Top PDF Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

The cell samples washed with phosphate buffered saline (PBS, Lonza Group Ltd., Basal, Switzerland) and cells were lysed in M- PER lysis buffer (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Protein concentra- tions of samples were analyzed using the Bradford method [20]. The amount of ARPE-19 and hESC samples was 10 m g. The samples were run in 7% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels and then wet-blotted to nitrocellulose mem- branes (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Blocking was done with 3% fat-free dry milk in 0.3% Tween 20/ PBS at room temperature (RT) for 1 h. Thereafter, membranes were incubated in primary antibody dilutions anti-MRP1 (1:2000, overnight at 4uC), anti-MRP4 (1:5000, 1 h at RT), or anti-MRP5 (1:2000, 1 h at RT) rat monoclonal MRP antibodies (all from Abcam, Cambridge, UK) or alpha-tubulin (1:4000, 30 min at RT, Sigma-Aldrich) that was used as a loading control. All primary antibodies were diluted in 0.5% bovine serum albumin (BSA) in 0.3% Tween 20/PBS. After 365 minutes washes with 0.3% Tween 20/PBS the membranes were incubated in horseradish peroxidase-conjugated anti-mouse IgG or antibody (GE Health- care), diluted in 3% fat-free dry milk in 0.3% Tween 20/PBS (1:10 000 for MRP1, 1:10 000 for MRP4, and 1:2000 for MRP5) for 1 h at RT, and 30 min at RT for alpha-tubulin (1:10 000). Protein- antibody-complexes both in MRP and alpha-tubulin labeling were detected using an enhanced chemiluminescence method (Milli- pore, Billerica, MA, USA).
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Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

an accelerated ageing process in AMD [24,46,47,48]. We have previously shown that sublethal concentrations of hydrogen peroxide induced senescence-associated ß-Galactosidase (SA-ß- Gal) activity in primary cultured RPE cells [29]. In the experiments of the current study, treatment of primary human RPE cultures with CSE could significantly increase the proportion of SA-ß-Gal positive cells. Positive staining of SA-ß-Gal has also been detected in vitro in late passage RPE cultures [49,50] and in vivo in the RPE cells of old primate eyes [51]. In human RPE cells, an increased expression of SA-ß-Gal staining could be triggered by mild hyperoxia-mediated ROS release [52]. Furthermore, cellular senescence can also be identified by increased expression of senescence-associated biomarkers such as Apo J, CTGF, and fibronectin. All three biomarkers are inducible by oxidative stress [16,18]. In our experiments, exposure of primary human RPE cells to CSE could lead to a significant elevation of Apo J, CTGF, and fibronectin expression. The cellular chaperone Apo J has been previously detected in the RPE of AMD donor eyes, although its role and function in the RPE is still unclear [19,53]. In contrast, CTGF and fibronectin have been found in the Bruch’s membrane, in drusen and in basal linear deposits of AMD eyes [20,21,22]. Furthermore, we could show that treatment of human RPE cells with CSE also increased the secretion of fibronectin and laminin into the culture media. Laminin is a basement membrane protein, which is involved in the formation of basal laminar deposits of the ageing macula [24]. Both laminin and fibronectin have been shown to be secreted by senescent human RPE cells [23]. In the pathogenesis of AMD, it is assumed that cellular senescence and dysfunction of the RPE lead to an increased aggregation of ECM [15,54]. Therefore, CSE-induced levels of CTGF and fibronectin
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Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

