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

Summary Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture,raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV,we show that those containing this amplicon have higher population doubling rates,attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs,only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells,whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells,establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas,linking this mutation with malignant transformation. View Publication

$\$-Thalassemia ($\$-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in $\$-globin gene (HBB). It is estimated that 4.5% of the population in the world carry $\$-Thal mutants (1),posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However,homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here,we describe a robust process combining efficient generation of integration-free $\$-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly,the gene-corrected $\$-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal $\$-globin. Our studies provide an efficient and universal strategy to correct different types of $\$-globin mutations in $\$-Thal iPSCs for disease modeling and applications. View Publication

The Sox6 transcription factor plays critical roles in various cell types,including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells,we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar,evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo,and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied,in erythroid cells,by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation,mediated by the double Sox6 binding site within its own promoter,may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. View Publication

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotidebackslashnrepeat (CAG) expansion in the coding region of the ATAXN2 gene onbackslashnchromosome 12,which produces an elongated,toxic polyglutamine tract,backslashnleading to Purkinje cell loss. There is currently no effective therapy.backslashnOne of the main obstacles that hampers therapeutic development is lackbackslashnof an ideal disease model. In this study,we have generated andbackslashncharacterized SCA2-induced pluripotent stem (iPS) cell lines as an inbackslashnvitro cell model. Dermal fibroblasts (FBs) were harvested from primarybackslashncultures of skin explants obtained from a SCA2 subject and a healthybackslashnsubject. For reprogramming,hOct4,hSox2,hKlf4,and hc-Myc werebackslashntransduced to passage-3 FBs by retroviral infection. Both SCA2 iPS andbackslashncontrol iPS cells were successfully generated and showed typical stembackslashncell growth patterns with normal karyotype. All iPS cell lines expressedbackslashnstem cell markers and differentiated in vitro into cells from threebackslashnembryonic germ layers. Upon in vitro neural differentiation,SCA2 iPSbackslashncells showed abnormality in neural rosette formation but successfullybackslashndifferentiated into neural stem cells (NSCs) and subsequent neuralbackslashncells. SCA2 and normal FBs showed a comparable level of ataxin-2backslashnexpression; whereas SCA2 NSCs showed less ataxin-2 expression thanbackslashnnormal NSCs and SCA2 FBs. Within the neural lineage,neurons had thebackslashnmost abundant expression of ataxin-2. Time-lapsed neural growth assaybackslashnindicated terminally differentiated SCA2 neural cells were short-livedbackslashncompared with control neural cells. The expanded CAG repeats of SCA2backslashnwere stable throughout reprogramming and neural differentiation. Inbackslashnconclusion,we have established the first disease-specific human SCA2backslashniPS cell line. These mutant iPS cells have the potential for neuralbackslashndifferentiation. These differentiated neural cells harboring mutationsbackslashnare invaluable for the study of SCA2 pathogenesis and therapeutic drugbackslashndevelopment. View Publication

We performed gene expression microarray analysis coupled with spherical self-organizing map (sSOM) for artificially developed cancer stem cells (CSCs). The CSCs were developed from human induced pluripotent stem cells (hiPSCs) with the conditioned media of cancer cell lines,whereas the CSCs were induced from primary cell culture of human cancer tissues with defined factors (OCT3/4,SOX2,and KLF4). These cells commonly expressed human embryonic stem cell (hESC)/hiPSC-specific genes (POU5F1,SOX2,NANOG,LIN28,and SALL4) at a level equivalent to those of control hiPSC 201B7. The sSOM with unsupervised method demonstrated that the CSCs could be divided into three groups based on their culture conditions and original cancer tissues. Furthermore,with supervised method,sSOM nominated TMED9,RNASE1,NGFR,ST3GAL1,TNS4,BTG2,SLC16A3,CD177,CES1,GDF15,STMN2,FAM20A,NPPB,CD99,MYL7,PRSS23,AHNAK,and LOC152573 genes commonly upregulating among the CSCs compared to hiPSC,suggesting the gene signature of the CSCs. View Publication

