技术资料

The development of the mature epidermis requires a coordinated sequence of signaling events and transcriptional changes to specify surface ectodermal progenitor cells to the keratinocyte lineage. The initial events that specify epidermal keratinocytes from ectodermal progenitor cells are not well understood. Here,we use both developing mouse embryos and human embryonic stem cells (hESCs) to explore the mechanisms that direct keratinocyte fate from ectodermal progenitor cells. We show that both hESCs and murine embryos express p63 before keratin 14. Furthermore,we find that Notch signaling is activated before p63 expression in ectodermal progenitor cells. Inhibition of Notch signaling pharmacologically or genetically reveals a negative regulatory role for Notch signaling in p63 expression during ectodermal specification in hESCs or mouse embryos,respectively. Taken together,these data reveal a role for Notch signaling in the molecular control of ectodermal progenitor cell specification to the epidermal keratinocyte lineage. View Publication

Genomic abnormalities may accumulate in human embryonic stem cells (hESCs) during in vitro maintenance. Characterization of the mechanisms enabling survival and expansion of abnormal hESCs is important due to consequences of genetic changes for the therapeutic utilization of stem cells. Furthermore,these cells provide an excellent model to study transformation in vitro. We report here that the histone deacetylase proteins,HDAC1 and HDAC2,are increased in karyotypically abnormal hESCs when compared to their normal counterparts. Importantly,similar to many cancer cell lines,we found that HDAC inhibitors repress proliferation of the karyotypically abnormal hESCs,whereas normal cells are more resistant to the treatment. The decreased proliferation correlates with downregulation of HDAC1 and HDAC2 proteins,induction of the proliferation inhibitor,cyclin-dependent kinase inhibitor 1A (CDKN1A),and altered regulation of tumor suppressor protein Retinoblastoma 1 (RB1). Through genome-wide transcriptome analysis we have identified genes with altered expression and responsiveness to HDAC inhibition in abnormal cells. Most of these genes are linked to severe developmental and neurological diseases and cancers. Our results highlight the importance of epigenetic mechanisms in the regulation of genomic stability of hESCs,and provide valuable candidates for targeted and selective growth inhibition of karyotypically abnormal cells. textcopyright 2013 Elsevier B.V. View Publication

Human (h) pluripotent stem cells (PSC) such as embryonic stem cells (ESC) can be directed into cardiomyocytes (CMs),representing a potential unlimited cell source for disease modeling,cardiotoxicity screening and myocardial repair. Although the electrophysiology of single hESC-CMs is now better defined,their multi-cellular arrhythmogenicity has not been thoroughly assessed due to the lack of a suitable experimental platform. Indeed,the generation of ventricular (V) fibrillation requires single-cell triggers as well as sustained multi-cellular reentrant events. Although native VCMs are aligned in a highly organized fashion such that electrical conduction is anisotropic for coordinated contractions,hESC-derived CM (hESC-CM) clusters are heterogenous and randomly organized,and therefore not representative of native conditions. Here,we reported that engineered alignment of hESC-VCMs on biomimetic grooves uniquely led to physiologically relevant responses. Aligned but not isotropic control preparations showed distinct longitudinal (L) and transverse (T) conduction velocities (CV),resembling the native human V anisotropic ratio (AR=LCV/TCV=1.8-2.0). Importantly,the total incidence of spontaneous and inducible arrhythmias significantly reduced from 57% in controls to 17-23% of aligned preparations,thereby providing a physiological baseline for assessing arrhythmogenicity. As such,promotion of pro-arrhythmic effect (e.g.,spatial dispersion by ?? adrenergic stimulation) could be better predicted. Mechanistically,such anisotropy-induced electrical stability was not due to maturation of the cellular properties of hESC-VCMs but their physical arrangement. In conclusion,not only do functional anisotropic hESC-VCMs engineered by multi-scale topography represent a more accurate model for efficacious drug discovery and development as well as arrhythmogenicity screening (of pharmacological and genetic factors),but our approach may also lead to future transplantable prototypes with improved efficacy and safety against arrhythmias. ?? 2013. View Publication

Although activated inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs) are potent T cell suppressors,nonactivated IMCs and IDCs promote T cell activation and Th1/Th17 cell differentiation. In this study,we investigated how to reduce the proinflammatory properties of IMCs and IDCs and further convert them into immune regulatory dendritic cells (DCs). We found that IL-4 and retinoic acid (RA) cotreatment of GM-CSF-differentiated IDCs synergistically induced the expression of aldehyde dehydrogenase family 1,subfamily A2,a rate-limiting enzyme for RA synthesis in DCs. IL-4 plus RA-treated IDCs upregulated CD103 expression and markedly reduced the production of proinflammatory cytokines upon activation. IL-4 plus RA-treated IDCs strongly induced CD4�?�Foxp3�?� regulatory T cell differentiation and suppressed Th1 and Th17 differentiation. Mechanistically,the transcription factors Stat6 and RA receptor $$ play important roles in aldehyde dehydrogenase family 1,subfamily A2,induction. In addition,IL-4 and RA signaling pathways interact closely to enhance the regulatory function of treated DCs. Adoptive transfer of IL-4 plus RA-treated DCs significantly increased regulatory T cell frequency in vivo. Direct treatment with IL-4 and RA also markedly suppressed actively induced experimental autoimmune encephalomyelitis. Our data demonstrate the synergistic effect of IL-4 and RA in inducing a regulatory phenotype in IDCs,providing a potential treatment strategy for autoimmune diseases. View Publication

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells,but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC₅₀ = ˜15 ng/mL) by inducing apoptosis,and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations,BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes,such as glucose-regulated protein 78 (GRP78),X-box binding protein 1 (XBP1),and C/EBP homologous protein (CHOP). Finally,we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer. View Publication

