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

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24(low/�?�)). Since then,CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties,including aldehyde dehydrogenase (ALDH) activity,have also been used to isolate CSCs from malignant tissues. Recently,we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24,and CD133. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic,but ALDH+ cells are relatively more invasive. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,a fluorescent substrate of ALDH. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells. View Publication

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1,ALDH3A1,and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity,and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells,commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together,these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential,that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis,and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component,implicating Notch signaling in lung cancer stem cell maintenance. View Publication

For antibody-based therapy of cancer,monoclonal antibodies (mAbs) of human origin are superior to mouse,mouse/human chimeric or humanized mAbs,because of their minimum immunogenicity to humans and their efficient collaboration with human effector cells. In the present study,human mAbs were prepared against a pancarcinoma antigen,MK-1 (Ep-CAM),using a genetically-engineered mouse (KM mouse) that contains the human immunoglobulin genes. Spleen cells from KM mice,immunized with recombinant MK-1,were fused with P3-U1 mouse myeloma cells. Of 44 anti-MK-1 clones analyzed,two were of IgG4 and the others of IgM clones. Although the two IgG4 clones were suggested to recognize the same antigenic determinant or two closely located determinants,their VK regions were encoded by different light-chain genes while their VH sequences were identical. The two IgG4 and one of the IgM clones tested revealed antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity,respectively,against MK-1-expressing cells in vitro,suggesting that these fully human mAbs produced against MK-1 and their V-region genes,which are applicable for the preparation of engineered antibody fragments that may be useful for antibody-based therapy of cancer. View Publication

OBJECTIVE: The aim of this narrative review was to evaluate the role of cancer stem cells (CSCs) and sex steroids in the pathophysiology of human breast cancer. METHODS: A key-word search was performed using the Scopus database. Preference was given to studies using human cells and tissues. RESULTS: Long-term estrogen-progestin hormone therapy is known to increase breast cancer risk,although the mechanisms are poorly understood. In the last few years,it has become clear that many human breast cancers contain CSCs,which may be responsible for much of the tumor's malignant behavior. Very recently,the impact of estrogen,progesterone,and progestins on breast CSCs and their progeny has been studied and clarified. Most breast CSCs are estrogen receptor negative and progesterone receptor negative,although some intermediary progenitor forms have hormone receptors,especially progesterone receptor. Most mature human breast cancer cellsare estrogen receptor positive and can thus be stimulated by estrogen. Breast CSCs usually elaborate CD44+,CD24j/low and/or ALDEFLUOR+ cell markers and are lineage markers negative. One of the main roles of progesterone and progestin seems to be on certain breast cancer stem intermediate forms,inducing them to revert back to a more primitive breast CSC form. CONCLUSIONS: As the pathophysiology of human breast CSC is clarified,it is probable that this will lead to novel,effective breast cancer treatments and,perhaps,new breast cancer preventive agents. This research may also lead to safer hormone therapy regimens. View Publication

Thyroid carcinoma is the most common endocrine malignancy and the first cause of death among endocrine cancers. We show that the tumorigenic capacity in thyroid cancer is confined in a small subpopulation of stem-like cells with high aldehyde dehydrogenase (ALDH(high)) activity and unlimited replication potential. ALDH(high) cells can be expanded indefinitely in vitro as tumor spheres,which retain the tumorigenic potential upon delivery in immunocompromised mice. Orthotopic injection of minute numbers of thyroid cancer stem cells recapitulates the behavior of the parental tumor,including the aggressive metastatic features of undifferentiated thyroid carcinomas,which are sustained by constitutive activation of cMet and Akt in thyroid cancer stem cells. The identification of tumorigenic and metastagenic thyroid cancer cells may provide unprecedented preclinical tools for development and preclinical validation of novel targeted therapies. View Publication

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly,the BCR/ABL fusion gene,which is present in chronic myelogenous leukemia (CML),was also detected in the endothelial cells of patients with CML,suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells,thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease,and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly,as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv,and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together,these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library,which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. View Publication

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular,primitive leukemia cells,often termed leukemia stem cells,are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins,presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation,CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise,we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly,these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism,which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1),as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism,an intrinsic property of primary human AML cells. View Publication

Fluorescence in situ hybridization (FISH) is superior to routine chromosome analysis (RCA) in detecting important prognostic genetic abnormalities in plasma cell dyscrasia (PCD); however,its sensitivity is hampered due to paucity of plasma cells (PC) in whole bone marrow (BM). Studies showed that the abnormality detection rate in enriched plasma cells (EPC) is greater than unselected plasma cells (UPC),but purification techniques are limiting to only FISH when sample volumes are inadequate. Not performing RCA may compromise patient care since RCA is equally important for detecting non-PC related abnormalities when the diagnosis is undefined. To resolve this critical issue,we designed a study where an immuno-magnetic CD138 enriched positive selection was used for FISH while the negative fraction (NF) was used to retrieve other myeloid elements for RCA. Parallel FISH studies were performed using UPC and CD138 EPC,while karyotyping was achieved using whole BM and discarded myeloid elements from the NF. Results showed that the abnormality rate of EPC was doubled compared to UPC for FISH,and CA displayed 100% success rate using the NF. PCD related chromosome abnormalities were confined to whole BM while non-PCD related abnormalities were found in both whole BM and NF. Our results demonstrate the feasibility of using the NF for RCA. View Publication

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies,but is absent on normal tissues,including hematopoietic progenitor cells,and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity,PRAME-specific cytotoxic T lymphocytes (CTLs),we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal,PRAME-specific CTL lines and elicited high-avidity CTLs,with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope,P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts,but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays,which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors,indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies. View Publication

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program,by reprogramming of a more differentiated cell type by oncogenic insults,or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5,K14,and vimentin),luminal (E-cadherin,K8,K18,or K19),and stem/progenitor (CD49f,CD29,CD44,and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways,such as Notch,Wnt,Hedgehog,and LIF,in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers,these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer. View Publication