Human embryonic stem cells (hESCs)

Top PDF Human embryonic stem cells (hESCs):

Preclinical safety studies of human embryonic stem cell‐derived retinal pigment epithelial cells for the treatment of age‐related macular degeneration

Preclinical safety studies of human embryonic stem cell‐derived retinal pigment epithelial cells for the treatment of age‐related macular degeneration

Abstract As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bed- side, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in devel- opment, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined dif- ferentiation protocol to generate functional human embryonic stem cells (hESCs)- derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and bio- distribution and tumorigenicity analysis to rule out potential migratory or tumori- genic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro cul- ture or differentiation, and enforces the importance to examine the genome integ- rity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology.
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Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentrations

Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentrations

SUMMARY The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demon- strate that (1) differentiation of hESCs induces ran- dom XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiolog- ical oxygen.
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A gene expression signature shared by human mature oocytes and embryonic stem cells.

A gene expression signature shared by human mature oocytes and embryonic stem cells.

Background Oocytes have the unique ability to remodel the chromatin of the germinal nuclei into a totipotent state. These mod- ifications are particularly striking for the male pro-nuclei: upon fertilization, the sperm chromatin packaging pro- tamines are stripped off and replaced by histones, the DNA is demethylated within 4 hours of fertilization, and the amino terminal tails of histones are modified includ- ing methylation of arginin 9 and phosphorylation of serin 10 of histone H3 (H3K9 and PhH3S10, respectively) [1,2]. Remarkably, the reprogramming properties of oocytes are not restricted to the very specialized germinal nuclei. Indisputably, the cloning of Dolly has shown that the oocyte cytoplasm is able to extensively reverse the chro- matin modifications associated with a differentiated state [3,4]. Somatic cell nuclear transplantation (SCNT) has since been extended to other species, including human cells, and to many cell types, including terminally differ- entiated cells such as granulocytes [5,6]. Thus differentia- tion is not anymore considered as an irreversible process, but rather as modifications of the cellular epigenome and transcriptome, that are amenable to complete reversal. In addition to oocytes, other cell types can reprogram somatic cells towards pluripotency. For example, using cell fusion strategies, it has been shown that hybrid cell clones obtained by fusion of a differentiated cell with either teratocarcinoma cells or embryonic stem cells dis- play features of pluripotent, undifferentiated cells with concomitant loss of the markers associated with differen- tiation [7,8]. More recently, and quite unexpectedly, Taka- hashi and Yamanaka have shown that the expression of only four selected transcription factors, OCT3/4, SOX2, CMYC and KLF4, is sufficient to drive a mouse fibroblast into an induced pluripotent stem cell (iPS) with all the features of embryonic stem cells, including a high growth rate and the ability to form a variety of tissues from all three germ layers in vitro and in vivo [9]. These results have been confirmed by other studies, extended to human cells, and applied to non-fibroblastic cells such as mesen- chymal stem cells (MSCs), gastric epithelial cells or hepa- tocytes [10-12]. At the center of cellular reprogramming lies the activation of the pluripotency transcriptional reg- ulatory circuitry involving POU5F1/OCT4, NANOG and SOX2 [13] and extensive chromatin-remodeling. How- ever, the details of this process, such as the exact media- tors of the chromatin modifications, remain ill defined. Data from xenopus egg experiments point to nucleosomal ATPases, but these findings await confirmation using mammalian oocytes [14,15].
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Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways.

Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways.

3 phenotype in vitro follows sequential activation of gene networks and epigenetic changes that closely mimic events occurring in vivo during embryogenesis [1-5]. Identification of culture conditions that specifically direct hES toward unique phenotypes provides new ways to analyze molecular correlates of early developmental transition phases. One of the major technical advances for the study of such transitions is the development of efficient technologies such as DNA microarray that enable monitoring gene expression at the level of mRNA on a genomic scale. Nevertheless, analysis of how genes are controlled during transition toward a dedicated developmental path is restricted mainly by the ability to obtain homogeneous populations of cells because their phenotypes are continuously changing over time. Despite this, several studies clearly demonstrate that such approaches applied to hES and their progenies can provided useful informations about gene expression involved in the developmental processes [6,7]. As an example of meta-analyze see [8] and for review on human embryonic stem cell transcriptome profile see also [9].
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Molecular signature of erythroblast enucleation in human embryonic stem cells