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs),the molecular mechanism involved remains to be fully elucidated. Here,we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover,BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together,our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy,however,is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs,implying that activation of JNKs is essential for BMP9 osteoinductive activity. View Publication

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor,cyclopamine,and the activators,Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes. View Publication

BACKGROUND/AIMS [corrected] Embryonic stem cells (ES cells) have the capacity to propagate indefinitely,maintain pluripotency,and differentiate into any cell type under defined conditions. As a result,they are considered to be the best model system for research into early embryonic development. AICA ribonucleotide (AICAR) is an activator of AMP-activated protein kinase (AMPK) that is thought to affect ES cell function,but its role in ES cell fate decision is unclear. METHODS In this study,we performed microarray analysis to investigate AICAR downstream targets and further understand its effect on ES cells. RESULTS Our microarray data demonstrated that AICAR can significantly up-regulate pluripotency-associated genes and down-regulate differentiation-associated transcription factors. Although AICAR cannot maintain ES cell identity without LIF,it can antagonize the action of RA-induced differentiation. Using those differentially expressed genes identified,we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the Database for Annotation,Visualization and Integrated Discovery (DAVID) online system. AICAR was not only shown to influence the AMPK pathway,but also act on other signaling pathways such as BMP,MAPK and TGF-β,to maintain the stemness of J1 ES cells. Furthermore,AICAR modulated ES cell epigenetic modification by altering the expression of epigenetic-associated proteins,including Dnmt3a,Dnmt3b,Smarca2,Mbd3,and Arid1a,or through regulating the transcription of long intervening non-coding RNA (lincRNA). CONCLUSION Taken together,our work suggests that AICAR is capable of maintaining ES cell self-renewal and pluripotency,which could be useful in future medical treatment. View Publication

BACKGROUND To investigate the expression status of PIWIL2 and piR-932 in breast cancer stem cells and the role they could play in tumor cell growth and metastasis through Latexin. METHODS CD44(+)/CD24(-) tumor cells (CSC) from clinical specimens were sorted using flow cytometry. PIWIL2 expression status was detected in CSC cells by microarray analysis and 1086 breast cancer specimens by Western blot and immunohistochemistry staining. piR-932 expression was also detected in CSC cells by piRNA microarray assay. The relationship between the PIWIL2 protein and clinico-pathological parameters and prognosis was subsequently determined. RESULTS CSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. PIWIL2 protein was expressed higher in CSC cells compared to the control cells. In total,334 (30.76%) of the 1086 breast cases showed high PIWIL2 expression. PIWIL2 was observed to be related to age,tumor size,histological type,tumor stage,and lymph node metastasis (all P textless 0.05). Furthermore,we have found that one of the Piwi-interacting RNAs (piRNAs) called piR-932 expressed significantly higher in the breast cancer cells that were induced to EMT,and it could form immune complexes through immunoprecipitation with PIWIL2; in PIWIL2+ breast cancer stem cells,Latexin expression significantly reduced because of its promoter region CpG island methylation. CONCLUSIONS These results suggest that the combination of piR-932 and PIWIL2 may be a positive regulator in the process of breast cancer stem cells through promoting the methylation of Latexin,and they both could be the potential targets for blocking the metastasis of breast cancer. View Publication

Genome-wide association studies (GWASs) have reported many single nucleotide polymorphisms (SNPs) associated with psychiatric disorders,but knowledge is lacking regarding molecular mechanisms. Here we show that risk alleles spanning multiple genes across the 10q24.32 schizophrenia-related locus are associated in the human brain selectively with an increase in the expression of both BLOC-1 related complex subunit 7 (BORCS7) and a previously uncharacterized,human-specific arsenite methyltransferase (AS3MT) isoform (AS3MT(d2d3)),which lacks arsenite methyltransferase activity and is more abundant in individuals with schizophrenia than in controls. Conditional-expression analysis suggests that BORCS7 and AS3MT(d2d3) signals are largely independent. GWAS risk SNPs across this region are linked with a variable number tandem repeat (VNTR) polymorphism in the first exon of AS3MT that is associated with the expression of AS3MT(d2d3) in samples from both Caucasians and African Americans. The VNTR genotype predicts promoter activity in luciferase assays,as well as DNA methylation within the AS3MT gene. Both AS3MT(d2d3) and BORCS7 are expressed in adult human neurons and astrocytes,and they are upregulated during human stem cell differentiation toward neuronal fates. Our results provide a molecular explanation for the prominent 10q24.32 locus association,including a novel and evolutionarily recent protein that is involved in early brain development and confers risk for psychiatric illness. View Publication

BACKGROUND Alcohol abuse produces an enormous impact on health,society,and the economy. Currently,there are very limited therapies available,largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. RESULTS Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs,but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition,a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover,a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells. CONCLUSIONS This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models. View Publication

HCMV is a highly sophisticated virus that has developed various mechanisms for immune evasion and viral dissemination throughout the body (partially mediated by neutrophils). NK cells play an important role in elimination of HCMV-infected cells. Both neutrophils and NK cells utilize similar sets of chemokine receptors to traffic,to and from,various organs. However,the mechanisms by which HCMV attracts neutrophils and not NK cells are largely unknown. Here,we show a unique viral protein,vCXCL1,which targets three chemokine receptors: CXCR1 and CXCR2 expressed on neutrophils and CXCR1 and CX3CR1 expressed on NK cells. Although vCXCL1 attracted both cell types,neutrophils migrated faster and more efficiently than NK cells through the binding of CXCR2. Therefore,we propose that HCMV has developed vCXCL1 to orchestrate its rapid systemic dissemination through preferential attraction of neutrophils and uses alternative mechanisms to counteract the later attraction of NK cells. View Publication