技术资料

Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here,we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages,we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage,we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes,formed large numbers of multilineage and myeloid colonies,and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit,greater than 95% CD45(+) hematopoietic cells could be generated. In addition,we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification,whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development. View Publication

In somatic cells,eroded telomeres can induce DNA double-strand break signaling,leading to a form of replicative senescence or apoptosis,both of which are barriers to tumorigenesis. However,cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis,enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction,and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs,2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably,these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions,or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together,our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction. View Publication

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However,our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore,we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types,n = 792) by immunohistochemical staining. Using the ALDEFUOR assay,ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition,an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct,and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers,we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439,p = 0.0036). Finally,ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker,ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast,lung,ovarian or colon cancer. View Publication

Accumulating evidence suggests that the regulation of gene expression by histone lysine methylation is crucial for several biological processes. The histone lysine methyltransferase G9a is responsible for the majority of dimethylation of histone H3 at lysine 9 (H3K9me2) and is required for the efficient repression of developmentally regulated genes during embryonic stem cell differentiation. However,whether G9a plays a similar role in adult cells is still unclear. We identify a critical role for G9a in CD4(+) T helper (Th) cell differentiation and function. G9a-deficient Th cells are specifically impaired in their induction of Th2 lineage-specific cytokines IL-4,IL-5,and IL-13 and fail to protect against infection with the intestinal helminth Trichuris muris. Furthermore,G9a-deficient Th cells are characterised by the increased expression of IL-17A,which is associated with a loss of H3K9me2 at the Il17a locus. Collectively,our results establish unpredicted and complex roles for G9a in regulating gene expression during lineage commitment in adult CD4(+) T cells. View Publication

First developed for hematologic disorders,the concept of cancer stem cells (CSCs) was expanded to solid tumors,including colorectal cancer (CRC). The traditional model of colon carcinogenesis includes several steps that occur via mutational activation of oncogenes and inactivation of tumor suppressor genes. Intestinal epithelial cells exist for a shorter amount of time than that required to accumulate tumor-inducing genetic changes,so researchers have investigated the concept that CRC arises from the long-lived stem cells,rather than from the differentiated epithelial cells. Colon CSCs were originally identified through the expression of the CD133 glycoprotein using an antibody directed to its epitope AC133. It is not clear if CD133 is a marker of colon CSCs-other cell surface markers,such as epithelial-specific antigen,CD44,CD166,Musashi-1,CD29,CD24,leucine-rich repeat-containing G-protein-coupled receptor 5,and aldehyde dehydrogenase 1,have been proposed. In addition to initiating and sustaining tumor growth,CSCs are believed to mediate cancer relapse after chemotherapy. How can we identify and analyze colon CSCs and what agents are being designed to kill this chemotherapy-refractory population? View Publication

The vast majority of cancer-related death is due to the metastatic spread of the primary tumour. Circulating tumour cells (CTC) are essential for establishing metastasis and their detection has long been considered as a possible tool to assess the aggressiveness of a given tumour and its potential of subsequent growth at distant organs. Conventional markers are not reliable in detecting occult metastasis and,for example,fail to identify approximately 40% of cancer patients in need of more aggressive or better adjusted therapies. Recent studies in metastatic breast cancer have shown that CTC detection can be used as a marker for overall survival and assessment of the therapeutic response. The benefits of CTC detection in early breast cancer and other solid tumours need further validation. Moreover,optimal CTC detection techniques are the subject of controversy as several lack reproducibility,sensitivity and/or specificity. Recent technical advances allow CTC detection and characterization at the single-cell level in the blood or in the bone marrow. Their reproducibility propels the use of CTC in cancer staging and real-time monitoring of systemic anticancer therapies in several large clinical trials. CTC assays are being integrated in large clinical trials to establish their potential in the management of cancer patients and improve our understanding of metastasis biology. This review will focus on the techniques currently used,the technical advancements made,the limitations of CTC detection and future perspectives in this field. View Publication

Trib1,Trib2,and Trib3 are mammalian homologs of Tribbles,an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice,whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members,we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia,whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor,CCAAT/enhancer-binding protein-alpha (C/EBPalpha),is important for leukemogenesis. Similar to Trib2,Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast,Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha,which account for their differential ability to induce leukemia. View Publication

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5,were cross resistant to other small-molecule CCR5 antagonists,and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4(+) T cells. The V3 loop contained residues essential for viral resistance to APL,while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However,these mutations were context dependent,being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N' terminus of CCR5 in the presence of APL. In addition,the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However,recognition of drug-bound CCR5 was less efficient,resulting in a tropism shift toward effector memory cells upon infection of primary CD4(+) T cells in the presence of APL,with relative sparing of the central memory CD4(+) T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses,then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T(CM) subset of CD4(+) T cells and result in improved T cell homeostasis and immune function. View Publication

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences,highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date,alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells,the transfer of intact viral genomes is unacceptable for clinical applications,due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins,which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover,intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements,we were able to generate a self-inactivating (SIN) alpharetroviral vector,whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection. View Publication

Using a high-throughput chemical screen,we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action,we discovered an essential role of E-cadherin signaling for ESC survival. Specifically,we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling,which then leads to a fatal perturbation of integrin signaling. Furthermore,we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally,we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology,growth factor requirement,and sensitivity to enzymatic cell dissociation. View Publication

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could,in theory,contribute to the increased frequency of tumor development observed in p53-deficient mice and humans. In our previous work,we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice,PFTb impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition,we showed that PFTb stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly,however,PFTb treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus,although PFTb administration led to increased numbers of HSCs and HPCs,it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis. View Publication

Microfluidic image cytometry (MIC) has been developed to study phenotypes of various hPSC lines by screening several chemically defined serum/feeder-free conditions. A chemically defined hPSC culture was established using 20 ng mL(-1) of bFGF on 20 microg mL(-1) of Matrigel to grow hPSCs over a week in an undifferentiated state. Following hPSC culture,we conducted quantitative MIC to perform a single cell profiling of simultaneously detected protein expression (OCT4 and SSEA1). Using clustering analysis,we were able to systematically compare the characteristics of various hPSC lines in different conditions. View Publication