Human cells

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An Adaptable Platform for Directed Evolution in Human Cells

An Adaptable Platform for Directed Evolution in Human Cells

AdPol gene, which should not be under any selection pressure in our trans-complementing system, and the neighboring pIX, IVa2, and pTP genes, suggesting that such selection only impacts our mutation rate at most two-fold. Because AdPol selectively replicates only adenoviral DNA, EP-Pol can only introduce mutations into the adenoviral genome. This mutagenesis technique thus represents an improvement over other strategies that evolve genes directly in the human genome. In such strategies, off-target mutations can arise through basal or through the enhanced mutagenesis rates, which can subvert selection pressure and generate false positives. Furthermore, even recent mutagenesis methods that target specific genes within the human genome by using somatic hypermutation 1112 or Cas9-fusion proteins 13-15 still display significant off-target genetic modification. 43-45 Especially given the large size of the human genome, many pathways to cheating selection may be available. Our use of an orthogonal replication system means that the human host cells are discarded and replaced with each passage, preventing mutation accumulation in the human cell that could potentially cheat selection pressure. As a result, false positives are restricted to the ~30 kb viral genome, providing much more limited escape options than might be found in the entire human genome. This advantage, combined with the more rapid expansion of adenovirus relative to human cells allowing a larger number of directed evolution rounds in a given time period, highlights the ability of our platform to quickly scan mutational space with minimal risk of selection subversion.
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GC content shapes mRNA storage and decay in human cells

GC content shapes mRNA storage and decay in human cells

an intrinsic property of these mRNAs and not simply the result of their sequestration in PBs ( Hubstenberger et al., 2017 ). Interestingly, the mRNAs of haplo-insufficiency genes, which by defi- nition are expected to have a limited protein yield, are indeed enriched in PBs ( Figure 6C ). In addition to the GC-dependent codon bias, we also observed some GC-independent codon bias in PB-enriched mRNAs. Interestingly, some important post-transcriptional regulation programs involve codon usage. This was shown for proliferation- versus differentiation-specific transcripts in human cells ( Gingold et al., 2014 ) and for maternal mRNA clearance during the maternal-to-zygotic transition in zebrafish, Xenopus, mouse, and Drosophila ( Bazzini et al., 2016 ). Codon usage could also enable the regulation of small subsets of mRNAs, depending on cellular requirements. In man, half of GO sets show more variability in codon usage than expected by chance ( Rudolph et al., 2016 ). Based on GO analysis, we previously demonstrated that PB mRNAs tend to encode regula- tory proteins, while PB-excluded mRNAs encode basic functions. Furthermore, proteins of the same complexes tended to be encoded by mRNAs co-regulated in or out of PBs, in so-called post-tran- scriptional regulons ( Hubstenberger et al., 2017 ; Standart and Weil, 2018 ). We speculate that spe- cific codon usage could also underlie these post-transcriptional regulons.
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GC content shapes mRNA decay and storage in human cells

GC content shapes mRNA decay and storage in human cells

* Corresponding author: dominique.weil@upmc.fr Summary Control of protein expression results from the fine tuning of mRNA synthesis, decay and translation. These processes, which are controlled by a large number of RNA-binding proteins and by localization in RNP granules such as P-bodies, appear often intimately linked although the rules of this interplay are not well understood. In this study, we combined our recent P-body transcriptome with various transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors. This analysis revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA decay. It also rationalized why PBs mRNAs have a strikingly low protein yield. We report too the existence of distinct mRNA decay pathways with preference for AU-rich or GC-rich transcripts. Compared to this impact of the GC content, sequence-specific RBPs and miRNAs appeared to have only modest additional effects on their bulk targets. Altogether, these results lead to an integrated view of post-transcriptional control in human cells where most regulation at the level of translation is dedicated to AU-rich mRNAs, which have a limiting protein yield, whereas regulation at the level of 5’ decay applies to GC-rich mRNAs, whose translation is optimal.
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Tropism of Tanapox virus infection in primary human cells.

Tropism of Tanapox virus infection in primary human cells.

