技术资料

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or,alternatively,induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects,we focused on SNON (SKIL),a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast,enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus,our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment. View Publication

Human induced pluripotent stem cells (HIPSC) have tremendous value as a source of autologous cells for cellular transplantation in the treatment of degenerative diseases. The protocols described here address methods for large-scale genetic modification of HIPSCs. The first is an optimized method for transfecting HIPSCs cultured in feeder-free conditions. The second method allows nucleofection of trypsinized HIPSCs at an optimal cell density. Both methods enable robust generation of stable HIPSC transfectants within two weeks. Our protocols are highly reproducible and do not require optimization for individual HIPSC and human embryonic stem cell (HESC) lines. View Publication

Human embryonic stem cells (hESC) are difficult to adapt to 96-well plate assays,such as the MTT assay,because they survive best when plated as colonies,which are not easily counted and plated accurately. Two methods were developed to address this problem. In the first,ROCK inhibitor (ROCKi) was used,which allows accurate counting and plating of single hESC. In the second,small colonies were plated without ROCKi but with adaptations for accurate counting and plating. The MTT assay was also adapted for use with mouse neural stem cells. These methods allow the MTT assay to be conducted rapidly and accurately with high reproducibility between replicate experiments. When screening volatile chemicals in a 96-well plate,vapor effects may occur and dose ranges must be carefully defined. The methods were validated using the NIH assay guidance tool. These methodss could readily be translated to other 96-well plate assay. View Publication

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find,unexpectedly,that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2,3,5,6,7 and 11),whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP,whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results,hESCs contain a previously undescribed complex comprising TAFs 2,6,7,11 and TBP. Altering the composition of hESC TAFs,either by depleting TAFs that are present or ectopically expressing TAFs that are absent,results in misregulated expression of pluripotency genes and induction of differentiation. Thus,the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001. View Publication

Aim: Reprogramming of somatic cells utilizing viral free methods provide a remarkable method to generate human induced pluripotent stem cells (hiPSCs) for regenerative medicine. In this study,we evaluate developmental ontogeny of cardiomyocytes following induced differentiation of hiPSCs. Main Methods: Fibroblasts were reprogrammed with episomal vectors to generate hiPSC and were subsequently differentiated to cardiomyocytes. Ontogenic development of cardiomyocytes was studied by real-time PCR. Key findings: Human iPSCs derived from episomal based vectors maintain classical pluripotency markers,generate teratomas and spontaneously differentiate into three germ layers in vitro. Cardiomyogenic induction of these hiPSCs efficiently generated cardiomyocytes. Ontogenic gene expression studies demonstrated that differentiation of cardiomyocytes was initiated by increased expression of mesodermal markers,followed by early cardiac committed markers,structural and ion channel genes. Furthermore,our correlation analysis of gene expression studies with human heart demonstrated that pivotal structural genes like cardiac troponin,actinin,myosin light chain maintained a high correlation with ion channel genes indicating coordinated activation of cardiac transcriptional machinery. Finally,microelectrode recordings show that these cardiomyocytes could respond aptly to pharmacologically active drugs. Cardiomyocytes showed a chronotropic response to isoproterenol,reduced Na+ influx with quinidine,prolongation of beating rate corrected field potential duration (cFPD) with E-4031 and reduced beating frequency and shortened cFPD with verapamil. Significance: Our study shows that viral free hiPSCs efficiently differentiate into cardiomyocytes with cardiac-specific molecular,structural,and functional properties that recapitulate developmental ontogeny of cardiogenesis. These results,coupled with the potential to generate patient-specific hiPSC lines hold great promise for the development of in vitro platform for drug pharmacogenomics; disease modeling and regenerative medicine. textcopyright 2012 Elsevier Inc. All rights reserved. View Publication

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet,acquiring donor cells is,in most instances,an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances,as the isolation of urinary cells is simple (30 ml of urine are sufficient),cost-effective and universal (can be applied to any age,gender and race). Moreover,the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential,and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases,including those affecting the kidney. View Publication