Molecular signature of erythroblast enucleation in human embryonic stem cells

For erythroid induction and differentiation we used a two-step protocol adapted from our previous proce- dures [20, 21, 23]. To allow hEB formation, undifferen- tiated hESCs were treated with collagenase IV (1 mg/mL; Invitrogen) and transferred to low attachment plates (Nunc) in liquid culture medium (LCM: IMDM [Biochrom], 450 μg/mL holo-human transferrin [Scipac], 10 µg/mL recombinant human insulin [Incelligent SG, CellGen], 2 IU/mL heparin and 5% human plasma) in the presence of stem cell factor (SCF, 100 ng/mL), throm- bopoietin (TPO, 100 ng/mL), FLT3 ligand (FL, 100 ng/mL), recombinant human bone morphogenetic pro- tein 4 (BMP4, 10 ng/mL), recombinant human vascular endothelial growth factor (VEGF-A165, 5 ng/mL), inter- leukin-3 (IL-3, 5 ng/mL), interleukin-6 (IL-6, 5 ng/mL) (Peprotech) and erythropoietin (Epo, 3 U/mL) (Eprex, kindly provided by Janssen-Cilag, France). hEBs were cultured for 9 days or 20 days at 37°C in a humidified 5% CO 2 atmosphere. The cells were then dissociated
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A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas.

A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas.

found to be highly enriched in hESCs in at least ten or publications, among 5567 genes found in at least one. Some of these differences may be explained by platform-to-platform or lab-to- lab variability, but this is likely not the main explanation as suggested by transcriptome platform comparisons [22]. Rather, the differences between the hESC cells lines used, the control samples, the specific caveats of each transcriptome analysis technique and the statistical methodologies likely contribute to these disparities (see Supplemental Table S2). For instance, the homeobox transcription factor NANOG, which is universally expressed in hESC, was not reported by Sperger et al. because no probe for NANOG was present on their microarray [29], nor was it reported by Brandenberger et al. because the differential regulation of NANOG in their in vitro differentiation model did not met their stringent statistical criteria [30]. ZFP42 (the human homolog of murine rex1) was never listed by MPSS studies because its MPSS signature has repeat sequences [31]. Another pitfall impacting on differentially expressed genes lists comparisons and contributing to the “small intersection” problem [32] is that in order to be at the intersection of 20 lists, a gene must have fulfilled 20 times the statistical filter, which it does with a probability equal to the product of the probabilities of each test. A way to circumvent this difficulty would be to obtain the raw data from these studies and to apply specific statistical tests [33]. However, the raw data were not available for many studies analyzed here, which prevented us from applying this approach in our study. Nevertheless, the 1076 hESC genes list provides the opportunity to the scientific community to examine the genes that have found over- or underexpressed in hESC by several authors. These lists provide further molecular insight into the biology of this unique stem cell model and are now starting points for many new research directions in the field of hESC. In the future, it will be interesting to extend this list by investigating additional hESC lines. The collection of additional transcriptome data is
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Efficient aseptic and automatable vitrification of human Embryonic Stem Cells using bio-safe & chemically defined media

Efficient aseptic and automatable vitrification of human Embryonic Stem Cells using bio-safe & chemically defined media

insurance of biological safety since cells are stored in containers that are predisposed to leakage when plunged into liquid nitrogen (LN2). We describe our newly developed hPSCs (hESCs [human embryonic SCs – RCM-1 cell line] and hiPSCs [human induced PSCs – dKips & gRips cell lines]) cryopreservation method based on aseptic VIT (no direct contact with LN2) using only chemically defined materials and media, and amenable to automation. 1: Vitrification is a cryopreservation method based on the conversion of a liquid into a glass-like state by an infinite enhancement of its viscosity and without formation of ice crystals.
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Aseptic and automatable vitrification of human embryonic stem cells using  defined media

Aseptic and automatable vitrification of human embryonic stem cells using defined media

