技术资料

Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However,existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study,we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis,overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression,target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore,the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications. View Publication

Nuclear factor,erythroid 2-like 2 (Nrf2) is a master transcription factor for cellular defense against endogenous and exogenous stresses by regulating expression of many antioxidant and detoxification genes. Here,we show that Nrf2 acts as a key pluripotency gene and a regulator of proteasome activity in human embryonic stem cells (hESCs). Nrf2 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Nrf2 inhibition impairs both the self-renewal ability of hESCs and re-establishment of pluripotency during cellular reprogramming. Nrf2 activation can delay differentiation. During early hESC differentiation,Nrf2 closely colocalizes with OCT4 and NANOG. As an underlying mechanism,our data show that Nrf2 regulates proteasome activity in hESCs partially through proteasome maturation protein (POMP),a proteasome chaperone,which in turn controls the proliferation of self-renewing hESCs,three germ layer differentiation and cellular reprogramming. Even modest proteasome inhibition skews the balance of early differentiation toward mesendoderm at the expense of an ectodermal fate by decreasing the protein level of cyclin D1 and delaying the degradation of OCT4 and NANOG proteins. Taken together,our findings suggest a new potential link between environmental stress and stemness with Nrf2 and the proteasome coordinately positioned as key mediators. View Publication

INTRODUCTION Advances in the field of stem cells have led to novel avenues for generating induced pluripotent stem cells (iPSCs) from differentiated somatic cells. iPSCs are typically obtained by the introduction of four factors--OCT4,SOX2,KLF4,and cMYC--via integrating vectors. Here,we report the feasibility of a novel reprogramming process based on vectors derived from the non-integrating vaccine strain of measles virus (MV). METHODS We produced a one-cycle MV vector by substituting the viral attachment protein gene with the green fluorescent protein (GFP) gene. This vector was further engineered to encode for OCT4 in an additional transcription unit. RESULTS After verification of OCT4 expression,we assessed the ability of iPSC reprogramming. The reprogramming vector cocktail with the OCT4-expressing MV vector and SOX2-,KLF4-,and cMYC-expressing lentiviral vectors efficiently transduced human skin fibroblasts and formed iPSC colonies. Reverse transcription-polymerase chain reaction and immunostaining confirmed induction of endogenous pluripotency-associated marker genes,such as SSEA-4,TRA-1-60,and Nanog. Pluripotency of derived clones was confirmed by spontaneous differentiation into three germ layers,teratoma formation,and guided differentiation into beating cardiomyocytes. CONCLUSIONS MV vectors can induce efficient nuclear reprogramming. Given the excellent safety record of MV vaccines and the translational capabilities recently developed to produce MV-based vectors now used for cancer clinical trials,our MV vector system provides an RNA-based,non-integrating gene transfer platform for nuclear reprogramming that is amenable for immediate clinical translation. View Publication

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs),induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique,we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly,most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements,including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators,including the reprogramming factors Klf4,Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered,exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes. View Publication

Nucleosome position,density,and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis,we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly,extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters,suggesting developmental stage-specific organization of histone methylation states. View Publication

The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA,nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently,there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions,we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription� View Publication

Human cell transformation is a key step for oncogenic development,which involves multiple pathways; however,the mechanism remains unclear. To test our hypothesis whether cell oncogenic transformation shares some mechanisms with the process of reprogramming non-stem cells to induced pluripotent stem cells (iPSC),we studied the relationship among the key factors for promoting or inhibiting iPSC in radiation-transformed human epithelial cell lines derived from different tissues (lung,breast and colon). We unexpectedly found that p63 and OCT4 were highly expressed (accompanied by low expressed p53 and miR-34a) in all transformed cell lines examined when compared with their non-transformed counterparts. We further elucidated the relationship of these factors: the 3p strand of miR-34a directly targeted OCT4 by binding to the 3′ untranslated region (3′-UTR) of OCT4 and,OCT4,in turn,stimulated p63 but inhibited p53 expression by binding to a specific region of the p63 or p53 promoter. Moreover,we revealed that the effects of OCT4 on promoting cell oncogenic transformation were by affecting p63 and p53. These results support that a positive loop exists in human cells: OCT4 upregulation as a consequence of inhibition of miR-34a,promotes p63 but suppresses p53 expression,which further stimulates OCT4 upregulation by downregulating miR-34a. This functional loop contributes significantly to cell transformation and,most likely,also to the iPSC process. View Publication

Oct4 is critical to maintain the pluripotency of human embryonic stem cells (hESCs); however,the underlying mechanism remains to be fully understood. Here,we report that silencing of Oct4 in hESCs leads to the activation of tumor suppressor p53,inducing the differentiation of hESCs since acute disruption of p53 in p53 conditional knockout (p53CKO) hESCs prevents the differentiation of hESCs after Oct4 depletion. We further discovered that the silencing of Oct4 significantly reduces the expression of Sirt1,a deacetylase known to inhibit p53 activity and the differentiation of ESCs,leading to increased acetylation of p53 at lysine 120 and 164. The importance of Sirt1 in mediating Oct4-dependent pluripotency is revealed by the finding that the ectopic expression of Sirt1 in Oct4-silenced hESCs prevents p53 activation and hESC differentiation. In addition,using knock-in approach,we revealed that the acetylation of p53 at lysine 120 and 164 is required for both stabilization and activity of p53 in hESCs. In summary,our findings reveal a novel role of Oct4 in maintaining the pluripotency of hESCs by suppressing pathways that induce differentiation. Considering that p53 suppresses pluripotency after DNA damage response in ESCs,our findings further underscore the stringent mechanism to coordinate DNA damage response pathways and pluripotency pathways in order to maintain the pluripotency and genomic stability of hESCs. 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

The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols,immaturity,and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional,and thereby stable,optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons,and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level. View Publication

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm,which is manifested by rosette formation,with consecutive differentiation into neural progenitors and early glial-like cells. In this study,we examined the involvement of early neural markers - OTX2,PAX6,Sox1,Nestin,NR2F1,NR2F2,and IRX2 - in the onset of rosette formation,during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation,which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation,when rosettes comprise no more than 3-5 cells,and that its expression precedes that of established markers of early neuronal differentiation. Importantly,the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly,we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro,and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice. ?? 2013 Elsevier B.V. View Publication

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy,drug screening,physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods,techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results,we adopted a polycistronic cassette encoding Thomson's cocktail OCT4,NANOG,SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones,based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes,ONSL and OCT4,SOX2,KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly,in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM,we indeed observed a remarkable synergy,yielding a reprogramming efficiency of textgreater2%. We present here a drastic improvement of the reprogramming efficiency,which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore,non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency. View Publication