for further research of the pathogenesis and treatment of this disease. View Publication

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation,such as the TCR/CD3 complex and the costimulatory receptor CD28,are capable of mediating T-cell activation resulting in tumor cell killing. In this study,we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2,when used alone rather than in combination,markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore,these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and,in particular,of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients. View Publication

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However,it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3,a small molecule inhibitor of the p53/MDM2 interaction,is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19(+) B-CLL cells,but not on normal CD19(+) B lymphocytes,peripheral-blood mononuclear cells,or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined,only one was resistant to nutlin-3-mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway,nutlin-3 up-regulated the steady-state mRNA levels of PCNA,CDKN1A/p21,GDF15,TNFRSF10B/TRAIL-R2,TP53I3/PIG3,and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover,nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL. View Publication

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo,de novo osteoclastogenesis is observed but it is currently unknown whether,besides increased osteoclast differentiation from undifferentiated precursors,other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study,an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment,the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally,chromosomal painting identified nuclei from female DCs,previously injected into a male recipient,among the nuclei of giant cells at sites of osteolysis. Finally,osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice,which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether,our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases. View Publication

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease,characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene,resulting in the generation of progerin,a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin,and more importantly,lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs,progerin and its ageing-associated phenotypic consequences are restored. Specifically,directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally,our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs,also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing,our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing. View Publication

MicroRNAs (miRNAs) are small,noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-,maturation-,and disease-specific miRNA expression has been described,miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics,biochemical analysis,and unbiased and hypothesis-driven miRNA target prediction,we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells,miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression,respectively. However,gene-specific RNAi reveals that this reduction,alone and in combination,is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression,DNA-binding,and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression,indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed,we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly,gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. View Publication

Estrogens regulate osteoblast differentiation and mineralization. We identified GATA4 as a transcription factor expressed in osteoblasts and directly regulated by 17β-estradiol in this cell type but not in breast cancer cells,another estrogen-responsive tissue. Chromatin immunoprecipitation sequencing (chromatin immunoprecipitation sequencing) reveals that estrogen receptor α (ERα) binds to chromatin near GATA4 at five different enhancers. GATA4 and ERα are both recruited to ERα binding sites near genes that are specifically expressed in osteoblasts and control osteoblast differentiation. Maximal binding of GATA4 precedes ERα binding,and GATA4 is necessary for histone 3 lysine 4 dimethylation at ERα binding sites,suggesting that GATA4 is a pioneer factor for ERα. As such,knockdown of GATA4 reduced recruitment of ERα to DNA. Our study illustrates that GATA4 is a pioneer factor for ERα recruitment to osteoblast-specific enhancers. View Publication

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here,we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein,decreased survival in longitudinal studies,and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening. View Publication

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC),which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation,and this hydroxymethylation change is critical for optimal epigenetic reprogramming,but does not compromise primed pluripotency. Compared with hESCs,we find that iPSCs tend to form large-scale (100 kb–1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions,most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly,these 5hmC aberrant hotspots largely coincide (∼ 80%) with aberrant iPSC–ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC–hESC differences. View Publication

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm,which is manifested by rosette formation,with consecutive differentiation into neural progenitors and early glial-like cells. In this study,we examined the involvement of early neural markers - OTX2,PAX6,Sox1,Nestin,NR2F1,NR2F2,and IRX2 - in the onset of rosette formation,during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation,which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation,when rosettes comprise no more than 3-5 cells,and that its expression precedes that of established markers of early neuronal differentiation. Importantly,the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly,we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro,and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice. ?? 2013 Elsevier B.V. View Publication

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However,modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator,VP64-p65-Rta (VPR),fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes,targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs). View Publication