Proliferation curves after warming of slow frozen vs vitrified cells ¼ well of a 6-well plate. hESCs proliferation curves after SF or V.[r]

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Aseptic and automatable vitrification of human embryonic stem cells using defined media

Aseptic and automatable vitrification of human embryonic stem cells using defined media

Proliferation curves after warming of slow frozen vs vitrified cells ¼ well of a 6-well plate. hESCs proliferation curves after SF or V[r]

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Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency

Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency

. Specifically, which is the precise correlation of these different pluripotency states with the in vivo equivalents is still a question of debate. Known molecular markers of such plasticity are mainly transcription factors operating within a pluripotency gene regulatory network 9 . More recently, metabolites are emerging as key regulators of stem cell plasticity, acting as epigenetic modifiers 10,11 ; however, much less is known on the role of microenvironment. Indeed, elucidation of the extrinsic mechanisms that control stem cell plasticity is crucial for understanding both early embryo development and controlling the differentiation potential of pluripotent stem cells 12 . In the attempt to shed lights on this issue, we focused on the glycosylphosphatidylinositol (GPI)-anchored extracellular protein Cripto. Cripto is a key developmental factor and a multifunctional signalling molecule 13 . In the mouse embryo, Cripto is essential for primitive streak formation and patterning of the anterior–posterior axis during gastrulation 14 and it negatively regulates ESC neural differentiation while permitting cardiac differentiation 15 . Although largely considered as a stem cell surface marker 16 , no studies so far have directly investigated its functional role in pluripotency. In this study, we report the consequences of genetic and pharmacological modulation of Cripto signalling on the generation and/or maintenance of mEpiSCs and hESCs.
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en fr Human embryonic and neural stem cells : when PrP and APP are mixed Cellules souches embryonnaires et neurales humaines : quand la PrP et l'APP "s'en mêlent" ou "s’emmêlent"

Cellules souches embryonnaires et neurales humaines : quand la PrP et l’APP ”s’en mêlent” ou ”s’emmêlent”.. Félicie Radreau.[r]

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Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells

Chromatin Structure and Gene Expression Programs of Human Embryonic and Induced Pluripotent Stem Cells

We then applied this method to identify statistically significant differences in chromatin structure between ESCs and iPSCs and found 50 genomic regions (29 genes) with differential H3K4me3 occupancy and four regions (two genes) with differential H3K27me3 occupancy ( Table S3 ). These regions of differential occupancy represent a tiny fraction of the genome (0.003%), and although there was no obvious theme associated with them, we considered several possible causes for the differential modification. First, we investigated whether these differences were due to the presence of exogenous reprogramming factors in iPSCs, but there were no significant differences in these chro- matin modifications between transgene-containing and trans- gene-excised iPSCs ( Table S2 ; Soldner et al., 2009 ). Second, we investigated whether the chromatin differences between ESCs and iPSCs were due to residual epigenetic signatures left from the parental fibroblast cell line, but found no evidence that iPSCs contain H3K4me3 or H3K27me3 signatures that reflect their cell of origin ( Table S4 ). Lastly, we examined whether any gene expression changes were associated with differences in histone modification between ESCs and iPSCs, but found that this was not the case ( Figure S1 ). We conclude that there are a small number of regions in these human ESCs and iPSCs that show differences in H3K4me3- and H3K27me3-modified nucleosomes. These differences involve a small fraction of the genome and have little or no influence on gene expression. However, we cannot exclude the possibility that these small chromatin differences observed in undifferentiated cells may exert subtle effects on cells upon differentiation.
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Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues.

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues.