In a previous study we have shown that breast epithelial cells exhibiting stem cell properties spontaneously fuse with breast cancer cells, thereby giving rise to hybrid cell lines exhibiting novel properties, such as an altered migratory activity and an enhanced drug resistance [4,5,23]. Flow cytometric analysis of M13HS-2 and M13HS-8 hybrid cell lines demonstrated expression of the chemokine receptor CCR7 [5], which is a member of the seven transmembrane G protein-coupled receptor family that has two ligands: CCL19 and CCL21 [24]. CCL19 is expressed by lymphatic endothelial cells, whereas CCL21 is constitutively expressed on specialized high endothelial venules (HEVs) of lymph nodes, Peyer’s patches, thymus, spleen and mucosal tissue [25,26]. CCR7 is prevalent in various subsets of T lymphocytes and activated dendritic cells and the interaction with its ligand CCL21 recruits these cell populations to the lymph nodes [24,25]. In accordance to other G protein-coupled receptors CCR7 activates signal transduction via G protein-dependent and independent mechanisms, whereby CCL19 and CCL21 elicit different cellular functions in various cell types (for review see [27]). For instance, both ligands induce G protein activation and calcium mobilization, indicating PLC-b/c activation, with equal potency, but only activation by CCL19, but not CCl21, promotes robust desensitization of endogenous CCR7 due to receptor phosphorylation and b-arrestin recruitment in a human T cell lymphoma cell line [28]. The differential effects of both ligands on CCR7 signaling and desensitization might be attributed to striking differences in activation of the G protein-coupled receptor (GRK)/ b-arrestin system [29]. CCL19 dependent b-arrestin2 recruitment is catalyzed by both GRK3 and GRK6, whereas CCL21 activates GRK6 alone indicating that GRK3 activity is involved in CCR7 desensitization [29]. In dendritic cells CCL19/CCL21 mediated CCR7 G protein-dependent signaling leads to activation of MAPK family members ERK1/2 (MAPK p42/44 ), p38, and JNK as well as PI3K (for review see [27]). In CD4 T-cells CCL21 modulates T-cell receptor signaling through Ras and Rac dependent pathways concomitant with increased phosphorylation levels of AKT, MEK, and MAPK p42/44 [30]. Interestingly, neither p38 nor JNK were phosphorylated in CCL21 co-stimulated CD4 T-cells [30] indicating CCR7 specific signal transduction cascades vary among different cell types. Likewise, a linkage of G protein- coupled receptors to the MAPK signaling pathway through class IB PI3Kc and phosphotyrosine kinase (PTK), SHC, GRB2, SOS, RAS and RAF signaling has been reported [31]. In contrast to G protein dependent signaling, MAPK activation is also facilitated via a G protein-independent mechanisms due to CCL19/CCL21 mediated engagement of GRK6/b-arrestin 2 [29], which may point to a pivotal role of MAPK activity in CCR7 signaling.
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Use of Ferritin Expression, Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells, to Monitor Differentiation with Magnetic Resonance Imaging In Vitro.

Use of Ferritin Expression, Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells, to Monitor Differentiation with Magnetic Resonance Imaging In Vitro.

The purpose of this study was to establish a method for monitoring the neural differentiation of stem cells using ferritin transgene expression, under the control of a neural-differentia- tion-inducible promoter, and magnetic resonance imaging (MRI). Human adipose tissue- derived mesenchymal stem cells (hADMSCs) were transduced with a lentivirus containing the human ferritin heavy chain 1 (FTH1) gene coupled to one of three neural cell-specific promoters: human synapsin 1 promoter (SYN1p, for neurons), human glial fibrillary acidic protein promoter (GFAPp, for astrocytes), and human myelin basic protein promoter (MBPp, for oligodendrocytes). Three groups of neural-differentiation-inducible ferritin- expressing (NDIFE) hADMSCs were established: SYN1p-FTH1, GFAPp-FTH1, and MBPp- FTH1. The proliferation rate of the NDIFE hADMSCs was evaluated using a Cell Counting Kit-8 assay. Ferritin expression was assessed with western blotting and immunofluorescent staining before and after the induction of differentiation in NDIFE hADMSCs. The intracellu- lar iron content was measured with Prussian blue iron staining and inductively coupled plasma mass spectrometry. R2 relaxation rates were measured with MRI in vitro. The prolif- eration rates of control and NDIFE hADMSCs did not differ significantly (P > 0.05). SYN1p- FTH1, GFAPp-FTH1, and MBPp-FTH1 hADMSCs expressed specific markers of neurons, astrocytes, and oligodendrocytes, respectively, after neural differentiation. Neural differenti- ation increased ferritin expression twofold, the intracellular iron content threefold, and the R2 relaxation rate two- to threefold in NDIFE hADMSCs, resulting in notable hypointensity in T2-weighted images (P < 0.05). These results were cross-validated. Thus, a link between neural differentiation and MRI signals (R2 relaxation rate) was established in hADMSCs.
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Male germ-like cell differentiation potential of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells in co-culture with human placenta cells in presence of BMP4 and retinoic acid

