Embryonic Stem Cell

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Embryonic stem cell-derived cardiomyocytes for the treatment of doxorubicin-induced cardiomyopathy

Embryonic stem cell-derived cardiomyocytes for the treatment of doxorubicin-induced cardiomyopathy

ECG: Electrocardiogram; ECHO: Echocardiogram; EDV: End-diastolic volume; EF: Ejection fraction; ESV: End-systolic volume; FBS: Fetal bovine serum; FDR: False discovery rate; GMEM: Glasgow Minimum Essential Medium; HR: Heart rate; IAM: Iodoacetamide; IC: Intracavitary route; LC-MS/MS: Liquid chromatography tandem mass spectrometry; LIF: Leukemia inhibitory factor; Luc2: Luciferase 2; mESC: Mouse embryonic stem cell; OCT3/4: Octamer-binding transcription factor 3/4; PCR: Polymerase chain reaction; PSM: Peptide sequence match; QTc: Corrected QT; SSEA-1: Stage-specific embryonic antigen-1; SV: Stroke volume; TCM: Total conditioning medium; TUNEL: Deoxynucleotidyl transferase-mediated 2 ′ -deoxyuridine 5 ′ -triphosphate nick end labeling
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Human Embryonic Stem Cell (hESC) and Human Embryo Research

Human Embryonic Stem Cell (hESC) and Human Embryo Research

created embryonic stem cell lines are needed to serve as a comparison with the newly developed lines to establish whether they are indeed equivalent to traditionally developed lines. More- over, although a single cell biopsy may be performed in IVF cases to test for genetic diseases, it is unclear whether it would be appropriate to transfer to a uterus an embryo that underwent such biopsy for the creation of stem cell lines. It is not known whether the biopsy makes the embryo less likely to implant. Women undergoing IVF typically choose to transfer to their uterus embryos with the highest likelihood of implantation and, even- tually, healthy birth. If embryos that have undergone a biopsy for purposes unrelated to health are not going to be chosen for implantation and will be eventually discarded, then the single biopsy procedure does not result in “ sparing ” embryos (although it may result in a delay in destruction). Re- search is ongoing to identify novel and more ef fi cient methods of obtaining stem cells from human embryos, and it is anticipated that this area will continue to evolve.
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Human Embryonic Stem Cell (hESC) and Human Embryo Research

Human Embryonic Stem Cell (hESC) and Human Embryo Research

created embryonic stem cell lines are needed to serve as a comparison with the newly developed lines to establish whether they are indeed equivalent to traditionally developed lines. More- over, although a single cell biopsy may be performed in IVF cases to test for genetic diseases, it is unclear whether it would be appropriate to transfer to a uterus an embryo that underwent such biopsy for the creation of stem cell lines. It is not known whether the biopsy makes the embryo less likely to implant. Women undergoing IVF typically choose to transfer to their uterus embryos with the highest likelihood of implantation and, even- tually, healthy birth. If embryos that have undergone a biopsy for purposes unrelated to health are not going to be chosen for implantation and will be eventually discarded, then the single biopsy procedure does not result in “ sparing ” embryos (although it may result in a delay in destruction). Re- search is ongoing to identify novel and more ef fi cient methods of obtaining stem cells from human embryos, and it is anticipated that this area will continue to evolve.
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Realizing Potential: A Pragmatic Look at Human Embryonic Stem Cell Research

Realizing Potential: A Pragmatic Look at Human Embryonic Stem Cell Research

n the United States, there are over 400,000 cryogenically frozen embryos (Hoffman, et al., 2003). These frozen embryos are almost exclusively produced from in vitro fertilization (IVF) and related treatments. Much debate centers on the fate of these embryos. Among the current options available to the parents of leftover embryos are embryo adoption, keeping the embryos frozen for future use, destroying them, faux-implantations to let the embryos „naturally‟ die (Grady, 2008), and donating them for human embryonic stem cell (hES cell) research. While not all of the embryos are destroyed, many are, and it is wasteful for those embryos to simply be discarded. With the consent of the parents, researchers should use the embryos slated to be destroyed or discarded for the purpose of realizing the great potential they possess, rather than letting them be wasted.
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LongSAGE profiling of nine human embryonic stem cell lines

