技术资料

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1,one of 19 ALDH isoforms expressed in humans,was generally believed to be responsible for the ALDH activity of CSCs. More recently,experiments with murine hematopoietic stem cells,murine progenitor pancreatic cells,and human breast CSCs indicate that other ALDH isoforms,particularly ALDH1A3,significantly contribute to aldefluor positivity,which may be tissue and cancer specific. Therefore,potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators. View Publication

AIM: Dendritic cell (DC)-based vaccines are designed to exploit the intrinsic capacity of these highly effective antigen presenting cells to prime and boost antigen-specific T-cell immune responses. Successful development of DC-based vaccines will be dependent on the ability to utilize and harness the full potential of these potent immune stimulatory cells. Recent advances to generate DCs derived from human embryonic stem cells (hESCs) that are suitable for clinical use represent an alternative strategy from conventional approaches of using patient-specific DCs. Although the differentiation of hESC-derived DCs in serum-free defined conditions has been established,the stimulatory potential of these hESC-derived DCs have not been fully evaluated. METHODS: hESC-derived DCs were differentiated in serum-free defined culture conditions. The delivery of antigen into hESC-derived DCs was investigated using mRNA transfection and replication-deficient adenoviral vector transduction. hESC-derived DCs modified with antigen were evaluated for their capacity to stimulate antigen-specific T-cell responses with known HLA matching. Since IL-12 is a key cytokine that drives T-cell function,further enhancement of DC potency was evaluated by transfecting mRNA encoding the IL-12p70 protein into hESC-derived DCs. RESULTS: The transfection of mRNA into hESC-derived DCs was effective for heterologous protein expression. The efficiency of adenoviral vector transduction into hESC-derived DCs was poor. These mRNA-transfected DCs were capable of stimulating human telomerase reverse transcriptase antigen-specific T cells composed of varying degrees of HLA matching. In addition,we observed the transfection of mRNA encoding IL-12p70 enhanced the T-cell stimulation potency of hESC-derived DCs. CONCLUSION: These data provide support for the development and modification of hESC-derived DCs with mRNA as a potential strategy for the induction of T-cell-mediated immunity. View Publication

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4-Hydroxy-5-methoxy-2,3-dihydro-1H-[1,3]benzodioxolo[5,6-c]pyrrolo[1,2-f]-phenanthridium chloride (NK314) is a benzo[c] phenanthridine alkaloid that inhibits topoisomerase IIα,leading to the generation of DNA double-strand breaks (DSBs) and activating the G(2) checkpoint pathway. The purpose of the present studies was to investigate the DNA intercalating properties of NK314,to evaluate the DNA repair mechanisms activated in cells that may lead to resistance to NK314,and to develop mechanism-based combination strategies to maximize the antitumor effect of the compound. A DNA unwinding assay indicated that NK314 intercalates in DNA,a property that likely cooperates with its ability to trap topoisomerase IIα in its cleavage complex form. The consequence of this is the formation of DNA DSBs,as demonstrated by pulsed-field gel electrophoresis and H2AX phosphorylation. Clonogenic assays demonstrated a significant sensitization in NK314-treated cells deficient in DNA-dependent protein kinase (DNA-PK) catalytic subunit,Ku80,ataxia telangiectasia mutated (ATM),BRCA2,or XRCC3 compared with wild-type cells,indicating that both nonhomologous end-joining and homologous recombination DNA repair pathways contribute to cell survival. Furthermore,both the DNA-PK inhibitor 8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (NU7441) and the ATM inhibitor 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933) significantly sensitized cells to NK314. We conclude that DNA-PK and ATM contribute to cell survival in response to NK314 and could be potential targets for abrogating resistance and maximizing the antitumor effect of NK314. View Publication

By mimicking embryonic development of the hematopoietic system,we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines,extra cellular matrix components,and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%,from seven pluripotent lines) from the differentiation culture,including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover,the numbers of hematopoietic progenitor cells generated,as measured by colony forming unit assays,were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors,it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability. View Publication

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this,we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts,acinar cells,and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. View Publication

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors,very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel,aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally,array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes,thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis,potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4,a GTPase regulator located in the commonly deleted 7q31 region,was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets,providing further validation of our findings. Finally,DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis,thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region. View Publication

Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones,three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation,but it does not require culture preadaptation,use of microcarriers or any other matrices. Over a time course of 4-7 d,hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly,hPSCs maintain pluripotency and karyotype stability for more than ten passages. View Publication

Pluripotent stem cells can be genetically labeled to facilitate differentiation studies. In this paper,we describe a gene-targeting protocol to knock in a GFP cassette into key gene loci in human pluripotent stem cells (hPSCs),and then use the genetically tagged hPSCs to guide in vitro differentiation,immunocytochemical and electrophysiological profiling and in vivo characterization after cell transplantation. The Olig transcription factors have key roles in the transcription regulatory pathways for the genesis of motor neurons (MNs) and oligodendrocytes (OLs). We have generated OLIG2-GFP hPSC reporter lines that reliably mark MNs and OLs for monitoring their sequential differentiation from hPSCs. The expression of the GFP reporter recapitulates the endogenous expression of OLIG genes. The in vitro characterization of fluorescence-activated cell sorting-purified cells is consistent with cells of the MN or OL lineages,depending on the stages at which they are collected. This protocol is efficient and reliable and usually takes 5-7 months to complete. The genetic tagging-differentiation methodology used herein provides a general framework for similar work for differentiation of hPSCs into other lineages. View Publication

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically,hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and,in the long-term,stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and,more importantly,developmental stage-specific differentiation propensity. Here,we report synergistic inhibition of glycogen synthase kinase 3 (GSK3),transforming growth factor β (TGF-β),and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor,GSK3 inhibitor (CHIR99021),and TGF-β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs. View Publication

Chromosomal translocations of the mixed lineage leukemia (MLL) gene are a common cause of acute leukemias. The oncogenic function of MLL fusion proteins is,in part,mediated through aberrant activation of Hoxa genes and Meis1,among others. Here we demonstrate using a tamoxifen-inducible Cre-mediated loss of function mouse model that DOT1L,an H3K79 methyltransferase,is required for both initiation and maintenance of MLL-AF9-induced leukemogenesis in vitro and in vivo. Through gene expression and chromatin immunoprecipitation analysis we demonstrate that mistargeting of DOT1L,subsequent H3K79 methylation,and up-regulation of Hoxa and Meis1 genes underlie the molecular mechanism of how DOT1L contributes to MLL-AF9-mediated leukemogenesis. Our study not only provides the first in vivo evidence for the function of DOT1L in leukemia,but also reveals the molecular mechanism for DOT1L in MLL-AF9 mediated leukemia. Thus,DOT1L may serve as a potential therapeutic target for the treatment of leukemia caused by MLL translocations. View Publication

In addition to the well-recognized role in extracellular matrix remodeling,the tissue inhibitor of metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biologic functions,including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of HSCs,whose biologic behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1(-/-) mice have decreased BM cellularity and,consistent with this finding,TIMP-1(-/-) HSCs display reduced capability of long-term repopulation. Interestingly,the cell cycle distribution of TIMP-1(-/-) stem cells appears distorted,with a dysregulation at the level of the G(1) phase. TIMP-1(-/-) HSCs also display increased levels of p57,p21,and p53,suggesting that TIMP-1 could be intrinsically involved in the regulation of HSC cycling dynamics. Of note,TIMP-1(-/-) HSCs present decreased levels of CD44 glycoprotein,whose expression has been proven to be controlled by p53,the master regulator of the G(1)/S transition. Our findings establish a role for TIMP-1 in regulating HSC function,suggesting a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu. View Publication