Male germ-like cell differentiation potential of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells in co-culture with human placenta cells in presence of BMP4 and retinoic acid

cells and germ cells, which can be stimulated by RA. Retinoids are involved in the regulation of testicular functions, which appear to be necessary for spermatogenesis and the development of spermatocytes through early stages of meiosis. RA promotes differentiation of ESCs and bone marrow cells into germ (24), Furthermore, BMP4 leads to expression of PGCs specific genes such the Dpp3a (Stella), Fragilis, mouse vase homologue (Mvh) gene (25, 26). In the present study, we found hUMSCs changed morphologically to shiny clusters after incubation in this culture system, although we could not observe the round cell type that had been reported by other groups. Perhaps long-term culture is needed for the round cell shape. However, the expression of germ-cell specific genes such as c- Kit/DDX4/Piwil2/alfa6 and beta 1 integrin in the differentiated hUMSCs confirmed that hUMSCs can differentiate into germ cells. To analyze germ cell characteristics of induced hUMSCs we examined the expression of mRNA and protein markers diagnostic of germ cell development at different stages of HUMSC differentiation. C-Kit is a germ-cell enriched gene highly expressed in PGCs. It is expressed in early spermatogenic cells and in later stages of spermatogenesis, particularly in the acrosomal particles of the round spermatids and the acrosomal region of testicular spermatozoa (27). Intergrin a6 is the surface marker of spermatogonial stem cells (28). Integrins such as v, 6, and 1 are also expressed on adult spermatogonia (28, 29). However, not only prospermatogonia and adult undifferentiated spermatogonia but also somatic cells in the testis express all of these markers (30).
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Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

TGF-b1 has been previously demonstrated to have a direct effect on retinal pericytes and influence their potential interactions with endothelial cells. Its defined roles include helping maintain retinal vascular barrier function [51], increasing expression of smooth muscle actin [52], and enhancing pericyte contraction [53], which have been suggested to help stabilize endothelial cells [54,55]. We wished to evaluate the extent to which hASC- pericytes might also be influenced by TGF-b1 pre-treatment, if these treatment effects were analogous those observed with retinal pericytes, and if TGF-b1 might enhance hASC functionality within in vitro and in vivo assays of pericyte function and vascular stabilization. We first performed an hASC and bovine retinal endothelial cell (BREC) co-culture assay previously described for evaluating retinal pericytes [25]. Using this assay, we tested whether pre-conditioning the hASC and/or retinal pericytes with TGF-b1 would enhance these cells’ ability to inhibit BREC cell cycle entry in a contact-dependent manner. After 24–48 hours of Figure 2. Human adipose-derived stem cells (hASCs) stabilize oxygen-induced retinopathy (OIR). A–C, Eyes of NOD SCID mice, injected intravitreally with hASCs at P12 following OIR, and then harvested at P14, demonstrate improved revascularization of central retina as compared to contralateral PBS injected (blue) carrier controls (16.4% reduction, n = 17, p = 0.03). D–F, NOD SCID eyes injected intravitreally with hASCs at P2, prior to OIR, exhibited dramatically less central vascular ablation at P12 than contralateral PBS injected carrier controls (blue) (52.9% reduction, n = 6, p = 0.03). Lines in C and F connect data points for hASC injected and contralateral PBS injected eyes in the same mouse.
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Dissertação para obtenção do Grau de Doutor em Bioengenharia Orientador: Paula Marques Alves, PhD, ITQB-UNL Co-orientador: Manuel Carrondo, Professor, FCT-UNL Co-orientador: Daniel Wang, Professor, MIT

