技术资料

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However,it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here,we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days,at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis,thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional,morphological and functional signatures of differentiated neurons,with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons,suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types. View Publication

Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here,we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air-liquid interface for 19-21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology,gene expression,and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and,therefore,offers a promising approach to studying vocal fold biology and biomechanics in health and disease. View Publication

The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore,we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs,RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129-5p may play a role in promoting differentiation,while down-regulated miRNAs such as miR-367,miR-18b,and miR-20b are implicated in cell proliferation. Subsequent miRNA-target and network analysis revealed that these miRNAs are involved in cellular development,cell cycle progression,cell death,and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis,eye differentiation and development. View Publication

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism,pantothenate and CoA biosynthesis,glutathione metabolism,and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information,including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. View Publication

Cryopreservation is an essential technique to preserve stem cells,semipermanently sustaining their potentials. There are two main approaches of cryopreservation for human pluripotent stem cells (hPSCs). The first is the vitrification,which involves instantaneous freeze and thaw of hPSCs. The second is the conventional slow-cooling method and a rapid thaw. Both cryopreservation protocols have been standardized and optimized to yield high survivability of hPSCs. View Publication

Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study,we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures,single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells,corresponding to an increased expression of pluripotency markers OCT4 and NANOG,and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed,aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols,with induction of primitive streak cells using bone morphogenetic protein 4 and activin A,followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5,thus indicating the production of large numbers of immature cardiomyocytes (˜65,000/cm(2) or ˜1.5 per input hESC). This protocol was shown to be effective in HES3,H9,and,to a lesser,extent,MEL1 hESC lines. In addition,we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression,whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation,and potentially for the future treatment of heart failure. View Publication

Avian species are important model animals for developmental biology and disease research. However,unlike in mice,where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function,clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs),the first nonmammalian iPSCs,which were clonally isolated and propagated,important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes,indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes,oligodendrocytes,and neurons. Ultimately,qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers,extraembryonic tissues,and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals. View Publication

MED13L is a component subunit of the Mediator complex,an important regulator of transcription that is highly conserved across eukaryotes. Here we report MED13L disruption in a translocation t(12;19) breakpoint of a patient with Pierre-Robin syndrome,moderate intellectual disability (ID),craniofacial anomalies,and muscular defects. The phenotype is similar to previously described patients with MED13L haploinsufficiency. Knockdown of MED13L orthologue in zebrafish,med13b,showed early defective migration of cranial neural crest cells (NCCs) that contributed into cartilage structure deformities in the later stage,recapitulating craniofacial anomalies seen in human patients. Notably,we observed abnormal distribution of developing neurons in different brain regions of med13b morphant embryos,which could be rescued upon introduction of full-length human MED13L mRNA. To compare with mammalian system,we suppressed MED13L expression by short-hairpin RNA in ES-derived human neural progenitors,and differentiated them into neurons. Transcriptome analysis revealed differential expression of components of Wnt and FGF signalling pathways in MED13L-deficient neurons. Our finding provides a novel insight into the mechanism of overlapping phenotypic outcome targeting NCCs derivatives organs in patients with MED13L haploinsufficiency,and emphasizes a clinically recognizable syndromic phenotype in these patients. This article is protected by copyright. All rights reserved. View Publication

BackgroundX-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome,the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates,triggering silencing of the chromosome. In mouse,an alternative Xist promoter,P2 is also the site of YY1 binding,which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation,including absence of a functional antisense regulator Tsix,and absence of strictly paternal inactivation in extraembryonic tissues,prompting us to examine regulatory regions for the human XIST gene.ResultsWe demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However,YY1 binding is insufficient to drive P2 expression or establish the DHS,which may require a development-specific factor. Furthermore,reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.ConclusionsThe differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter,P2,that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition,this region binds YY1 on the unmethylated inactive X chromosome,and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST. View Publication

Human pluripotent stem cells (hPSCs) are proving to be a valuable source of endothelial cells (ECs),pericytes,and vascular smooth muscle cells (vSMCs). Although an increasing number of phenotypic markers are becoming available to determine the phenotypes of these cells in vitro,the ability to integrate and form functional vessels in the host organism,typically mouse,remains critical for the assessment of EC functional competence. However,current mouse models require relatively large numbers of cells that might be difficult to derive simultaneously from multiple hPSCs lines. Therefore,there is an urgent need for new functional assays that are robust and can be performed with small numbers of cells. Here we describe a novel zebrafish xenograft model to test functionality of hPSC-derived ECs. The assay can be performed in 10 days and requires only ˜100-400 human cells per embryo. Thus,the zebrafish xenograft model can be useful for the accurate and rapid assessment of functionality of hPSC-derived ECs in a lower vertebrate model that is widely viewed by regulatory authorities as a more acceptable alternative to adult mice. View Publication

Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass,which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos,as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor,small interfering RNAs,or a dominant-negative approach suppresses Nanog expression,and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1,aka Smarca4 (SWI/SNF-related,matrix-associated,actin-dependent regulator of chromatin,subfamily a,member 4)),a key regulator of ESC self-renewal and pluripotency,in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene,providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.Cell Death and Differentiation advance online publication,22 August 2014; doi:10.1038/cdd.2014.124. View Publication

Incurable neurological disorders such as Parkinson's disease (PD),Huntington's disease (HD),and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases,we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor,valproic acid (VPA),and inhibitor of p160-Rho associated coiled-coil kinase (ROCK),Y-27632,after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology,cell surface antigens,pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542,inhibitors of the SMAD signaling pathway,HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction,neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture,which had the ability to differentiate further into NPCs and neurons,as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays. View Publication