LongSAGE profiling of nine human embryonic stem cell lines

The following additional data are available with the online version of this paper. Additional data file 1 is a summary of mouse specific tag types identified. Additional data file 2 is a table of genomic mappings for 268,515 unique tag sequences found in nine independent human embryonic stem cell lines. Additional data file 3 is a Gene Ontology analysis of nine independent human embryonic stem cells. Tag counts are expressed for each GO category for the top 1,000 by tag count. Additional data file 4 lists statistically significant differen- tially expressed LongSAGE tags found between embryonic stem cells and terminally differentiated tissues. Additional data file 5 is a table listing the 4,337 genes found in common across 8 undifferentiated human embryonic stem cell lines. Additional data file 6 is a table listing the 20,047 LongSAGE tags exclusively expressed in embryonic stem cell lines. Addi- tional data file 7 is a table listing the 634 LongSAGE tags exclusively expressed in ESCs that uniquely map to the human genome at least 2 kb away from an annotated tran- script. Additional data file 8 is a table listing the 301 Long- SAGE tags exclusively expressed in ESCs that uniquely map to species conserved regions of the human genome at least 2 kb away from an annotated transcript. Additional data file 9 is a table listing the 52 ESC specific transcripts identified by 5' RACE. Additional data file 10 lists the RACE and qPCR primer sequences used in this study.
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Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors

Varicella-Zoster Virus Infects Human Embryonic Stem Cell-Derived Neurons and Neurospheres but Not Pluripotent Embryonic Stem Cells or Early Progenitors

shown to be a cell-intrinsic inhibition of VZV replication (20, 27). Interestingly, naïve hESC have PML bodies with an atypical dis- tribution and composition (3), and it is possible that they have a role in the inability of VZV genomes to express viral genes in hESC, albeit by a different mechanism from that described for fibroblasts. An alternative explanation for the lack of EGFP ex- pression in hESC after transfection with genome-containing BACs is that the virus immediately enters a latent state and does not transcribe even IE genes in the process. Future experiments FIG 3 VZV is unable to replicate in hESC when infection is bypassed by transfection of BAC DNA containing the VZV genome. hESC and ARPE cells were transfected with BACs containing VZV-GFP62 or PrV-GFP and photographed 6 days later. None of the hESC colonies displayed GFP, indicating VZV-GFP62 infection (A and B) in contrast to the many fluorescent foci resulting from viral replication in the ARPE cells (C and D). Transfection of PrV genome-containing BACs into hESC results in viral replication, as demonstrated by the GFP fluorescence after transfection with PrV-GFP (E and F). Transfection of PrV-GFP BACs in parallel to MeWo cells also leads to viral replication (G and H). Panels A, C, E, and G are phase-contrast micrographs, and panels B, D, F, and H are fluorescence photomicrographs of the same fields, with hESC colonies delineated by yellow dashed lines. Scale bars, 100 ␮ m.
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Embryonic stem cell-derived extracellular vesicles enhance the therapeutic effect of mesenchymal stem cells

Embryonic stem cell-derived extracellular vesicles enhance the therapeutic effect of mesenchymal stem cells

MSCs have an signaficant effect on wound-healing and some other diseases [29, 30]. To investigate whether ES-EVs could enhance the therapeutic effects of MSCs in vivo, luciferase-labeled senescent MSCs were pre-treated with ES-EVs, and implanted into the injury area of cutaneous wound mice model. The cells were next determined by measuring luminescence signals within 7 days. In line with the observed repression of cellular decay in vitro, ES-EVs treatment also effectively restored the viability of senescent MSCs in vivo (Figure 5A). On day 5 and 7, in the ES-EVs treatment group, the amount of viable cells at the injury site was significantly higher than in that of control groups (Figure 5B). We also tested the effect of senescent MSCs with different treatment on wound healing in vivo, and found that ES-EVs treatment improved the healing process of a full-thickness excisional skin wound-healing model compared with control group (Figure 5C). On day 12 the wound had been healed in ES-EVs treatment, but which were not in other two groups (Figure 5C). Histologic analysis of the wounded areas demonstrated that ES-EVs treatment enhanced the thickness of epithelium as well as promoted the synthesis and regeneration dermal collagen (Figure 5D, 5E). Even for the early-passaged MSCs (young MSCs), pretreated with ES-EVs aslo improved its therapeutic effects in vivo (Figure S5). Taken together, these data indicated that ES-EVs enhanced the therapeutic effects of MSCs in vivo, by increasing epithelial and dermal cell proliferation, angiogenesis, dermal collagen synthesis, and further accelerate skin wound healing.
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Role of CD133 in human embryonic stem cell proliferation and teratoma formation