Dissertação para obtenção do Grau de Doutor em Bioengenharia Orientador: Paula Marques Alves, PhD, ITQB-UNL Co-orientador: Manuel Carrondo, Professor, FCT-UNL Co-orientador: Daniel Wang, Professor, MIT

levels of fetal the CYP450 (53, 54) the HepaRG cell line holds promise for hepatic drug induction tests. This latter hepatoma cell line was shown to express nuclear receptors and CYP450 transcripts at levels comparable with primary human hepatocytes (55) and its CYP450 activity induction has been proven for the CYP450 isoforms 1A2, 2C9 and 3A4, among others, as well as the presence of phase II and III drug metabolizing enzymes (57, 60); such results make this cell line an alternative to larger throughput drug metabolism studies, since it can proliferate up to several passages. The main disadvantages of using the HepaRG cell line are its clonal origin (it originated from a hepatocellular carcinoma from one single individual (55)) and the co-culture nature of this system, since HepaRG cell culture contain both hepatocytes and cholangiocytes, making it difficult to quantify the metabolic activity per hepatocyte. The former clonality issue limits the assessment of the naturally occurring inter-individual variability in CYP450 metabolism (42, 61) when using the HepaRG model. For a mid to long term perspective, the differentiation of human pluripotent stem cell lines to hepatocytes is a promising strategy which can potentially deliver an unlimited number of terminally differentiated hepatocytes. Hepatic differentiation protocols have already been established (62, 63) and adapted to large scale culture (64), but the obtained hESC derived hepatocyte-like cells have a strong fetal liver phenotype, namely the expression of Alfa-Fetoprotein and CYP7A1. Further maturation of these cells has been a subject of several publications (65-69) showing the expression of mature mRNAs and protein, such as CYP3A4 (70), thus proving the potential of pluripotent stem cell hepatic differentiation for providing hepatocytes for drug development tests.
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Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration.

Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration.

Stem cells derived directly from uncontaminated Wharton’s jelly are less heterogenous and possess unique beneficial properties over other mesenchymal stem cells[11–15]. Human Whar- ton’s Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) in its pure form have many advan- tages over other type of stem cells including higher proliferation rates, stemness characteristics that lasts several passages in vitro, wide multipotency, hypoimmunigenicity and anticancer properties[12]. hWJ-MSCs evoked minimal immune reactivity with low expression of MHC I molecules and no expression of MCH II molecules; rendering them a good source of allogeneic cell transplantation[14,16]. hWJ-MSCs has greater differentiation potential [10,12,17] than other tissues in umbilical cord. The potential of hWJ-MSCs to differentiate into neurons[17– 20] especially retinal progenitor cells[21] is a promising feature in cell therapy for conditions such as retinal degeneration. Apart from that, hWJ-MSCs can also synthesize and secrete tro- phic factors or cytokines and to support the expansion and function of other neural cells [17,18,20,22]. Trophic factors secreted by hWJ-MSCs showed a better neural differentiation and neural cell migration when compared with trophic factors by bone marrow-derived mes- enchymal stem cells [23].
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Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