Figure 3. Sequential ChIP. Chromatin was extracted from 4 human ES cell lines and ChIP was performed sequentially using anti H3K4me3 and then anti-H3K27me3 antibodies. Please click here to view a larger version of this figure. Figure 4. ChIP from Chromatin Extracted from Specific Embryonic Cardiac Regions. Chromatin was extracted from 25 ventricles of E9.5 mouse embryos (2 litters) lysed and sonicated. The anti-H3K27ac antibody was used for immunoprecipitation. The DNA was used for sequencing. The figure shows genomic regions of the cardiac genes Nkx2.5 and Tbx5 enriched in the modified histone and one genomic region of Shox2 pacemaker-specific gene not enriched and not expressed in the ventricle. Please click here to view a larger version of this figure.
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Cell-cell and cell-medium interactions in the growth of mouse embryonic stem cells

Cell-cell and cell-medium interactions in the growth of mouse embryonic stem cells

Nevertheless, smaller‐scale studies have implicated a large number of genes as being required for the  maintenance  of  pluripotency.  Perhaps  not  surprisingly,  almost  all  major  signal  transduction  pathways  seem  to  play  a  role  (reviewed  in  Liu  et  al. 23 ).  For  historical  reasons,  one  pathway  that  has  received  considerable  attention  is  the  Stat3  pathway.  mESCs  were  initially  propagated  using  a  feeder  layer  consisting  of  embryonic  fibroblast  cells 16,  17   (in  a  medium  supplemented  with  bovine  calf  serum).  Subsequently,  it  was  found  that  the  requirement  for  the  feeder  layer  could  be  replaced  by  adding  leukemia inhibitory factor (LIF) to the medium 24, 25 . LIF binds to a heterodimer complex consisting of the  LIF  receptor  (LIF‐R)  and  another  receptor  subunit  called  Membrane  glycoprotein  130  (gp130).  This  binding  event  leads  to  the  activation  of  several  pathways,  of  which,  the  activation  of  Stat3  has  been  shown to be  essential for the  maintenance of a pluripotent state in mouse  ES cells 26, 27 , though not in  human  ES  cells 28 .  Besides  Stat3,  Oct‐4 29,  152 ,  Sox‐2 30,  31 ,  and  Nanog 32‐34   are  three  other  transcription  factors shown to be essential for developing and maintaining the pluripotent state. These factors appear  to be essential in mouse as well as human ES cells (see above references).  
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A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells

A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells

Cell Death and Disease (2013) 4, e737; doi:10.1038/cddis.2013.246; published online 25 July 2013 Subject Category: Experimental Medicine Because of their pluripotency and unlimited proliferation properties, embryonic stem cells (ESCs) have been proposed as cellular therapy for human diseases. However, this potential is impaired by the poor efficiency of ESC differentia- tion into mature differentiated cells and by the persistence of undifferentiated cells with a tumorigenic capacity within differentiated cultures. Therefore, there is a crucial need for a better understanding of molecular mechanisms governing ESC commitment into specific lineages. Self-renewal of mouse ESCs is dependent on the intracellular pathways involving a complex interplay between specific epigenetic processes, miRNAs and transcription factors, such as Oct4, Nanog or Sox2 (see for reviews 1,2 ). In vitro ESC differentiation can be induced by various experimental protocols resulting in the commitment into a variety of mature differentiated cell types (see for reviews 3–5 ). This process can be modulated between the 2nd and 5th day of differentiation by the potent morphogen retinoic acid (RA) that induces differentiation into neurons and adipocytes 6–8 and, conversely, inhibits cardiomyocyte and skeletal myotube formation. 6,9,10 Although ESC differentiation experimental models recapitulate the
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Synergistic targeting of breast cancer stem-like cells by human γδ T cells and CD8+ T cells

Synergistic targeting of breast cancer stem-like cells by human γδ T cells and CD8+ T cells

non-CSCs failed to survive under such culture conditions (Figure 1f). Tumours derived from CSC-like cells exhibited a capacity to differentiate (Figure 2d), especially after prolonged periods of tumour development (Supplementary Tables S1 and S2). In contrast, tumours derived from non-CSCs showed no signs of differentiation or enrichment of contaminant CSC-like cells (Figure 2d). Histologically, 7/11 tumours arising from CSC-like cells were intimately associated with native mouse mammary ducts, cuffing the vessels with areas of necrosis distal to the vessels. The majority of such tumours showed at least moderate levels of epithelioid differentiation as confirmed by their expression of pan-cytokeratin (AE1/AE3) (Figure 2e); lung metastases showed predominant epithelioid differentiation with no residual features of CSC-like cells (data not shown). However, tumours derived from CSC-like cells uniformly stained for vimentin (Figure 2e), indicative of an only partial reverse EMT process during tumour development in vivo. No adenocarcinoma differentiation was identified morphologically, as judged by the absence of carcinoma embryonic antigen expression (Supplementary Table S2).
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Genome-wide analysis of ATP-dependent chromatin remodeling functions in embryonic stem cells