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation,proliferation,angiogenesis,and apoptosis. In this study,we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular,we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors,NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells,while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302,which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly,higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4,C-MYC,OCT4,and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as KMOS3") when compared to using four factors ("KMOS"). Furthermore View Publication

Retinitis pigmentosa,other inherited retinal diseases,and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here,we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx+ photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells,facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling. STEM CELLS2013;31:408–414 View Publication

FTIR micro-spectroscopy is a sensitive,non-destructive and label-free method offering diffraction-limited resolution with high signal-to-noise ratios when combined with a synchrotron radiation source. The vibrational signature of individual cells was used to validate an alternative strategy for reprogramming induced pluripotent stem cells generated from amniocytes. The iPSC lines PB09 and PB10,were reprogrammed from the same amniocyte cell line using respectively the Oct54,Sox2,Lin28,and Nanog and the Oct4 and Sox2 transcription factor cocktail. We show that cells reprogrammed by the two different sets of transfection factors have similar spectral signatures after reprogramming,except for a small subpopulation of cells in one of the cell lines. Mapping HeLa cells at subcellular resolution,we show that the Golgi apparatus,the cytoplasm and the nucleus have a specific spectral signature. The CH(3):CH(2) ratio is the highest in the nucleus and the lowest in the Golgi apparatus/endoplasmic reticulum,in agreement with the membrane composition of these organelles. This is confirmed by specific staining of the organelles with fluorescent dyes. Subcellular differentiation of cell compartments is also demonstrated in living cells. View Publication

In metazoans,the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes,by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific,while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps,we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover,cLADs are depleted of synteny breakpoints,pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically,the overall sequence conservation is low for cLADs. Instead,cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this A/T rule" in embryonic stem cells View Publication

Development of pharmaceutical agents for cardiac indication demands elaborate safety screening in which assessing repolarization of cardiac cells remains a critical path in risk evaluations. An efficient platform for evaluating cardiac repolarization in vitro significantly facilitates drug developmental programs. In a proof of principle study,we examined the effect of antiarrhythmogenic drugs (Vaughan Williams class I-IV) and noncardiac active drugs (terfenadine and cisapride) on the repolarization profile of viral-free human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Extracellular field potential (FP) recording using microelectrode arrays demonstrated significant delayed repolarization as prolonged corrected FP durations (cFPDs) by class I (quinidine and flecainide),class III (sotalol and amiodarone),and class IV (verapamil),whereas class II drugs (propranolol and nadolol) had no effects. Consistent with their sodium channel-blocking ability,class I drugs also significantly reduced FPmin and conduction velocity. Although lidocaine (class IB) had no effects on cFPDs,verapamil shortened cFPD and FPmin by 25 and 50%,respectively. Furthermore,verapamil reduced beating frequencies drastically. Importantly,the examined drugs exhibited dose-response curve on prolongation of cFPDs at an effective range that correlated significantly with therapeutic plasma concentrations achieved clinically. Consistent with clinical outcomes,drug-induced arrhythmia of tachycardia and bigeminy-like waveforms by quinidine,flecainide,and sotalol was demonstrated at supraphysiological concentrations. Furthermore,off-target effects of terfenadine and cisapride on cFPD and Na( + ) channel blockage were similarly revealed. These results suggest that hiPSC-CMs may be useful for safety evaluation of cardioactive and noncardiac acting drugs for personalized medicine. View Publication

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine,especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells,but these predominantly rely on biochemical cues. Recently,differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However,the means by which these topographical cues improve neuronal yield remains unknown. In this study,we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 ??m gratings increase the rate of neural differentiation,and that an additional culture period of 2 ??m gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added,thus generating hPSC-derived neural cells in a more cost effective and efficient manner. ?? 2012 Elsevier Ltd. View Publication