Role of CD133 in human embryonic stem cell proliferation and teratoma formation

interacting pentaspan transmembrane glycoprotein, we speculated that the destruction of CD133 may disturb some basic biological processes and related signal trans- mission, which was also demonstrated by changes in protein transport, cell division, cell proliferation, apop- tosis, and other processes (Fig. 5a, b). To further glean biological insight from the transcript-level responses to loss of CD133, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to ex- plore functional pathways enriched for the differentially expressed genes (DEGs) between KO and WT cells. Among the 1746 differentially expressed genes, 63.52% were downregulated, and most of these downregulated genes were associated with metabolic pathways (Fig. 5c) and metabolic changes, including changes in biosyn- thetic processes, catabolic processes, cellular nitrogen compound metabolic processes, nucleic acid metabolic processes, and other processes (Fig. 5d), coinciding with decreased proliferation. According to the interaction network, the metabolic changes were closely related to cellular biological regulation and were also accompanied by dysregulation of essential signaling pathways, such as the PI3K-Akt, AMPK, and p53 pathways (Fig. 5c). The significantly changed genes enriched in these KEGG pathways are shown in heatmaps (Fig. 6a–e). Although the upregulated genes were enriched for the cGMP-PKG and PI3K-Akt signaling pathways, the downregulated genes were enriched for the AMPK signaling pathway (Fig. 6a–d), and the downstream changes were not sim- ply corresponding upregulation or downregulation. Tak- ing Wnt signaling as an example, there were genes enriched both for positive and negative regulation of the Wnt signaling pathway; however, the downstream genes of the Wnt signaling pathway were mostly downregu- lated (Fig. 6e, f). Overall, the final changes after CD133 deletion resulted from coregulation of all those path- ways, and these pathways also interacted with each other not only on the regulation level but also on the gene level (Fig. 6g). The downstream changes in the cell cycle were also consistent with changes in the S phase popula- tion of CD133 KO hESCs (Fig. 3b), and significant DEGs associated with the cell cycle were mostly downregulated (Fig. 6h). Notably, neither GO/KEGG terms nor specific genes were found to be significantly related to hESC pluripo- tency or three-germ layer differentiation (Additional file 6:
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The Epigenomics of Embryonic Stem Cell Differentiation

The Epigenomics of Embryonic Stem Cell Differentiation

The PcG complex is not the only chromatin modifier that antagonizes Brg1. Mbd3, a subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex co-occupies with Brg1 several hundred promoters associated with cell signaling and adhesion [71]. The NuRD complex contains both a Swi2/Snf2-related ATPase (Mi-2) and HDAC1 and HDAC2. Expression of Brg1/Mbd3 co-occupied genes is fine-tuned through positive regulation by Brg1 and negative regulation through NuRD (Fig. 2C). The role of Mbd3 has become clearer recently after its enrich- ment was mapped and found to co-localize on biva- lent genes with the methylcytosine modification 5’hydroxyl methylcytosine (5’hmC), whose function remained elusive for some time [71, 72]. 5’hmC is created by the TET family of Fe(II) + 2 oxoglu- tarate-dependent enzymes. Both TET1 and TET2 are required for ESC self-renewal [73, 74]. TET-mediated 5’hmC facilitates the recruitment of Mbd3, which via its HDAC activity decreases H4 acetylation. Reduced histone acetylation in turn results in nucleosome oc- cupation and gene silencing. TET1 further mediates CpG-containing gene repression by facilitating PRC2 recruitment [75]. Bivalent H3K4me3/H3K27me3 promoters are typically decorated with 5’hmC and carry TET1 [72, 76]. Hence, TET1/5’hmC have dual roles in gene repression by recruiting both NuRD and PcG complexes to developmental genes.
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MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate

There is a single Mras gene from nematodes to mammals and in the ascidian embryo the orthologue has a role in neural differentiation, suggesting an evolutionarily conserved function (Keduka et al., 2009). The Xenopus embryo has contributed substantially to the deciphering of the molecular mechanisms that control embryo patterning, which relies on signalling pathways that are highly conserved between mammals and amphibians (Harland and Grainger, 2011; Heasman, 2006). LIF and its receptors (gp130 and gp190), along with the Pluri genes (such as Esrrb, Sod2, Cd9, Fzd5, Susd2 and Smarcd3), are conserved in Xenopus laevis (our unpublished data). In addition, cells from the animal part of the Xenopus blastula embryo (animal cap cells) are similar to mammalian ESCs. They are pluripotent and able to differentiate toward the lineage-specific cell types of the three germ layers (Lan et al., 2009; Okabayashi and Asashima, 2003). Moreover, the molecular mechanisms that govern self-renewal mediated by Oct4 are conserved between mammals and Xenopus (Morrison and Brickman, 2006; Abu-Remaileh et al., 2010). This makes Xenopus a suitable model in which the basic conserved functions of these genes can be studied during early embryogenesis and organogenesis, two processes not easily amenable in the mouse embryo. For instance, our recent work using a combined approach of mESCs and Xenopus embryonic cells has demonstrated a conserved function for a histone methyltransferase adaptor protein in smooth muscle cell differentiation (Gan et al., 2011).
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Embryonic stem cell research and the moral status of human embryos

Embryonic stem cell research and the moral status of human embryos

We have now seen that the attempts to circumvent the moral status problem all fail. This does not show that some new attempt might not succeed, but I think it unlikely. We are therefore left with the moral status problem. But maybe there is another way around this problem that takes it seriously, but still leads to the conclusion that ES cell research is morally acceptable. What I am thinking of here is a direct consequentialist approach, which, in being consequentialist sees moral status not in terms of rights, but in terms of value. Could we not argue that if the benefits of doing ES cell research are likely to outweigh the harms, including the harms created by destroying a given number of embryos with moral status, then we should do ES cell research?
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Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition

Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition

Understanding the genetic and epigenetic mechanisms that control the initial state and differentiation capability of pluripotent stem cells is essential for the comprehension of mammalian development and for the design of experimental protocols for the controlled generation of cell types of therapeutic interest (Hanna et al., 2010; Rossant, 2008; Murry and Keller, 2008; Niwa, 2007). Mouse embryonic stem cells (ESCs) have been isolated from the inner cell mass (ICM) of the blastocyst, may generate chimeric embryos at high efficiency, and their undifferentiated state depends on a self-maintaining network of core transcription factors [Oct4 (Pou5f1), Sox2, Nanog and Klf4/2/5] and signaling pathways (LIF, WNT and BMP4), which ensure self-renewal and protection from FGF-mediated lineage commitment (Silva and Smith, 2008; Lanner and Rossant, 2010; Hanna et al., 2010; Silva et al., 2009; Chambers et al., 2007; Niwa, 2011; Niwa et al., 2009; Ying et al., 2008; Ying et al., 2003a; Wray et al., 2011; Yi et al., 2011; Lyashenko et al., 2011; ten Berge et al., 2011; Nichols et al., 2009; Matsuda et al., 1999). The ESC population is not homogeneous, but rather is characterized by cell-to-cell spontaneous and reversible differences in the expression level and sensitivity to specific transcription factors and signaling pathways, which together maintain the balance between self-renewal and susceptibility to differentiation. This condition has been defined as the metastable state of ESCs (Niwa et al., 2009; Miyanari and
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Identification of embryonic stem cell–derived midbrain dopaminergic neurons for engraftment

Identification of embryonic stem cell–derived midbrain dopaminergic neurons for engraftment

promising cell source for future translational studies. The use of BAC transgenic reporter lines may not be suitable for human clini- cal translation due to the need for transgene insertion and reporter gene expression. However, our profiling studies present multiple candidate surface markers that may enable comparable antibody- mediated cell isolation strategies. The ultimate goal for translation may be, similar to that of drug-based therapies, to establish fully characterized, scalable, and ready-to-use populations of NURR1- stage DA neurons that can be properly dosed and standardized for therapeutic applications. While many hurdles remain on the road toward this goal, we recently described a novel strategy for deriving midbrain DA neurons from hESCs that resulted in excel- lent in vivo survival and function (34). The choice of DA neuron differentiation stage used in this study was influenced by the cur- rent BAC reporter data, as we specifically aimed at transplanting hESC-derived DA neurons at the Nurr1 expression stage. However, formal comparisons using hESC-based BAC or knockin reporter lines will be required to formally address these questions in puri- fied human cells. While DA neuron replacement in PD represents a symptomatic treatment unlikely to affect all disease-related symptoms, access to pure populations of engraftable DA neurons will be a major step forward toward testing the full potential of DA neuron replacement therapies in PD. Finally, we believe that SSRLA is a powerful application of ESCs for gene discovery that could be used for generating refined genetic signatures in other specific ESC-derived cell types.
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A molecular scheme for improved characterization of human embryonic stem cell lines