Abstract Tanapox virus (TPV) belongs to the genus Yatapoxvirus and causes a relatively benign zoonotic disease in man, with symptoms that resemble a mild version of human monkeypox. In order to investigate the underlying mechanisms of TPV pathogenesis, the tropism and replication characteristics of TPV were examined in a variety of primary human cells. A GFP expressing TPV (TPV-GFP) was constructed and used to infect primary human dermal fibroblasts (pHDFs) and peripheral blood mononuclear cells (PBMCs), both of which are believed to be major in vivo targets of poxvirus infection. pHDFs fully supported productive replication and cell–cell spread of TPV-GFP. However, induction of cell cycle arrest in pHDFs by contact mediated inhibition or rapamycin treatment eliminated the ability of TPV to fully stimulate cell cycle progression and dramatically reduced viral replication. TPV-GFP-infected human PBMCs were screened for permissiveness by FACS analysis. CD14+ cells (monocytes) were the primary cellular target for TPV infection. A small proportion of CD3+ cells (T cells) were positive for GFP expression, yet TPV was not able to replicate and spread in cultured peripheral blood lymphocytes, regardless of their state of activation. Primary human monocytes, however, demonstrated robust TPV replication, yet these cells no longer supported replication of TPV once they differentiated into macrophages. This unique ex vivo tropism of TPV gives key insights into the basis for the self-limiting pathogenicity of TPV in man. © 2007 Elsevier Inc. All rights reserved.
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Genetic analysis of cell size homeostasis in human cells

Genetic analysis of cell size homeostasis in human cells

iii Abstract All organisms are essentially structured and fitness defined by cell number, type and size. Composed of around 30 trillion cells, humans have cells with remarkably varied functions and size, ranging from a neuron that can reach one meter in length to a lymphoid cell that is around 16 µm in diameter. At a fundamental level, size is determined by the balance between cell growth and cell division. The molecular networks that control and maintain optimal cell size are yet to be deciphered. The mTORC1 pathway is a major regulator of cell growth that plays a central role in integrating intra- and extra-cellular stimuli. This study addresses the investigation and characterization of the molecular players and processes that orchestrate cell size in human cells, as determined by chemical-genetic size screens and epistasis analysis. I undertook a CRISPR/Cas9 extended-knockout (EKO) genome-wide library screen in the NALM-6 pre-B lymphoma cell line, followed by cell size fractionation by counter flow elutriation in the presence of the mTOR inhibitor rapamycin, and compared the screen data to a similar screen performed in the absence of rapamycin. The analysis indicates that upstream of mTOR, the loss of genes that are related to nutrient sensing, results in size changes in the presence of mTOR inhibition. Also, several gene knockouts in ribosome biogenesis and calcium homeostasis led to size alterations, suggesting a possible a pivotal role of these processes in cell size control in a mTOR-dependent fashion. This study provides insights into the genetic networks that regulate cell size in a mTOR- dependent fashion and establishes new hypotheses for future experimental tests.
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Bacterial FtsZ induces mitochondrial fission in human cells

Bacterial FtsZ induces mitochondrial fission in human cells

672 C 673 Semiautomated quantification (MiNA plugin) of mitochondrial morphology expressed 674 by the amount of individual mitochondria/mitochondrial area in U2OS cells transfected [r]

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Proteome alteration induced by hTERT transfection of human fibroblast cells

Proteome alteration induced by hTERT transfection of human fibroblast cells

Background Telomeres are specialized functional DNA-protein com- plexes that cap the end of linear chromosomes. Their role is to protect chromosomes from degradation, recombina- tion, or fusion, and to prevent the chromosome ends from being detected as strand breaks. At each cell division, tel- omeres shorten until they reach a critical size that drives eukaryotic cells into replicative senescence. Telomere length therefore acts as a biological life clock. Telomerase, a ribonucleoprotein complex, is involved in telomere length maintenance in eukaryotic cells by adding telom- eric repeats to the 3' end of chromosomes. Telomerase activity is downregulated in most human cells during embryogenesis, thereby limiting their proliferative capac- ity. However, the reactivation of telomerase activity is observed in 90% of all human tumor cells, making this enzyme an attractive target for selective cancer therapy. Reconstitution of telomerase activity by ectopic expres- sion of the catalytic telomerase subunit (hTERT) cDNA stabilizes the telomere length of fibroblasts and other cell types that therefore acquire immortality [1]. Such hTERT- transfected cells have been proposed as immortal versions of normal human cells model with the advantage of indef- inite proliferation. This strategy is applied for biochemical and physiological studies of normal cell growth, differen- tiation, genetic manipulation, etc [1-4]. More recently, experiments using transplanted telomerase-immortalized cells have been conducted in immunodeficient mice [5]. Adenocortical hTERT immortalized cells were able to replace cells with deficient functions. However such cell- based therapies raise important interrogations about medium and long term incidence of hTERT-immortalized cell autotransplantation cells. Indeed, several studies have evidenced that hTERT has other physiological roles beyond maintaining telomere such as potent incidence on malignant transformation of human fibroblasts by a tel- omere length-independent mechanisms [6-8]. Further- more, the key role of telomerase in cancer cell immortalization led to the development of therapeutic strategies based on telomerase inhibition. A detailed char- acterization of hTERT-related transfection processes is therefore of outstanding interest to monitor anti-telomer- ase drug therapy.
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3D Chromosome Regulatory Landscape of Human Pluripotent Cells

