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

BACKGROUND Induced pluripotent stem cells (iPSCs) hold tremendous potential,both as a biological tool to uncover the pathophysiology of disease by creating relevant human cell models and as a source of cells for cell-based therapeutic applications. Studying the reprogramming process will also provide significant insight into tissue development. OBJECTIVE We sought to characterize the derivation of iPSC lines from nasal epithelial cells (NECs) isolated from nasal mucosa samples of children,a highly relevant and easily accessible tissue for pediatric populations. METHODS We performed detailed comparative analysis on the transcriptomes and methylomes of NECs,iPSCs derived from NECs (NEC-iPSCs),and embryonic stem cells (ESCs). RESULTS NEC-iPSCs express pluripotent cell markers,can differentiate into all 3 germ layers in vivo and in vitro,and have a transcriptome and methylome remarkably similar to those of ESCs. However,residual DNA methylation marks exist,which are differentially methylated between NEC-iPSCs and ESCs. A subset of these methylation markers related to epithelium development and asthma and specific to NEC-iPSCs persisted after several passages in vitro,suggesting the retention of an epigenetic memory of their tissue of origin. Our analysis also identified novel candidate genes with dynamic gene expression and DNA methylation changes during reprogramming,which are indicative of possible roles in airway epithelium development. CONCLUSION NECs are an excellent tissue source to generate iPSCs in pediatric asthmatic patients,and detailed characterization of the resulting iPSC lines would help us better understand the reprogramming process and retention of epigenetic memory. View Publication

Purpose: The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients. Experimental design: Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45-) with a stem-like" phenotype (CD44+CD24low/-� View Publication

Green tea has shown remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays,cell culture systems,and epidemiological studies. Many of these biological effects of green tea are mediated by epigallocatechin 3-gallate (EGCG),the major polyphenol present therein. We have earlier shown that EGCG treatment results in apoptosis of several cancer cells,but not of normal cells (J. Natl. Cancer Inst. 89,1881-1886 (1997)). The mechanism of this differential response of EGCG is not known. In this study,we investigated the involvement of NF-kappaB during these differential responses of EGCG. EGCG treatment resulted in a dose-dependent (i) inhibition of cell growth,(ii) G0/G1-phase arrest of the cell cycle,and (iii) induction of apoptosis in human epidermoid carcinoma (A431) cells,but not in normal human epidermal keratinocytes (NHEK). Electromobility shift assay revealed that EGCG (10-80 microM) treatment results in lowering of NF-kappaB levels in both the cytoplasm and nucleus in a dose-dependent manner in both A431 cells and NHEK,albeit at different concentrations. EGCG treatment was found to result in a dose-based differential inhibition of TNF-alpha- and LPS-mediated activation of NF-kappaB in these cells. The inhibition of NF-kappaB constitutive expression and activation in NHEK was observed only at high concentrations. The immunoblot analysis also demonstrated a similar pattern of inhibition of the constitutive expression as well as activation of NF-kappaB/p65 nuclear protein. This inhibition of TNF-alpha-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha. Taken together,EGCG was found to impart differential dose-based NF-kappaB inhibitory response in cancer cells vs normal cells; i.e.,EGCG-mediated inhibition of NF-kappaB constitutive expression and activation was found to occur at much higher dose of EGCG in NHEK as compared to A431 cells. This study suggests that EGCG-caused cell cycle deregulation and apoptosis of cancer cells may be mediated through NF-kappaB inhibition. View Publication

The cell surface marker CD133 has been proposed as a brain tumor stem cell marker. However,there have been substantial controversies regarding the necessity and role of CD133 in tumorigenesis. This study aimed to characterize CD133(+) cells in brain tumors. Human brain tumor specimens and whole blood were collected from the same patients (N=12). We carried out dual FACS staining for CD133/CD34 and functional tumorigenesis and angiogenesis analyses of CD133(+) cells from different origins. We also investigated the in vivo tumorigenic potential and histological characteristics of four distinct groups on the basis of expression of CD133/CD34 markers (CD133(+),CD133(+)/CD34(+),CD133(+)/CD34(-),and CD133(-)). CD133(+) brain tumor cells coexpressed significantly higher positivity for CD34 (70.7±5.2% in CD133(+) vs. 12.3±4.2% in CD133(-) cells,P<0.001). CD133(+) brain tumor cells formed neurosphere-like spheroids and differentiated into multiple nervous system lineages unlike CD133(+) blood cells. They showed biological characteristics of endothelial cells,including vWF expression,LDL uptake and tube formation in vitro,unlike CD133(-) brain tumors cells. Pathologic analysis of brains implanted with CD133(+) cells showed large,markedly hypervascular tumors with well-demarcated boundary. CD133(+)/CD34(-) cells produced smaller but highly infiltrative tumors. Notably,pure angiogenic cell fractions (CD133(+)/CD34(+)) and CD133(-) tumor cells did not generate tumors in vivo. Our data suggest the presence of a distinct subpopulation of CD133(+) cells isolated from human brain tumors,with characteristics of endothelial progenitor cells (EPCs). View Publication

Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors,which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype,loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds,which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently,the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore,Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally,we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors. View Publication

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice,establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation,half of those with essential thrombocythemia or primary myelofibrosis do not,suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg,interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly,we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424,the first potent,selective,oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC(50)] = 281nM),and proliferation of JAK2V617F(+) Ba/F3 cells (IC(50) = 127nM). In primary cultures,INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F(+) polycythemia vera patients (IC(50) = 67nM) versus healthy donors (IC(50) textgreater 400nM). In a mouse model of JAK2V617F(+) MPN,oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines,and preferentially eliminated neoplastic cells,resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs. View Publication

5-Fluorouracil (5-FU) is a small,very membrane permeable drug that is poorly retained within the aqueous compartment of liposomal nanoparticles (LNP). To address this problem a novel method relying on formation of a ternary complex comprising copper,low molecular weight polyethylenimine (PEI) and 5-FU has been developed. More specifically,in the presence of entrapped copper and PEI,externally added 5-FU can be efficiently encapsulated (textgreater95%) in DSPC/Chol (1,2-Distearoyl-sn-Glycero-3-Phosphocholine/cholesterol; 55:45 mol%) liposomes (130-170 nm) to achieve drug-to-lipid ratios of 0.1 (mol:mol). Drug release studies completed using this LNP formulation of 5-FU demonstrated significant improvements in drug retention in vitro and in vivo. Plasma concentrations of 5-FU were 7- to 23-fold higher when the drug was administered intravenously to mice as the LNP 5-FU formulation compared to free 5-FU. Further,the therapeutic effects of the LNP 5-FU formulation,as determined in a HT-29 subcutaneous colorectal cancer model where treatment was given QDx5,was greater than that which could be achieved with free 5-FU when compared at equivalent doses. This is the first time an active loading method has been described for 5-FU. The use of ternary metal complexation strategy to encapsulate therapeutic agents may define a unique platform for preparation of LNP drug formulations. View Publication

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation,we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range,enabling targeting of many previously inaccessible PAMs. On average,enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a,and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing,endogenous gene activation and C-to-T base editing,and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively,enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing. View Publication

BACKGROUND: Cancer stem cells (CSCs) have been shown to be a small stem cell-like cell population which appears to drive tumorigenesis,tumor recurrence and metastasis. Thus,identification and characterization of CSCs may be critical to defining effective anticancer therapies. In prostate cancer (PCa),the CD44(+) cell population appears to have stem cell-like properties including being tumorigenic. The enzyme aldehyde dehydrogenase (ALDH) has been found to identify hematopoietic stem cells and our aim was to determine the utility of ALDH activity and CD44 in identifying PCa stem cell-like cells in PCa cell lines. MATERIALS AND METHODS: LNCaP cells and PC-3 cells were sorted based on their expression of CD44 and ALDH activity. The cell populations were investigated using colony-forming assays,invasion assays,sphere formation experiments in a non-adherent environment and 3-D Matrigel matrix culture to observe the in vitro stem-cell like properties. Different sorted cell populations were injected subcutaneously into NOD/SCID mice to determine the corresponding tumorigenic capacities. RESULTS: ALDH(hi) CD44(+) cells exhibit a higher proliferative,clonogenic and metastatic capacity in vitro and demonstrate higher tumorigenicity capacity in vivo than did ALDH(lo) CD44(-) cells. The tumors recapitulated the population of the original cell line. However,ALDHlo CD44(-) cells were able to develop tumors,albeit with longer latency periods. CONCLUSION: ALDH activity and CD44 do not appear to identify PCa stem cells; however,they do indicate increased tumorigenic and metastatic potential,indicating their potential importance for further exploration. View Publication