A molecular scheme for improved characterization of human embryonic stem cell lines

The lack of comparative analysis of hESC lines matters, because the properties and behavior of each line are uniquely shaped by their histories. It has become clear that different derivations produce hESC lines that are sim- ilar overall, but with inherent differences in gene expres- sion, methylation status, X-chromosome inactivation, rate of self-renewal, and ability to differentiate [2-4]. More importantly, the behavior of cells and their phenotypic state changes as culture conditions and the stress to which they are subjected is altered, and permanent genomic changes frequently occur as passage numbers increase [5- 7]. This has led to great difficulty in comparing results from one laboratory with another and even comparing results with different passages of the same cell line. Therefore, thorough and routine characterization of hESC lines is essential to avoid compromising the validity of results. The most common characterization method for hESC is immunocytochemical analysis of a handful of markers, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-80, and OCT-3/4 [8]. The next most frequent is reverse tran- scription PCR, which is used for those group of genes whose expression is involved in maintenance of the undif- ferentiated state [9,10]. While these assays certainly give indications of the undifferentiated state of the cells, they do not address other issues such as pluripotentiality or the degree of culture adaptation and genomic instability. To facilitate comparisons among lines, the hESC research community has begun to develop a number of tools. Work is proceeding toward conditions that support the propagation of all lines [11], sets of markers that truly define the undifferentiated and unadapted state of the cells [7,12-14], and markers predictive of the differentia- tion capacity of the cells [15]. The work presented here is part of efforts to create a database of the properties of each line and to identify a reference standard for comparisons between laboratories. To this end, we have assembled a set of molecular tests for hESC lines that assess identity, sta- bility of the nuclear and mitochondrial genomes, histo- compatibility profile, and the undifferentiated state of the cells. Some of these assays have been previously per- formed on individual lines, but to our knowledge, no sin- gle group has used all of these tests on any single line, and
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Dissecting hematopoietic differentiation using the embryonic stem cell differentiation model

Dissecting hematopoietic differentiation using the embryonic stem cell differentiation model

display an embryonic and fetal globin profile in culture. Further studies are needed to study the multilineage potential of these cells. Common to this and other studies on ESC-derived repopu- lating cells is the direct injection of cells into the femur when performing mouse transplantations. This highlights the concern that differentiation protocols still have to provide the appropriate maturation of cells for successful engraftment and bypass prob- lems such as cell aggregation based emboli formation. While it is helpful to work with stroma that mimic the embryonic HSC niche, efforts to identify the stromal components that support the main- tenance and differentiation of hematopoietic cells will aid in developing defined differentiation protocols that do not require cell coculture steps. The study by Ledran and colleagues identi- fied TGF β 1 and TGF β 3 as factors involved in positively regulating the hematopoietic differentiation from hESCs. Thus far multilineage reconstitution in the mouse has been achieved with ESC-derived hematopoietic cells generated by overexpression of the Hox family member, HoxB4, and caudal-related transcription factor Cdx4 (Wang et al., 2005b). In brief, there are protocols available to generate different hematopoietic lineages. However defined, serum-free and coculture-free protocols to reproducibly generate robust levels of adult repopulating HSCs from genetically un- manipulated ESCs are still lacking.
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Human embryonic stem cell for the treatment of multiple sclerosis: A case report

Human embryonic stem cell for the treatment of multiple sclerosis: A case report

Introduction: Multiple sclerosis (MS) is a chronic demyelinating disease with inflammatory neurodegeneration. It is caused by the genesis of autoimmune response to self-antigens in a genetically susceptible individual. Currently, no remedy is available for treatment of MS. However, corticosteroids and selective immunomodulators are used. Human embryonic stem cells (hESC) have been investigated in animal models which showed the potency to mitigate the signs and symptoms of MS. We present a case of patient with MS treated with hESC therapy. Case Report: A 34-year-old female with MS was referred to our facility. On presentation, the patient was unable to walk properly due to stiffness and paralysis in lower limbs and had significant weight loss in the last two years. The hESC therapy consisted of treatment phases separated by gap phases. After the hESC therapy, there was a remarkable improvement in the muscle bulk, tone and power of the patient. The patient experienced an increased energy level and power of upper limbs. She gained weight during the treatment, but there was no change in her walking status. Conclusion: We observed a significant improvement in the symptoms of MS with the hESC treatment. There was no adverse event observed during the treatment.
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Retrotransposons shape species specific embryonic stem cell gene expression

