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

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus,35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells,primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures,and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells,5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly,the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated,indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders. View Publication

Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However,the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study,we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette,we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly,more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification,and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional,as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore,hESCs stably modified with the CD1d gene may serve as a convenient,unlimited,and competent DC source for iNKT cell-based cancer immunotherapy. View Publication

Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX),the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene ( LRPPRC ). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions,tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study,we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC,homozygous for the LRPPRC *354V allele,and from a control,homozygous for the LRPPRC *A354 allele. Specifically,for both of these fibroblast lines we generated hiPSC,hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines,as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells,with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC,reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts,this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC. View Publication

Induced pluripotent stem (iPS) cells are being used increasingly to complement their embryonic counterparts to understand and develop the therapeutic potential of pluripotent cells. Our objectives were to identify an efficient cardiac differentiation protocol for human iPS cells as monolayers,and demonstrate that the resulting cardiac progenitors could provide a therapeutic benefit in a rodent model of myocardial infarction. Herein,we describe a 14-day protocol for efficient cardiac differentiation of human iPS cells as a monolayer,which routinely yielded a mixed population in which over 50% were cardiomyocytes,endothelium,or smooth muscle cells. When differentiating,cardiac progenitors from day 6 of this protocol were injected into the peri-infarct region of the rat heart; after coronary artery ligation and reperfusion,we were able to show that human iPS cell-derived cardiac progenitor cells engrafted,differentiated into cardiomyocytes and smooth muscle,and persisted for at least 10 weeks postinfarct. Hearts injected with iPS-derived cells showed a nonsignificant trend toward protection from decline in function after myocardial infarction,as assessed by magnetic resonance imaging at 10 weeks,such that the ejection fraction at 10 weeks in iPS treated hearts was 62%±4%,compared to that of control infarcted hearts at 45%±9% (Ptextless0.2). In conclusion,we demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart,and reduced remodeling of the heart after ischemic damage. View Publication

The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells,including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy,we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs,we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells,and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy. View Publication

Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types,and that this variation is likely determined by a combination of the chromatin landscape,cell-specific DNA-binding transcription factors,and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells,there is presently little understanding of this aspect of the Pol III system in human ES cells. Here,we determine the occupancy profiles of Pol III components in human H1 ES cells,and also induced pluripotent cells,and compare to known profiles of chromatin,transcription factors,and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly,we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells,especially NANOG. Notably,in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases,a peak of H3K4me3 and/or RNA Pol II,between the H3K27me3 and Pol III binding peaks,suggesting that H3K4me3 and Pol II activity may “insulate? Pol III from neighboring repressive H3K27me3. Further,we find iPSCs have a larger Pol III repertoire than their precursors. Finally,the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together,our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity. View Publication

BACKGROUND Self-renewing,chemoresistant breast cancer stem cells are believed to contribute significantly to cancer invasion,migration and patient relapse. Therefore,the identification of signaling pathways that regulate the acquisition of stem-like qualities is an important step towards understanding why patients relapse and towards development of novel therapeutics that specifically target cancer stem cell vulnerabilities. Recent studies identified a role for the aryl hydrocarbon receptor (AHR),an environmental carcinogen receptor implicated in cancer initiation,in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in the acquisition of cancer stem cell-like qualities. RESULTS To test this hypothesis,AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells were modulated with AHR ligands,shRNA or AHR-specific inhibitors,and phenotypic,genomic and functional stem cell-associated characteristics were evaluated. The data demonstrate that (1) ALDH(high) cells express elevated levels of Ahr and Cyp1b1 and Cyp1a1,AHR-driven genes,(2) AHR knockdown reduces ALDH activity by 80%,(3) AHR hyper-activation with several ligands,including environmental ligands,significantly increases ALDH1 activity,expression of stem cell- and invasion/migration-associated genes,and accelerates cell migration,(4) a significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of stem cell- and invasion/migration-associated gene sets is seen with genomic data obtained from 79 human breast cancer cell lines and over 1,850 primary human breast cancers,(5) the AHR interacts directly with Sox2,a master regulator of self-renewal; AHR ligands increase this interaction and nuclear SOX2 translocation,(6) AHR knockdown inhibits tumorsphere formation in low adherence conditions,(7) AHR inhibition blocks the rapid migration of ALDH(high) cells and reduces ALDH(high) cell chemoresistance,(8) ALDH(high) cells are highly efficient at initiating tumors in orthotopic xenografts,and (9) AHR knockdown inhibits tumor initiation and reduces tumor Aldh1a1,Sox2,and Cyp1b1 expression in vivo. CONCLUSIONS These data suggest that the AHR plays an important role in development of cells with cancer stem cell-like qualities and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of these properties. View Publication

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene,whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly,the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations),though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover,gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence,our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future,once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved,hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease. View Publication

BACKGROUND In colorectal carcinoma (CRC),activation of the Raf/MEK/ERK signaling pathway is commonly observed. In addition,the commonly used 5FU-based chemotherapy in patients with metastatic CRC was found to enrich a subpopulation of CD26(+) cancer stem cells (CSCs). As activation of the Raf/MEK/ERK signaling pathway was also found in the CD26(+) CSCs and therefore,we hypothesized that an ATP-competitive pan-Raf inhibitor,Raf265,is effective in eliminating the cancer cells and the CD26(+) CSCs in CRC patients. METHODS HT29 and HCT116 cells were treated with various concentrations of Raf265 to study the anti-proliferative and apoptotic effects of Raf265. Anti-tumor effect was also demonstrated using a xenograft model. Cells were also treated with Raf265 in combination with 5FU to demonstrate the anti-migratory and invasive effects by targeting on the CD26(+) CSCs and the anti-metastatic effect of the combined treatment was shown in an orthotopic CRC model. RESULTS Raf265 was found to be highly effective in inhibiting cell proliferation and tumor growth through the inhibition of the RAF/MEK/ERK signaling pathway. In addition,anti-migratory and invasive effect was found with Raf265 treatment in combination with 5FU by targeting on the CD26(+) cells. Finally,the anti-tumor and anti-metastatic effect of Raf265 in combination with 5FU was also demonstrated. CONCLUSIONS This preclinical study demonstrates the anti-tumor and anti-metastatic activity of Raf265 in CRC,providing the basis for exploiting its potential use and combination therapy with 5FU in the clinical treatment of CRC. View Publication

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis,biological activity,and further profiling of these compounds are described. This work resulted in the discovery of 17,GDC-0941,which is a potent,selective,orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer. View Publication