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

This study investigated the factors responsible for migration and homing of magnetically labeled AC133(+) cells at the sites of active angiogenesis in tumor. AC133(+) cells labeled with ferumoxide-protamine sulfate were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. An MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB),platelet-derived growth factor (PDGF),hypoxia-inducible factor-1alpha (HIF-1alpha),stromal cell derived factor-1 (SDF-1),matrix metalloproteinase-2 (MMP-2),vascular endothelial growth factor (VEGF),and endothelial markers. Fresh snap-frozen strips from the central and peripheral parts of the tumor were collected for Western blotting. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB(+)/AC133(+) cells were positive for endothelial cells markers. At the sites of PB(+)/AC133(+) cells,both HIF-1alpha and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB(+)/AC133(+) cell localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1,MMP-2,and PDGF at the peripheral parts of the tumors. HIF-1alpha was expressed at both the periphery and central parts of the tumor. This work demonstrates that magnetically labeled cells can be used as probes for MRI and histological identification of administered cells. View Publication

Focal adhesion kinase (FAK) has been implicated in the development of cancers,including those of the breast. Nevertheless,the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here,we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers,including aldehyde dehydrogenase,CD24,CD29,and CD61,we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their self-renewal and migration in vitro. Finally,through transplantation in NOD-SCID mice,we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together,these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. View Publication

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function,as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally,we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines,establish techniques that can facilitate the characterization of regulatory pathways of CSCs,and identify potential stem cell markers and therapeutic targets. View Publication

Chemokine receptor CX3CR1(+) dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103(+) DCs in the mucosa,which,through their ability to synthesize retinoic acid (RA),appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103(+) DC,CX3CR1(+) cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103(+) DCs,but not CX3CR1-expressing cells,migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover,CX3CR1(+) cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103(+) DC. These findings indicate that selectively CD103(+) DCs serve classical DC functions and initiate adaptive immune responses in local LNs,whereas CX3CR1(+) populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens. View Publication

BACKGROUND: Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples. METHODS: HPC-A samples from 44 patients were incubated with Aldefluor with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1(bright)) were analyzed for stem/progenitor markers CD34,CD90,CD117,and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression. RESULTS: In ALDH1(bright)/SSC(low) cells,0.13% of the cells had CD34(+) expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1(bright)/SSC(low)/CD90(dim) cells amongst the nonlineage depleted samples was 0.04%. CD117(dim-bright) expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1(bright)/SSC(low) nonlineage depleted cells with a frequency of 0.28%. The ALDH1(bright)/CD90(dim) cells had the smallest mean electronic volume of 264.9 microm(3) when compared with cells with CD34(bright) expression (270.2 microm(3)) and ALDH1(dim)/CD90(dim) cells (223 microm(3)). CONCLUSIONS: ALDH1(bright)/SSC(low) cells show heterogeneity in expression of the four stem cell markers studied. The CD90 cells in both the ALDH1(bright) and ALDH1(dim) populations had the smallest mean electronic volume when compared with similar cells with CD117 expression. View Publication

BACKGROUND: The use of functional genomics has largely increased our understanding of cell biology and promises to help the development of systems biology needed to understand the complex order of events that regulates cellular differentiation in vivo. One model system clearly dependent on the integration of extra and intra cellular signals is the development of B-lymphocytes from hematopoietic stem cells in the bone marrow. This developmental pathway involves several defined differentiation stages associated with specific expression of genes including surface markers that can be used for the prospective isolation of the progenitor cells directly from the bone marrow to allow for ex vivo gene expression analysis. The developmental process can be simulated in vitro making it possible to dissect information about cell/cell communication as well as to address the relevance of communication pathways in a rather direct manner. Thus we believe that B-lymphocyte development represents a useful model system to take the first steps towards systems biology investigations in the bone marrow. RESULTS: In order to identify extra cellular signals that promote B lymphocyte development we created a database with approximately 400 receptor ligand pairs and software matching gene expression data from two cell populations to obtain information about possible communication pathways. Using this database and gene expression data from NIH3T3 cells (unable to support B cell development),OP-9 cells (strongly supportive of B cell development),pro-B and pre-B cells as well as mature peripheral B-lineage cells,we were able to identify a set of potential stage and stromal cell restricted communication pathways. Functional analysis of some of these potential ways of communication allowed us to identify BMP-4 as a potent stimulator of B-cell development in vitro. Further,the analysis suggested that there existed possibilities for progenitor B cells to send signals to the stroma. The functional consequences of this were investigated by co-culture experiments revealing that the co-incubation of stromal cells with B cell progenitors altered both the morphology and the gene expression pattern in the stromal cells. CONCLUSIONS: We believe that this gene expression data analysis method allows for the identification of functionally relevant interactions and therefore could be applied to other data sets to unravel novel communication pathways. View Publication

