技术资料

Dosage compensation of the X chromosomes in mammals is performed via the formation of facultative heterochromatin on extra X chromosomes in female somatic cells. Facultative heterochromatin of the inactivated X (Xi),as well as constitutive heterochromatin,replicates late during the S-phase. It is generally accepted that Xi is always more compact in the interphase nucleus. The dense chromosomal folding has been proposed to define the late replication of Xi. In contrast to mouse pluripotent stem cells (PSCs),the status of X chromosome inactivation in human PSCs may vary significantly. Fluorescence in situ hybridization with a whole X-chromosome- specific DNA probe revealed that late-replicating Xi may occupy either compact or dispersed territory in human PSCs. Thus,the late replication of the Xi does not depend on the compactness of chromosome territory in human PSCs. However,the Xi reactivation and the synchronization in the replication timing of X chromosomes upon reprogramming are necessarily accompanied by the expansion of X chromosome territory. View Publication

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Cancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor 1 (ANTXR1) has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f(+)/EpCAM(-)/Lin(-)) expressed higher levels of ANTXR1 compared with mature luminal cells. CD49f(+)/EpCAM(-),CD44(+)/EpCAM(-),CD44(+)/CD24(-),or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44(+)/CD24(-)/ANTXR1(+) cells displayed enhanced self-renewal as measured by mammosphere assay compared with CD44(+)/CD24(-)/ANTXR1(-) cells. Activation of ANTXR1 by its natural ligand C5A,a fragment of collagen VI $$3,increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor-lipoprotein receptor-related protein 6 (LRP6),phosphorylation of GSK3$$/$$,and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression,self-renewal,invasion,tumorigenicity,and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A,which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Furthermore,ANTXR1 is among the 10% most overexpressed genes in breast cancer and is coexpressed with collagen VI. Thus,ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment,linking it to a stemness signaling network that drives metastatic progression. View Publication

PURPOSE: To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM).backslashnbackslashnMETHODS: Sixty-nine rats (38 male,31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1,6,and 12 months after surgery. Both ocular tissues and systemic organs (brain,liver,kidneys,spleen,heart,and lungs) were fixed in 4% paraformaldehyde,embedded in paraffin,and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker),anti-TRA-1-85 (human cell marker),anti-Ki67 (proliferation marker),anti-CD68 (macrophage),and anti-cytokeratin (epithelial marker).backslashnbackslashnRESULTS: The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P textless 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group.backslashnbackslashnCONCLUSIONS: hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects. View Publication

Metformin is the first-line drug treatment for type 2 diabetes. Globally,over 100 million patients are prescribed this drug annually. Metformin was discovered before the era of target-based drug discovery and its molecular mechanism of action remains an area of vigorous diabetes research. An improvement in our understanding of metformin's molecular targets is likely to enable target-based identification of second-generation drugs with similar properties,a development that has been impossible up to now. The notion that 5' AMP-activated protein kinase (AMPK) mediates the anti-hyperglycaemic action of metformin has recently been challenged by genetic loss-of-function studies,thrusting the AMPK-independent effects of the drug into the spotlight for the first time in more than a decade. Key AMPK-independent effects of the drug include the mitochondrial actions that have been known for many years and which are still thought to be the primary site of action of metformin. Coupled with recent evidence of AMPK-independent effects on the counter-regulatory hormone glucagon,new paradigms of AMPK-independent drug action are beginning to take shape. In this review we summarise the recent research developments on the molecular action of metformin. View Publication

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had textgreater1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization,two iPSC lines from each subject were selected,differentiated into postmitotic neurons,and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs,iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover,repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism. View Publication