3D Chromosome Regulatory Landscape of Human Pluripotent Cells

chromosomal frameworks in different cells and that transcriptional regulatory elements function within this context to produce cell-type specific gene expression programs. Conservation of 3D structure and associations with disease It has been estimated that approximately 65% of Hi-C derived chromosome structures are static among different cell types and different species (Dixon et al., 2015). The observation that chromosome structures are largely conserved in primates (Dixon et al., 2015; Dixon et al., 2012; Rao et al., 2014; Vietri Rudan et al., 2015) led us to investigate the extent to which CTCF binding is similarly conserved. Analysis of CTCF binding sites across 10 primates indicates that the DNA sequence in anchor regions of CTCF-CTCF loops in hESCs is more conserved in primates and vertebrates than in regions bound by CTCF that do not participate in loops (Figure 6A, S6A). A similar analysis showed that the CTCF DNA sequence motif in hESC loop anchor regions is highly conserved in primates and vertebrates (Figure 6B–C, S6B). CTCF is known to preferentially bind to hypomethylated DNA sequences (Wang et al., 2012), and further analysis revealed that the CTCF binding sequences in hESC loop anchor regions exhibit DNA hypomethylation across 37 human cell/tissue types (Figure S6C). Hypomethylation at CTCF-CTCF loop anchors persists in a broad spectrum of human cells, and is evident even during stages of embryogenesis when DNA is globally
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In vitro modeling of the neurovascular environment by coculturing adult human brain endothelial cells with human neural stem cells.

In vitro modeling of the neurovascular environment by coculturing adult human brain endothelial cells with human neural stem cells.

Although a variety of in vitro models have been developed to investigate angiogenesis [21] and the BBB [22], as well as the NVU using rodent cells [23], assays composed of only human cells investigating the formation of vasculature-like structures (VLS) of brain microvascular endothelial cells by interacting with brain cells, i.e. establishing a neurovascular environment, are generally lacking. Although co-culturing of hNSCs with non-CNS endothe- lial cells, such as Human Umbilical Vein Endothelial Cells (HUVECs), have been reported to identify paracrine factors that promote hNSCs proliferation [24], as well as angiogenesis- promoting factors [25], these do not model the interaction between hNSC and brain endothelium. Transwell experiments only investigate paracrine and autocrine factors, whereas extra- cellular matrix models, such as the Matrigel assay, are predom- inantly biased towards juxtacrine factors [26], hence these are more suitable to investigate a specific type of factors rather than their synergistic and iterative effects. However, given the appropriate conditions, endothelial cells will organize in vitro into a net of VLS, akin to a vascular bed in tissue. An in vitro model of the neurovascular environment therefore needs to involve direct cell-to-cell contact between endothelial and ‘‘brain’’ cells, while forming a network of VLS, hence affording the investigation of the dynamic interactions of the formation of a neurovascular environment, as well as the processes involved in angiogenesis in the brain.
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Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