Retrotransposons shape species specific embryonic stem cell gene expression

KAP1 repression of retroviruses was initially discov- ered in the context of murine leukaemia virus (MLV) [55], which led on from original observations that MLV was restricted in embryonic cells [66] through its primer binding site Pro (PBS-pro) sequence that binds proline tRNA [67, 68]. This sequence was later discovered to recruit KAP1 through the mouse KRAB-ZFP, Zfp809 [56]. MLV still serves as a practical model to explore the KAP1 repression pathway and it was recently uncovered that YY1 and EBP1 contribute to an MLV silencing com- plex [69–71], factors that may also repress ERVs with a PBS-pro and/or ERVs with unrelated PBS sites (reviewed in [72]). Elegant work has just revealed, through Zfp809 knockout mice and genetic and biochemical experiments in ESCs, that Zfp809 not only restricts exogenous MLV but also several ERVs that contain PBS-pro sequences, as predicted [73]. Strikingly, in Zfp809 knockout mice, disruption of silent chromatin marks normally estab- lished early in development at VL30-(virus-like 30) type PBS-pro ERVs leads to their overexpression in differenti- ated tissues, together with nearby genes. Of note, VL30 elements lack coding regions, illustrating how KAP1 represses not only coding but also non-coding ERVs that remain a threat because of their regulatory sequences. This new study, therefore, provides conclusive evidence that the KAP1/KRAB-ZFP pathway is necessary to repress retrotransposons and linked genes in vivo.
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Logical Fallacies in the Literature on Human Embryonic Stem Cell Research

Logical Fallacies in the Literature on Human Embryonic Stem Cell Research

scientific. For a scientist, the decision is fairly simple. If the research is scientifically sound and is likely to yield beneficial results, then one must go forward with the project [. . . .] However, anti-abortion forces argue that research should be restricted to adult stem cells, which can be harvested without destroying embryonic life. On the other hand, some conservatives in the Republican Party favor research using human embryos. They

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Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

To further delineate the hemangioblast population, a reporter ES cell line with the GFP coding sequences targeted into the Brachyury (Bry) locus was created by Fehling et al [3]. ES cells can differentiate in vitro to form spheroid cultures called embryonic bodies (EBs). These structures contain the derivatives of all the three germ layers, and it is possible to track mesoderm and hematopoietic lineage commitment in vitro with the temporal expression of two specific genes, restricted to the lineage of interest. Bry, a member of the T-box gene family, is a marker for the early mesoderm formation (the majority of the cells in the primitive streak are Bry positive). Bry expression decreases when cells migrate away from the primitive streak and further differentiate. Flk1, the vascular endothelial growth factor receptor 2, identifies a mesodermal population of cells further committed for differentiation and is commonly expressed with Bry in populations with hematopoietic potential. The analysis of Bry and Flk1 expression allowed for the detection of three subpopulations: the Bry - Flk1 - population
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Effect of substrate stiffness on early human embryonic stem cell differentiation

Effect of substrate stiffness on early human embryonic stem cell differentiation

Early proof of the influence of ECM stiffness on stem cell differentiation was provided by qualitative studies involving mouse mammary epithelial cells that showed increased differentiation when grown on soft gel colla- gen substrates as opposed to Tissue Culture Plastic [15]. ECM control of stem cell fate by regulating growth fac- tor diffusion has been demonstrated by artificially tether- ing a growth factor to a substrate, which increased survival of human mesenchymal stem cells (MSCs) [16]. In additional studies, the ECM was also found to be a more potent differentiation cue for MSCs than standard induction cocktails [17]. Tissue-level elasticity has been shown to be able to determine lineage and phenotype commitment in naïve MSCs. Later studies showed that human MSCs could be kept quiescent by growing them on polyacrylamide substrates that mimicked the proper- ties of marrow while preserving their multilineage po- tential [18]. When NIH/3 T3 cells were cultured on polydimethylsiloxane (PDMS) substrates patterned with varying stiffness, the cells accumulated preferentially on the stiffer regions of the substrates with differential re- modelling of ECM on stiff vs. compliant areas, which led to the suggestion that migration, and not proliferation, was responsible [19]. In seminal studies, Engler and col- leagues showed that matrices whose elasticities were comparable to brain tissue (“soft matrices”) were neuro- genic and stiffer and rigid matrices (with elasticities com- parable to muscle and bone tissue, respectively) were respectively myogenic and osteogenic [8]. Substrate com- pliance was also demonstrated to positively influence
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