We previously demonstrated that RARα2 expression is increased in CD138 selected plasma cells of relapsed multiple myelomas (MMs),and increased expression was linked to poor prognosis in newly diagnosed MM patients. In the present study,we demonstrate that increased RARα2 confers myeloma stem cell features. Higher expression of RARα2 was identified in the multiple myeloma stem cell (MMSC) fraction. Overexpression of RARα2 in bulk MM cell lines resulted in: 1) increased drug resistance; 2) increased clonogenic potential; 3) activation of both Wnt and Hedgehog (Hh) pathways; 4) increased side population and aldehyde dehydrogenase levels; and 5) increased expression of embryonic stem cell genes. The opposite effects were seen with RARα2 knockdown. We demonstrate that RARα2 induces drug resistance by activating the drug efflux pump gene ABCC3 and anti-apoptotic Bcl-2 family members. Inhibition of Wnt signaling or ABCC3 function could overcome drug resistance in RARα2 overexpressing MM cells. We also showed that in the 5TGM1 mouse model,targeting of the Wnt and Hh pathways using CAY10404,cyclopamine,or itraconazole significantly reduced the myeloma tumor burden and increased survival. Targeting RARα2 or its downstream signaling pathways provides a potential strategy to eliminate MMSC. View Publication

Neural crest (NC) cells contribute to the development of many complex tissues of all three germ layers during embryogenesis,and its abnormal development accounts for several congenital birth defects. Generating NC cells-including specific subpopulations such as cranial,cardiac,and trunk NC cells-from human pluripotent stem cells will provide a valuable model system to study human development and disease. Here,we describe a rapid and robust NC differentiation method called LSB-short" that is based on dual SMAD pathway inhibition. This protocol yields high percentages of NC cell populations from multiple human induced pluripotent stem and human embryonic stem cell lines in 8 days. The resulting cells can be propagated easily� View Publication

Here,we show for the first time,that the familial breast/ovarian cancer susceptibility gene BRCA1 activates the Notch pathway in breast cells by transcriptional upregulation of Notch ligands and receptors in both normal and cancer cells. We demonstrate through chromatin immunoprecipitation assays that BRCA1 is localized to a conserved intronic enhancer region within the Notch ligand Jagged-1 (JAG1) gene,an event requiring $$Np63. We propose that this BRCA1/$$Np63-mediated induction of JAG1 may be important the regulation of breast stem/precursor cells,as knockdown of all three proteins resulted in increased tumoursphere growth and increased activity of stem cell markers such as Aldehyde Dehydrogenase 1 (ALDH1). Knockdown of Notch1 and JAG1 phenocopied BRCA1 knockdown resulting in the loss of Estrogen Receptor-$$ (ER-$$) expression and other luminal markers. A Notch mimetic peptide could activate an ER-$$ promoter reporter in a BRCA1-dependent manner,whereas Notch inhibition using a $$-secretase inhibitor reversed this process. We demonstrate that inhibition of Notch signalling resulted in decreased sensitivity to the anti-estrogen drug Tamoxifen but increased expression of markers associated with basal-like breast cancer. Together,these findings suggest that BRCA1 transcriptional upregulation of Notch signalling is a key event in the normal differentiation process in breast tissue. View Publication

The SRY-related HMG-box family of transcription factors member SOX2 regulates stemness and pluripotency in embryonic stem cells and plays important roles during early embryogenesis. More recently,SOX2 expression was documented in several tumor types including ovarian carcinoma,suggesting an involvement of SOX2 in regulation of cancer stem cells (CSC). Intriguingly,however,studies exploring the predictive value of SOX2 protein expression with respect to histopathologic and clinical parameters report contradictory results in individual tumors,indicating that SOX2 may play tumor-specific roles. In this report,we analyze the functional relevance of SOX2 expression in human ovarian carcinoma. We report that in human serous ovarian carcinoma (SOC) cells,SOX2 expression increases the expression of CSC markers,the potential to form tumor spheres,and the in vivo tumor-initiating capacity,while leaving cellular proliferation unaltered. Moreover,SOX2-expressing cells display enhanced apoptosis resistance in response to conventional chemotherapies and TRAIL. Hence,our data show that SOX2 associates with stem cell state in ovarian carcinoma and induction of SOX2 imposes CSC properties on SOC cells. We propose the existence of SOX2-expressing ovarian CSCs as a mechanism of tumor aggressiveness and therapy resistance in human SOC. View Publication