Cell transplantation All animal surgical procedures were approved by The Institutional Animal Care and Use Committee, Centre anti-cancéreux, Val d’Aurelle-Paul Lamarque, Montpellier, France. SCID immunodeficient mice, aged 5 weeks (Charles River, L'arbresle, France) were anesthetized by intraperi- toneal injection of 100 mg/kg ketamin hydrochloride and 10 mg/kg xylasin. Human myoblasts were labelled with intravital fluorescence marker, PKH67 dye (Sigma). As determined by FACS analysis, more than 99% of cells were labelled by PKH67 (data not shown). Immediately after the injection or 2 days after implantation, muscle derived cells were iso- lated and plated for 24 hrs to recover implanted human cells and host mouse cells. Cells were then analysed by FACS and immunostaining. In a first set of experiments, human myoblasts were pre-treated with DEAB (50 $M) for 24 hrs and then 2 % 10 5 myoblasts were implanted. Control non-treated cells were injected into the right quadriceps and DEAB- treated cells into the left quadriceps of the same mice. Cells were recov- ered 2 days after implantation. In a second set of experiments, ALDH high (top 5% of the parent population) and ALDH low cells (bottom 5% of the parent population) were selected with FACS ARIA, labelled with PKH67 and then implanted into quadriceps. ALDH low cells were injected in the right quadriceps and ALDH high cells in the left quadriceps of the same mice. Due to the low amount of FACS-sorted myoblasts, we only grafted 1 % 10 4 cells. Implanted muscles were harvested immediately after injec- tion to determine accurately the real number of injected cells and then 2 days after implantation. According to the percentage of human cells (quantified by FACS) and the total number of cells (cell counter, Beckman Coulter, Roissy, France), we determined the number of human cells really injected into quadriceps. Statistical analysis was performed with Graph Pad Prism 4.0 Software using t-test.
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Multicellular human alveolar model composed of epithelial cells and primary immune cells for hazard assessment

Multicellular human alveolar model composed of epithelial cells and primary immune cells for hazard assessment

Working with untested human blood samples involves specific care to prevent the potential transmission of infectious diseases, such as HIV (human immunodeficiency virus), hepatitis B, and hepatitis C. Therefore, the use of personal protective measures such as gloves, gowns, masks, and eye protection are crucial and must be in accordance with good laboratory practice principles. These protections reduce the risk of exposing the skin or mucous membranes to potentially infectious fluids. Additionally, for those involved in handling buffy coats and PBMs, vaccination against the hepatitis B virus is mandatory, and blood titer levels of antibodies anti-hepatitis B must be above 100 IU/L (country- specific legislative requirements need to be addressed). In addition, all work must be performed in biosafety level 2 laboratories (country-specific legislative requirements need to be addressed). Standard health and safety precautions associated with working in a laboratory environment and handling mammalian cell culture, including handling of waste, should be adopted when conducting the entire protocol.
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αvβ3 integrin expression increases elasticity in human melanoma cells

αvβ3 integrin expression increases elasticity in human melanoma cells

b [ 4 ]. Each subunit is composed of a large extracellular N-terminal domain that allows speci fic ligand binding, a single transmembrane-spanning helix and a short C-terminal cytoplasmic sequence that provides docking for signaling, adaptors, and cytoskeletal-associated proteins. In mammalian tissues, subunits a and b combine in a restricted mode forming 24 different integrins, which are widely distributed. They integrate the extra -and intra- cellular environment through bidirectional signaling, and conse- quently are highly dynamic modulators of cell function, including cell adhesion, shape, motility, proliferation, differentiation, and survival. Through these processes, integrins are involved in many physiological aspects, including wound healing, immune response, hemostasis as well as in pathological dysfunctions, i.e. fibrosis, in- flammatory disease, bleeding, and thrombosis. On top of that, integrins are key elements for the modulation of the mechanics involved in the interaction between cells and the surrounding ECM. Importantly, expression of speci fic integrin types may have a different impact on the mechanical properties of the cell.
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Pannexin-1 in Human Lymphatic Endothelial Cells Regulates Lymphangiogenesis

Pannexin-1 in Human Lymphatic Endothelial Cells Regulates Lymphangiogenesis

* Correspondence: arnaud.monvoisin@univ-poitiers.fr; Tel.: +33-054-936-6385 Received: 27 April 2018; Accepted: 22 May 2018; Published: 24 May 2018    Abstract: The molecular mechanisms governing the formation of lymphatic vasculature are not yet well understood. Pannexins are transmembrane proteins that form channels which allow for diffusion of ions and small molecules (<1 kDa) between the extracellular space and the cytosol. The expression and function of pannexins in blood vessels have been studied in the last few decades. Meanwhile, no studies have been conducted to evaluate the role of pannexins during human lymphatic vessel formation. Here we show, using primary human dermal lymphatic endothelial cells (HDLECs), pharmacological tools (probenecid, Brilliant Blue FCF, mimetic peptides [ 10 Panx]) and siRNA-mediated knockdown that Pannexin-1 is necessary for capillary tube formation on Matrigel and for VEGF-C-induced invasion. These results newly identify Pannexin-1 as a protein highly expressed in HDLECs and its requirement during in vitro lymphangiogenesis.
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Topography of cancer-associated immune cells in human solid tumors.