Genome-wide analysis of ATP-dependent chromatin remodeling functions in embryonic stem cells

Heintzman, N.D., Hon, G.C., Hawkins, R.D., Kheradpour, P., Stark, A., Harp, L.F., Ye, Z., Lee, L.K., Stuart, R.K., Ching, C.W., et al. (2009). Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112. Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y.C., Laslo, P., Cheng, J.X., Murre, C., Singh, H., and Glass, C.K. (2010). Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell

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Differentiable PKC activation on pacemaking activity of cardiomyocytes derived from mouse embryonic stem cells

Differentiable PKC activation on pacemaking activity of cardiomyocytes derived from mouse embryonic stem cells

Cell-based approaches This approach involves transplanting a collection of pacemaker cells into the SA node to induce automaticity. Multiple candidate cell types have been used in preclinical studies to regenerate the injured heart, including embryonic stem cells (ESCs) [40] and induced pluripotent stem cells (iPSCs) [41][42]. ESCs cells are derived from the inner cell mass of the embryo and have the capacity to replicate and differentiation into any cell type, including cardiomyocytes. However, their unlimited differentiation can lead to forming teratomas once transplanted in their undifferentiated state [43]. They also are ethically problematic since they are obtained from early human embryo [44]. In addition, there is evidence that undifferentiated ESCs are rejected by host immune system [45]. Nonetheless ESCs are the most favored source for cardiac cell therapy. To overcome the ethical controversies, ESCs sources are used from species, other than human for experimental purposes and/or human iPSCs.
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Control of the Embryonic Stem Cell State

Control of the Embryonic Stem Cell State

Induced pluripotent stem cells (iPSCs) have been generated from a broad range of murine and human somatic cells by using forced expression of Oct4, Sox2, and other transcription factors ( Hanna et al., 2010; Stadtfeld and Hochedlinger, 2010; Yama- naka and Blau, 2010 ). Fully reprogrammed murine iPSCs are apparently equivalent to ESCs in developmental potency and gene expression, although some iPSCs can retain a memory of their somatic program. A few murine iPSCs have been shown to be capable of generating ‘‘all-iPSC’’ mice and thus have a developmental potency equivalent to ESCs ( Boland et al., 2009; Kang et al., 2009; Stadtfeld et al., 2010; Zhao et al., 2009 ). In one study with genetically matched murine ESCs and iPSCs, no consistent gene expression differences were observed, except for transcripts within the imprinted Dlk1–Dio3 gene cluster ( Stadtfeld et al., 2010 ). Similarly, few differences were observed in a comparison of gene expression and histone modifications in human ESCs and iPSCs ( Guenther et al., 2010 ). However, some iPSCs do retain an epigenetic memory of their donor cells ( Kim et al., 2010b; Polo et al., 2010; Lister et al., 2011 ). These results indicate that ESC state and thus ESC regu- latory circuitry is re-established in fully reprogrammed iPSCs, but that a limited memory of the gene expression program of the cell of origin can be observed in some iPSCs.
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Embryonic stem cell markers expression in cancers.

Embryonic stem cell markers expression in cancers.

Cancer stem cells refer to a subset of tumor cells that has the ability to self-renew and generate tumor heterogeneity [3; 4; 5]. Oct4 is a major transcription factor that is mandatory for the self-renewal and pluripotency characteristics of ES cells and germ cells. Rare cells that express Oct4 were identified in several somatic cancers[6]. Oct4A expressing cells are present in human benign and malignant prostate glands and the frequency of Oct4A expressing cells increases in prostate cancers[6]. A subpopulation of the Oct4A expressing cells co- expressed Sox2, an ES cell marker[6]. In the intestine, Oct4 expression causes dysplasia by inhibiting cellular differentiation in a manner similar to that in the ES cells[7].
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