BACKGROUND Humanized mouse models are still under development,and various protocols exist to improve human cell engraftment and function. METHODS Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34(+) hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM),spleen,and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS Most of the NSG mice tolerated the regimen well. The percentage of hCD45(+) and CD19(+) cells rose significantly in a time-dependent manner. The median percentage of hCD45(+)cells in the BM was 55.5% at week 8,and 67.2% at week 12. The median percentage of hCD45(+) cells in the spleen at weeks 8 and 12 was 42% and 51%,respectively. The median percentage of hCD19(+) cells in BM at weeks 8 and 12 was 21.5% and 39%,respectively (P=0.04). Similarly,the median percentage of hCD19(+) cells in the spleen at weeks 8 and 12 was 10% and 24%,respectively (P=0.04). The percentage of hCD19(+) B cells in PB was 23% at week 12. At week 8,hCD3(+) T cells were barely detectable,while hCD7(+) was detected in the BM and spleen. The percentage of hCD3(+) T cells was 2‒3% at week 12 in the BM,spleen,and PB of humanized NSG mice. CONCLUSION We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45(+) cells than earlier studies without any significant toxicity. And human CD45(+) cell engraftment at week 8 was comparable to that of week 12. View Publication

Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H2O2) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H2O2 showed increases in miR-1 expression,mitochondria dysfunction,cytochrome-c release and apoptosis,Addition of IGF-1 into the iPS cell cultures reduced the H2O2 cytotoxicity. Prediction algorithms showed that 3'-untranslated regions of IGF-1 gene as a target of miR-1. Moreover,miR-1 mimic,but not miR-1 mimic negative control,diminished the protective effect of IGF-1 on H2O2-induced mitochondrial dysfunction,cytochrome-c release and apoptosis in iPS cells. In conclusion,IGF-1 inhibits H2O2-induced mitochondrial dysfunction,cytochrome-c release and apoptosis. IGF-1's effect is,at least partially,regulated by miR-1 in iPS cells. ?? 2012 Elsevier Inc. View Publication

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2),a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly,whereas normal HSCs were rapidly exhausted after serial transplantations,overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast,similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a genetic genomics" screen� View Publication

The longevity of organisms is maintained by stem cells. If an organism loses the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment due to aging and/or stress,this may result in death or age-associated diseases,including cancer. Ewing sarcoma is the most lethal bone tumor in young patients and arises from primitive stem cells. Here,we demonstrated that endogenous Ewing sarcoma gene (Ews) is indispensable for stem cell quiescence,and that the ablation of Ews promotes the early onset of senescence in hematopoietic stem progenitor cells. The phenotypic and functional changes in Ews-deficient stem cells were accompanied by an increase in senescence-associated β-galactosidase staining and a marked induction of p16(INK4a) compared with wild-type counterparts. With its relevance to cancer and possibly aging,EWS is likely to play a significant role in maintaining the functional capacity of stem cells and may provide further insight into the complexity of Ewing sarcoma in the context of stem cells. View Publication

Bone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins,including SMAD1. In previous work,we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly,we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely,when Smad1 is depleted in FlK1(+) mesoderm,at a stage after hemangioblast commitment,the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis,including Gata1,Runx1 and Eklf,rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity,indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism,hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore,this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors. View Publication

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1,an inhibitor of cell cycle progression in yeast. At present,mDYRK-3 and mDYRK-2 have been cloned,and mDYRK-3 has been characterized with respect to kinase activity,expression among tissues and hematopoietic cells,and possible function during erythropoiesis. In sequence,mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a,but is 91.3% identical overall to hDYRK-3. Catalytically,mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a,mDYRK-2,and mDYRK-3 was high in testes,but unlike mDYRK1a and mDYRK 2,mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells,however,mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells,expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone),and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly,antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus,it is proposed that mDYRK-3 kinase functions as a lineage-restricted,stage-specific suppressor of red cell development. (Blood. 2001;97:901-910) View Publication

OBJECTIVE: To compare the suppressive effect of mesenchymal stem cells (MSC),derived from normal individuals or severe aplastic anemia patients (SAA),on T-cell activation. PATIENTS AND METHODS: We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n = 3),following immunosuppressive therapy (IS) (n = 16),or after a bone marrow transplant (BMT) (n = 4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR),phytohemaglutinin (PHA)-primed cultures,activation surface markers,gamma-IFN production,hematopoietic colony formation (CFC),production of cyclic ADP-ribose (cADPR). RESULTS: The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens (p = 0.009) or PHA (p = 0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells (p = 0.001); 3) impaired ability to suppress gamma-IFN production in PHA cultures,resulting in an 11-fold higher gamma-IFN concentration; 4) no preventive effect on T cell-mediated inhibition of CFC; and 5) significantly reduced (p = 0.009) production of cADPR,a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT. CONCLUSION: The ability of MSC to downregulate T-cell priming,proliferation,and cytokine release is deficient in patients with SAA,persists indefinitely after immunosuppressive therapy,but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined. View Publication