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Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration,conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC),which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation,and this hydroxymethylation change is critical for optimal epigenetic reprogramming,but does not compromise primed pluripotency. Compared with hESCs,we find that iPSCs tend to form large-scale (100 kb–1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions,most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly,these 5hmC aberrant hotspots largely coincide (∼ 80%) with aberrant iPSC–ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC–hESC differences. View Publication

A satisfactory in vitro model of vocal fold mucosa does not exist,thus precluding a systematic,controlled study of vocal fold biology and biomechanics. We sought to create a valid,reproducible three-dimensional (3D) in vitro model of human origin of vocal fold mucosa of human origin. We hypothesized that coculture of human embryonic stem cell (hESC)-derived simple epithelial cells with primary vocal fold fibroblasts under appropriate conditions would elicit morphogenesis of progenitor cells into vocal fold epithelial-like cells and creation of a basement membrane. Using an in vitro prospective study design,hESCs were differentiated into cells that coexpressed the simple epithelial cell marker,keratin 18 (K18),and the transcription factor,p63. These simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. The cells were cultured for 3 weeks in a keratinocyte medium at an air–liquid interface. After that time,the engineered mucosa demonstrated a stratified,squamous epithelium and a continuous basement membrane recapitulating the key morphologic and phenotypic characteristics of native vocal fold mucosa. hESC-derived epithelial cells exhibited positive staining for vocal fold stratified,squamous epithelial markers,keratin 13 (K13) and 14 (K14),as well as tight junctions,adherens junctions,gap junctions,and desmosomes. Despite the presence of components critical for epithelial structural integrity,the epithelium demonstrated greater permeability than native tissue indicating compromised functional integrity. While further work is warranted to improve functional barrier integrity,this study demonstrates that hESC-derived epithelial progenitor cells can be engineered to create a replicable 3D in vitro model of vocal fold mucosa featuring a multilayered,terminally differentiated epithelium. View Publication

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results,NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2,LMX1A,TH,GIRK2,PITX3,EN1,NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue,with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation,the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future. View Publication

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue,but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus,fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies,but that of BOECs was lower. In terms of invasiveness of biopsy sampling,biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs,but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative. View Publication

Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end,we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing,chromatin immunoprecipitation sequencing,and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition,we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches,leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions. ?? 2013 Elsevier Inc. View Publication

Neural stem cells (NSCs) possess immunosuppressive characteristics,but effects of NSCs on human dendritic cells (DCs),the most important antigen presenting cells,are less well studied. We used an in vitro approach to evaluate the effects of human NSCs on differentiation of human blood CD14+ monocytes into DCs. NSCs derived from H1 human embryonic stem cells (hESC-NSCs) and human ReNcell NSC line,as well as human bone marrow derived mesenchymal stem cells (MSCs),were tested. We observed that in response to treatment with interleukin-4 and granulocyte macrophage colony-stimulating factor CD14+ monocytes co-cultured with NSCs were able to down-regulate CD14 and up-regulate the differentiation marker CD1a,whereas MSC co-culture strongly inhibited CD1a expression and supported prolonged expression of CD14. A similar difference between NSCs and MSCs was noted when lipopolysaccharides were included to induce maturation of monocyte-derived DCs. However,when effects on the function of derived DCs were investigated,NSCs suppressed the elevation of the DC maturation marker CD83,although not the up-regulation of costimulatory molecules CD80,CD86 and CD40,and impaired the functional capacity of the derived DCs to stimulate alloreactive T cells. We did not observe any obvious difference between hESC-NSCs and ReNcell NSCs in inhibiting DC maturation and function. Our data suggest that although human NSCs are less effective than human MSCs in suppressing monocyte differentiation into DCs,these stem cells can still affect the function of DCs,ultimately regulating specific immune responses. View Publication

Aging is known to be the single most important risk factor for multiple diseases. Sirtuin 6,or SIRT6,has recently been identified as a critical regulator of transcription,genome stability,telomere integrity,DNA repair,and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging,we demonstrated that human dermal fibroblasts (HDFs) from older human subjects were more resistant to reprogramming by classic Yamanaka factors than those from younger human subjects,but the addition of SIRT6 during reprogramming improved such efficiency in older HDFs substantially. Despite the importance of SIRT6,little is known about the molecular mechanism of its regulation. We show,for the first,time posttranscriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop,which has implications for the modulation of SIRT6 expression in reprogramming of aging cells. View Publication