Topography of cancer-associated immune cells in human solid tumors.

Addressing the biological differences and the different responses to immunotherapy across tumor entities, we systematically compared different tumor types. We analyzed ten tumor entities in the framework of this classification and showed pronounced differences, but also unexpected cross- entity similarities: We found that lymphoid-hot tumors are more prevalent in immunotherapy-respon- sive tumor entities such as melanoma and lung adenocarcinoma than in other entities such as colo- rectal cancer. Immune-excluded tumors, such as lymphoid-excluded and myeloid-excluded tumors are common in head and neck tumors (HNSC). Lastly, we show that characteristic patterns of Figure 6. Overall similarity between tumor entities based on full immune topography. Hierarchical clustering based on all normalized cell densities of (A) PD1+exhausted lymphocytes; (B) Foxp3+regulatory T cells; (C) CD8+cytotoxic T lymphocytes; (D) CD68+monocytes/macrophages; (E) CD3 +lymphocytes and (F) CD163+pro tumor macrophages. Unit on the color scale: percentile-normalized cell density. (G) corresponding H & E image of the colorectal cancer sample used in this figure. (H) Hierarchical clustering of tumor types (N = 144 total patient samples).
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Intramuscular transplantation in non-human primates of human muscle-derived stem cells (MuStem cells), a promising candidate for cell therapy of muscular dystrophies

Intramuscular transplantation in non-human primates of human muscle-derived stem cells (MuStem cells), a promising candidate for cell therapy of muscular dystrophies

hMuStem cell isolation and characterization. Human muscle- derived cells were isolated from skeletal muscles of 9 to 15-year- old patients free of known muscle disease. Cells were isolated based on a modified version of preplating protocol initially described by Rouger et al., 2011. hMuStem cells were then cultivated on coated-plastic flasks in proliferation medium containing 10% human serum, PSF and human recombinant growth factors. For phenotypic characterization hMuStem cells were analyzed by FACS using a panel of myogenic, mesenchymal and pericytes markers. Myogenic differentiation was assessed based on cell morphology and the sarcomeric myosin heavy chain (sMyHC) expression. For in vivo myogenic potential demonstration, 3.10 5 hMuStem cells (n=4, batches)
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Human mucosal associated invariant T cells detect bacterially infected cells.

Human mucosal associated invariant T cells detect bacterially infected cells.

MR1-Restricted, Mtb-Reactive CD8 + T-Cell Clones are MAIT Cells We next performed phenotypic analyses of Mtb-reactive MR1- restricted T-cell clones to determine if they shared properties of previously characterized MR1-restricted MAIT cells. We selected a subset of clones representative of TB exposed (active, n = 5; LTBI, n = 4) and uninfected donors (n = 5) (Table 2). One defining feature of both mouse and human MR1-restricted MAIT cells is the expression of a semi-invariant TCR Va chain: Va7.2/Ja33 for humans and the highly homologous Va19/Ja33 for mice, respectively. Using an antibody that labels all T cells containing the Va7.2 chain including those that pair with the Ja33 region [32], the Va7.2 chain was detected by flow cytometry on all 14 MR1- restricted Mtb-specific T-cell clones as well as on an HLA-B08– restricted clone, but not on the HLA–E, or HLA-II–restricted clones (Figure 2F; Table 2). Given that 14 of 14 randomly selected clones were restricted by MR1, binomial analysis suggests a high prevalence of MR1 restriction among our panel of clones (.95%). Furthermore, we performed an analysis of Va7.2 TCR expression on an additional 28 NC clones. Here all 28 clones expressed the Va7.2 TCR suggesting that the remaining clones are MR1- restricted. The TCR of Va7.2-expressing MAIT cells from the gut has been associated with the expression of the Vb2 or Vb13 TCR b chains [31]. However, we found at least 10% of the Mtb-specific MR1-restricted T-cell clones did not express either Vb2 or Vb13 (Table 2 and unpublished data). To determine if Va7.2 + Mtb- reactive MR1-restricted T-cell clones expressed the canonical Va7.2/Ja33 CDR3 region, the TCR alpha encoding cDNA was cloned from six representative Mtb-reactive clones chosen on the basis of their distinct patterns of Vb TCR usage. All six T-cell clones were found to express the hAV72 segment as expected, and five of six expressed the hAJ33 segment (Table 3). Further, all six Mtb- reactive TCRs were found to have VJ junctional heterogeneity with two N additions, as previously reported for Va7.2/Ja33 TCRs [33,34]. Importantly all six TCRs from Mtb-reactive T-cell clones were found to encode CDR3a loops of the same length, which is highly conserved among all mammalian Va7.2/Ja33 + cells studied thus far. And finally, each of the sequences of the CDR3a loops of the five Va7.2/Ja33 + Mtb-reactive T cells matched a sequence from a previously reported Va7.2/Ja33 + cell of undefined restriction and antigen specificity (Table 3) [33,34].
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Role of XPC in the human cutaneous cancer cells invasion