A relatively rare aldehyde dehydrogenase 1 (ALDH1)-positive stem cell-like" subpopulation of tumor cells has the unique ability to initiate and perpetuate tumor growth; moreover� View Publication

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer worldwide. Signal transducers and activators of transcription 3 (STAT3) signaling is reported to promote tumor malignancy and recurrence in HNSCC. Cucurbitacins,triterpenoid derivatives,are strong STAT3 inhibitors with anticancer properties. Recent studies have shown aldehyde dehydrogenase 1 (ALDH1) to be a marker of cancer stem cells (CSC) in HNSCC. The aim of this study was to investigate the therapeutic effect of cucurbitacin I in HNSCC-derived CSCs. Using immunohistochemical analysis,we firstly showed that CD44,ALDH1,and phosphorylated STAT3 (p-STAT3) were higher in high-grade HNSCCs,and that triple positivity for CD44/ALDH1/p-STAT3 indicated a worse prognosis for HNSCC patients. Secondly,CD44(+)ALDH1(+) cells isolated from seven HNSCC patients showed greater tumorigenicity,radioresistance,and high expression of stemness (Bmi-1/Oct-4/Nanog) and epithelial-mesenchymal-transitional (Snail/Twist) genes as p-STAT3 level increased. Furthermore,we found that cucurbitacin I (JSI-124) can effectively inhibit the expression of p-STAT3 and capacities for tumorigenicity,sphere formation,and radioresistance in HNSCC-CD44(+)ALDH1(+). Notably,150 nmol/L cucurbitacin I effectively blocked STAT3 signaling and downstream survivin and Bcl-2 expression,and it induced apoptosis in HNSCC-CD44(+)ALDH1(+). Moreover,microarray data indicated that 100 nmol/L cucurbitacin I facilitated CD44(+)ALDH1(+) cells to differentiate into CD44�?�ALDH1�?� and enhanced the radiosensitivity of HNSCC-CD44(+)ALDH1(+). Xenotransplant experiments revealed that cucurbitacin I combined with radiotherapy significantly suppressed tumorigenesis and lung metastasis and further improved the survival rate in HNSCC-CD44(+)ALDH1(+)-transplanted immunocompromised mice. Taken together,our data show that cucurbitacin I,STAT3 inhibitor,reduces radioresistant,distant-metastatic,and CSC-like properties of HNSCC-CD44(+)ALDH1(+) cells. The potential of cucurbitacin I as a radiosensitizer should be verified in future anti-CSC therapy. View Publication

Th17 cells play a significant role in inflammatory and autoimmune responses. Although a number of molecular pathways that contribute to the lineage differentiation of T cells have been discovered,the mechanisms by which lineage commitment occurs are not fully understood. Transcription factors play a key role in driving T cells toward specific lineages. We have identified a role for the transcription factor Kruppel-like factor (KLF) 4 in the development of IL-17-producing CD4(+) T cells. KLF4 was required for the production of IL-17,and further,chromatin immunoprecipitation analysis demonstrated binding of KLF4 to the IL-17 promoter,indicating a direct effect on the regulation of IL-17. Further,KLF4-deficient T cells upregulated expression of retinoic acid-related orphan receptor γt similar to wild-type during the polarization process toward Th17,suggesting that these two transcription factors are regulated independently. View Publication

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma,an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis,bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2,respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors,and transduced cells were cultured in a semisolid medium permissive for the development of erythroid,myeloid,and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro,in contrast to Tax2-transduced cells,which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected,suggesting that Tax1 inhibited the maturation of relatively early,uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis,lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells,an activity that is not displayed by Tax2,and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo,the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2. View Publication

The phosphatidylinositol 3' kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway,which is a critical regulator of cell proliferation and survival,is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens,and hypothesised that KP372-1,an Akt inhibitor,would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells,leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling,further preclinical evaluation of this compound for treatment of thyroid cancer is warranted. View Publication

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells,and this is thought to be important in chemoresistance. A method for the assessment of ALDH activity in viable cells recently has been developed and made commercially available in a kit format. In this study,we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Via multicolor flow cytometry of cord blood ALDH+ cells,we have expanded on their phenotypic analysis. We then assessed the incidence,morphology,phenotype,and nonobese diabetic/ severe combined immunodeficiency engraftment ability of ALDH+ cells from acute myeloid leukemia (AML) samples. AML samples had no ALDH+ cells at all,an extremely rare nonmalignant stem/progenitor cell population,or a less rare,leukemic stem cell population. Hence,in addition to identifying nonmalignant stem cells within some AML samples,a high ALDH activity also identifies some patients' CD34+/ CD38- leukemic stem cells. The incidence of normal or leukemic stem cells with an extremely high ALDH activity may have important implications for resistance to chemotherapy. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis. View Publication