Human embryonic stem cells and mouse epiblast stem cells represent a primed pluripotent stem cell state that requires TGF-β/activin signaling. TGF-β and/or activin are commonly thought to regulate transcription through both Smad2 and Smad3. However,the different contributions of these two Smads to primed pluripotency and the downstream events that they may regulate remain poorly understood. We addressed the individual roles of Smad2 and Smad3 in the maintenance of primed pluripotency. We found that Smad2,but not Smad3,is required to maintain the undifferentiated pluripotent state. We defined a Smad2 regulatory circuit in human embryonic stem cells and mouse epiblast stem cells,in which Smad2 acts through binding to regulatory promoter sequences to activate Nanog expression while in parallel repressing autocrine bone morphogenetic protein signaling. Increased autocrine bone morphogenetic protein signaling caused by Smad2 down-regulation leads to cell differentiation toward the trophectoderm,mesoderm,and germ cell lineages. Additionally,induction of Cdx2 expression,as a result of decreased Smad2 expression,leads to repression of Oct4 expression,which,together with the decreased Nanog expression,accelerates the loss of pluripotency. These findings reveal that Smad2 is a unique integrator of transcription and signaling events and is essential for the maintenance of the mouse and human primed pluripotent stem cell state. View Publication

Reprogramming of somatic cells into patient-specific pluripotent analogues of human embryonic stem cells (ESCs) emerges as a prospective therapeutic angle in molecular medicine and a tool for basic stem cell biology. However,the combination of relative inefficiency and high variability of non-defined culture conditions precluded the use of this technique in a clinical setting and impeded comparability between laboratories. To overcome these obstacles,we sequentially devised a reprogramming protocol using one lentiviral-based polycistronic reprogramming construct,optimized for high co-expression of OCT4,SOX2,KLF4 and MYC in conjunction with small molecule inhibitors of non-permissive signaling cascades,such as transforming growth factor $\$(SB431542),MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin),in a defined extracellular environment. Based on human fetal liver fibroblasts we could efficiently derive induced pluripotent stem cells (iPSCs) within 14 days. We attained efficiencies of up to 10.97±1.71% resulting in 79.5- fold increase compared to non-defined reprogramming using four singular vectors. We show that the overall increase of efficiency and temporal kinetics is a combinatorial effect of improved lentiviral vector design,signaling inhibition and definition of extracellular matrix (Matrigel®) and culture medium (mTESR®1). Using this protocol,we could derive iPSCs from patient fibroblasts,which were impermissive to classical reprogramming efforts,and from a patient suffering from familial platelet disorder. Thus,our defined protocol for highly efficient reprogramming to generate patient-specific iPSCs,reflects a big step towards therapeutic and broad scientific application of iPSCs,even in previously unfeasible settings. View Publication

The nucleolus is a prominent subnuclear structure whose major function is the transcription and assembly of ribosome subunits. The size of the nucleolus varies with the cell cycle,proliferation rate and stress. Changes in nucleolar size,number,chemical composition,and shape can be used to characterize malignant cells. We used spontaneous Raman microscopy as a label-free technique to examine nucleolar spatial and chemical features. Raman images of the 1003 cm(-1) phenylalanine band revealed large,well-defined subnuclear protein structures in MFC-7 breast cancer cells. The 783 cm(-1) images showed that nucleic acids were similarly distributed,but varied more in intensity,forming observable high-intensity regions. High subnuclear RNA concentrations were observed within some of these regions as shown by 809 cm(-1) Raman band images. Principal component analyses of sub-images and library spectra validated the subnuclear presence of RNA. They also revealed that an actin-like protein covaried with DNA within the nucleolus,a combination that accounted for 64% or more of the spectral variance. Embryonic stem cells are another rapidly proliferating cell type,but their nucleoli were not as large or well defined. Estimating the size of the larger MCF-7 nucleolus was used to show the utility of Raman microscopy for morphometric analyses. It was concluded that imaging based on Raman microscopy provides a promising new method for the study of nucleolar function and organization,in the evaluation of drug and experimental effects on the nucleolus,and in clinical diagnostics and prognostics. View Publication

AbstractIntroduction
Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited,there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast,and express HLA-G,a marker of EVT. The goals of the present study were to further characterize the HLA-G+ cells derived from BMP4-treated hESC,and determine their suitability as a model.
Methods
HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G+ and HLA-G−/TACSTD2+ populations with immunomagnetic beads and expression profiles analyzed by microarray.
Results
There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent,stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7,and the majority were also HLA-G+. Gene expression profiles revealed that HLA-G+,BMP4-treated hESC were similar to,but distinct from,HLA-G+ cells isolated from first trimester placentas. Whereas HLA-G+ and HLA-G− cells from first trimester placentas had highly divergent gene expression profiles,HLA-G+ and HLA-G− cells from BMP4-treated hESC had somewhat similar profiles,and both expressed genes characteristic of early trophoblast development.
Conclusions
We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage. View Publication

BackgroundmicroRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years,the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.Methodology/ResultsUsing a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG,Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle,cell proliferation,p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p,hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).ConclusionOur findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers. View Publication