Role of XPC in the human cutaneous cancer cells invasion

91 Other data from the laboratory also indicated that inclusion of XP-C fibroblasts in the dermal equivalent resulted in higher contractile properties leading to a reduction of the diameter of collagen lattices of about 25 % (Frechet, Warrick et al. 2008). These observations were the first suggesting that cancer susceptibility of epidermal cells towards skin cancer could be elicited by the secretory phenotype of XP-C fibroblasts. In contrary, using an inhibitor of matrix metalloproteinases (MMPs) inhibits the contraction of collagen lattices by fibroblasts (Scott, Wood et al. 1998). Several studies reported on the role of MMPs in tumor progression and invasion (Curran and Murray 1999, Egeblad and Werb 2002) and in skin chronological aging (West 1994) and skin photoaging (Fisher, Datta et al. 1996); other studies have shown an overexpression of MMP1 in XP-C fibroblasts, suggesting that MMP1 could be an actors toward exacerbated tumor susceptibility in XP-C patients. My host laboratory decided to further focus his investigations on factors secreted by XP-C fibroblasts and implicated in the promotion of epidermal invasions. The analysis of secretome of XP-C fibroblasts in culture showed that XP-C fibroblasts secrete hepatocyte growth factor (HGF), a factor known to be implicated in cells morphogenesis, proliferation, malignant transformation and cancer progression (Kalluri and Zeisberg 2006). HGF also has been found to be increased in forestomach in the mice around invasive squamous cell carcinomas (Bhowmick, Chytil et al. 2004). As we mentioned before, XP-C patient present with high propensity to the development of aggressive skin cancers, most notably SCCs and melanoma. Our studies hence focus on the role of XP-C fibroblasts in the promotion of human squamous cell carcinoma with a particular attention to the following points:
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MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

images. Once the protocol has been validated by the user with his specific cells, mitochondria labeling will be left out for functional studies, to preclude possible biological biases. Dose-response analyses over a wide range of MSC mitochondria concentrations, with serial dilutions of the mitochondria preparation, is advisable. In fact, it should be noted that biological effects for the transferred mitochondria could be achieved for low mitochondria concentrations, in the lower range for detection by FACS or imaging. These functional studies, including investigations on GSC metabolism, proliferation and response to therapy, are carried out the day following the mitochondria transfer.
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Molecular signature of erythroblast enucleation in human embryonic stem cells

Molecular signature of erythroblast enucleation in human embryonic stem cells

M ATERIALS AND M ETHODS Cells and culture conditions The research on embryonic stem cells was authorized by the French Biomedicine Agency (R0500225A, R0500226A and R0500227A). The hESC lines H1 and H9 (National Institute of Health [NIH]: code WA01, passag- es 28-30 and code WA09, passages 27-33) were main- tained in an undifferentiated state by weekly mechani- cal passage on Matrigel (BD Biosciences, San Jose, CA, USA). Matrigel was diluted in KO-DMEM (1:100) and used to coat tissue culture dishes for 1 h at room tem- perature. After coating, the Matrigel solution was com- pletely removed and mTeSR-1 medium (Stemcell Tech- nologies, Vancouver, Canada) was added to the coated dishes. Thereafter the medium was changed on a daily basis. The normal karyotype of H1-hESCs and H9-hESCs was confirmed by performing karyotyping analysis at passage 24 and passage 26 respectively.
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Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells.

Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignemen[r]

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