技术资料

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells,which also expressed Oct4,SSEA-3 and SSEA-4. We also found another population expressing SSEA-4,but without Nanog,Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog,Oct4,SSEA-3,SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block,the cell cycle of hESC normalised,but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition,the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle,which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4. 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

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs),but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs,sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study,we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK,ATP in single hESC-VCMs was dormant under baseline conditions,but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ˜8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly,sarcIK,ATP displayed a ˜3-fold increase after treatment with hypoxia (5% O2). MitoIK,ATP was absent in hESC-VCMs. However,the thyroid hormone T3 up-regulated mitoIK,ATP,conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system,T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK,ATP. View Publication

Regenerative medicine,relying on human embryonic stem cell (hESC) technology,opens promising new avenues for therapy of many severe diseases. However,this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress,growth rates,and heterogeneous cellular states under current culture conditions. In this study,we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states,precisely control growth rates,significantly increase cell production,and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple,robust,scalable,and suitable for high-throughput screening and drug discovery. View Publication

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However,upgrading them to pluripotency confers refractoriness toward senescence,higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling,such as Down syndrome or $\$-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing,feeder-dependent culture. Here,we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium,a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4,Nanog,Sox2,SSEA-1,SSEA-4,TRA-1-60,TRA-1-81 in a pattern typical for human primed PSC. Additionally,the cells formed teratomas,and were deemed pluripotent by PluriTest,a global expression microarray-based in-silico pluripotency assay. However,we found that the PluriTest scores were borderline,indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology,non-integrating reprogramming and chemically defined culture are more acceptable. View Publication

Human pluripotent stem (hPS) cells such as human embryonic stem (hES) and induced pluripotent stem (hiPS) cells are vulnerable under single cell conditions,which hampers practical applications; yet,the mechanisms underlying this cell death remain elusive. In this paper,we demonstrate that treatment with a specific inhibitor of non-muscle myosin II (NMII),blebbistatin,enhances the survival of hPS cells under clonal density and suspension conditions,and,in combination with a synthetic matrix,supports a fully defined environment for self-renewal. Consistent with this,genetically engineered mouse embryonic stem cells lacking an isoform of NMII heavy chain (NMHCII),or hES cells expressing a short hairpin RNA to knock down NMHCII,show greater viability than controls. Moreover,NMII inhibition increases the expression of self-renewal regulators Oct3/4 and Nanog,suggesting a mechanistic connection between NMII and self-renewal. These results underscore the importance of the molecular motor,NMII,as a novel target for chemically engineering the survival and self-renewal of hPS cells. View Publication

Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity. After discrimination of species origins of the NGS reads based on SNVs,the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach,as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition,incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results,we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol,which will improve both accuracy and comparability of NGS results across different sample batches,laboratories and NGS platforms. View Publication

Human embryonic stem cells (hESCs) are a promising cell source for tissue engineering and regenerative medicine,but before they can be used in therapies,we must be able to accurately identify the state and progeny of hESCs. One of the most commonly used methods for identification is flow cytometry. Many flow cytometry applications use antibodies to detect the amount of antigen present on/in a cell. This allows for the identification of unique cell populations or the tracking of expression changes within a population during differentiation. The results are typically presented as a percentage of positively expressing cells (%Pos) for a marker of choice,relative to a negative control. However,this reporting term is vulnerable to distortion from outliers and inaccuracy from loss of information about the population's fluorescence intensity. In this article,we describe an alternate strategy that uses the normalized median fluorescence intensity (nMFI),in which the MFI of the stained sample is normalized to the MFI of the negative control,as the reporting term to more accurately describe a population of cells in culture. We observed that nMFI provides a more accurate representation for the quality of a starting population and comparing data of different experimental runs. In addition,we demonstrated that the nMFI is a more sensitive measure of pluripotent and differentiation markers expression changes during hESC differentiation into three germ layer lineages. View Publication

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However,human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here,we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus,restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs. View Publication

Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here,we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did,however,require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-catenin protein levels in stem and progenitor cells. View Publication

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

CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical,iPSC generation must be rapid and efficient. Therefore,we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector,rCLAE-R6,that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently,the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% ($$(A)/[$$(S)+$$(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected,patient-specific iPSCs for therapeutic applications. View Publication