medium for 4 days. The EBs were attached and spread onto a culture dish in non-treated or BMP4-only treated groups. However, in the RA only- or BMP4/RA-treated groups, the EBs were clustered together at the periphery of the cells and were swollen (Fig. 1A). For effective induction of EBs into GSC-like cells, the specification rate into the GC lineage was compared based on the expression of VASA for 2–8 days after treatment of the cells with BMP4 and/or RA. The VASA protein was expressed on the membrane and in the cytoplasm of developing PGCs at the genital ridge [28]. Although there was no VASA expression in undifferentiated hESCs (Fig. S2), the VASA-positive cells were most frequently localized on the surface of EBs (Fig. 1B). Figure 2 shows the percentages of VASA-positive putative GSC- like cells. The results also indicated that the specification in CHA- hES15 and H1 cells could potentially be enhanced through treatment with BMP4 or RA, which are important for differen- tiation into GCs in vitro. On day 2, VASA was detected in EBs cultured with and without BMP4/RA. The percentage of VASA- positive cells in EBs cultured with RA (9.0%60.7, 10.3%61.8 in CHA-hES15 and H1) and BMP4/RA (8.8%60.9, 10.3%61.1) was significantly greater than that in EBs cultured without factors (4.0%60.4, 4.3%61.1) and that in the BMP4 (5.8%60.5, 6.8%61.1)-treated group (p,0.05). After 4 days, a significant increase in percentage of VASA positive cells was detected in EBs cultured with BMP4 (15.5%62.6 and 17.5%62.9 in CHA-hES15 and H1), RA (20.4%63.6 and 26.3%64.0) or BMP4/RA (24.5%63.2 and 32.8%64.2) compared with the EB medium group (7.0%61.3 and 8.5%61.7)(p,0.05). In particular, on day 4, the EBs treated with BMP4/RA showed the highest expression of VASA. The VASA expression on day 8 (EB medium group: 5.8%61.4 and 4.5%61.6 in CHA-hES15 and H1, BMP-treated: 9.0%62.9 and 18.0%63.4, RA-treated: 21.3%61.7 and 20.5%62.8, BMP4/RA-treated: 23.0%63.5 and 28.5%63.8) was not increased compared with that of the 4-day culture groups. Indeed, the VASA expression was reduced in certain groups, and there were slightly more dead cells in the EBs (data not shown). The number of putative GSC-like cells was higher in BMP4/RA- treated EBs than in the other groups. Thus, we decided to use BMP4/RA in further experiments.
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Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

Zebrafish is a widely used animal model with well-characterized background in develop- mental biology. The fate of human adipose-derived stem cells (ADSCs) after their xeno- transplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viabili- ty and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3–4.3 hour post-fertilization (hpf). Green fluo- rescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical stain- ing. The results indicated that human ADSCs did not change their cell viability and the ex- pression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplan- tation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across- species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful tech- nical platform for the investigation of biology and physiology of stem cells in vivo.
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Cell Signaling and Differential Protein Expression in Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells with Hypermethylated Salvador/Warts/Hippo (SWH) Pathway Genes.

Cell Signaling and Differential Protein Expression in Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells with Hypermethylated Salvador/Warts/Hippo (SWH) Pathway Genes.

Human mesenchymal stem cells (MSCs) modified by targeting DNA hypermethylation of genes in the Salvador/Warts/Hippo pathway were induced to differentiate into neuronal cells in vitro. The differentiated cells secreted a significant level of brain-derived neurotrophy factor (BDNF) and the expression of BDNF receptor tyrosine receptor kinase B (TrkB) corre- lated well with the secretion of BDNF. In the differentiating cells, CREB was active after the binding of growth factors to induce phosphorylation of ERK in the MAPK/ERK pathway. Downstream of phosphorylated CREB led to the functional maturation of differentiated cells and secretion of BDNF, which contributed to the sustained expression of pERK and pCREB. In summary, both PI3K/Akt and MAPK/ERK signaling pathways play important roles in the neuronal differentiation of MSCs. The main function of the PI3K/Akt pathway is to maintain cell survival during neural differentiation; whereas the role of the MAPK/ERK pathway is probably to promote the maturation of differentiated MSCs. Further, cellular lev- els of protein kinase C epsilon type (PKC-ε) and kinesin heavy chain (KIF5B) increased with time of induction, whereas the level of NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1) decreased during the time course of differentiation. The correlation between PKC-ε and TrkB suggested that there is cross-talk between PKC-ε and the PI3K/Akt signal- ing pathway.
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Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.

Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.

total RNA obtained from approximately 5610 4 cells, is the most sensitive of the previously reported methods in detecting undiffer- entiated pluripotent stem cells. Lin28 is known to specifically inhibit the processing of let-7 miRNAs, which are involved in cell- fate decisions [28]. Interestingly, the aberrant expression of Lin28 transcripts has been recently reported in human germ-cell tumors [29], suggesting that Lin28 is a useful marker of germ-cell malignancy as well as of pluripotency of hiPSCs. Lin28 mRNA gradually decreased in the differentiation process and was completely diminished by passage 4 (Fig. 4C). These observations suggest that Lin28 transcripts are also available for presenting degree of differentiation in hiPSC-derived products because detection of residual Lin28 confirms the contamination of undifferentiated cells even at a late stage of differentiation. Needless to say, the distinct expression of Lin28 could possibly be observed in other normal somatic cells. However, Lin28 is, at least in part, one of the potent markers for detecting incompletely differentiated cells contained in RPE cells derived from pluripotent stem cells.
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Advances in Retinal Tissue Engineering

Advances in Retinal Tissue Engineering

in vitro and in the subretinal space. Early RPE studies cultured an established cell line (D407) on first generation copolymers made of PLLA and PLGA [37]. The D407 cell line is characterized by a robust proliferative capacity that, in this study was not hindered by a thin micropatterned PLGA composite polymer. In fact, after 7 days D407 cells had reached confluence, elaborated tight junctions and expressed the RPE’s iconic cobblestone morphology [37]. In an attempt to optimize this copolymer, a recent study fabricated 4 different polymers with 4 different ratios of PLLA and PLGA (10:90, 25:75, 50:50, 75:25 and 90:10) [38]. In each case cellular attachment and proliferation was achieved, however only the 25:75 PLLA-PLGA polymer was able to sustain cellular division and polymeric adherence for the course of the month long study [38]. Because the 25:75 PLLA-PLGA polymer was the thinnest and most porous polymer in the group, these findings suggest that the observed cellular viability resulted because of an optimization of these characteristics. Although immortalized cell lines display morphologic similarities to in vivo tissues, recent studies on tissue culture transwells have shown that ESC, iPS and fetal cell cultures more closely resemble actual RPE [39]. Transwell studies showed that all three primary cultures underwent melanization, attained high transepithelial resistance and mature protein expression [17,39,40]. These results have spurred investigations that have successfully cultured human embryonic stem cells on PLGA and parylene (a non-biodegradable polymer) [41–43]. These cells pigmented and produced mature RPE markers such as ZO-1, RPE65 and PEDF, bestophin and RDH5 on both substrates [41]. Further, human ESC-RPE seeded on parylene withstood surgical manipulation, maintained the appropriate morphology and survived for one month in the subretinal space of RCS rats [42,43].
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Towards the maturation and characterization of smooth muscle cells derived from human embryonic stem cells.

Towards the maturation and characterization of smooth muscle cells derived from human embryonic stem cells.

hVSMCs or hESC-derived SMPCs were treated with the inhibitor W-7 (12 m g/mL, Sigma) for 30 min and the agonist U46619 (1 m M, a CAM kinase-agonist) (Cayman Chemicals, http://www.caymanchem.com) in serum-free M199 medium for 3 days. At the end, cells were characterized for the expression of SMC markers by immunostaining. Finally, the contraction of U46619-treated cells was evaluated by embedding the cells in fibrin gels (3.5610 4 cells/50 m L) and measuring gel size at time 0 and 14 h by microscopy. This methodology was repeated to evaluate the effect of Rho kinase-agonist in cell contraction and maturation. In this case, the cells were exposed to the inhibitor Y27632 (13 m M) (Cayman Chemicals) and the agonist endothelin- 1 (End-1, 10 nM, Sigma).
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Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia.

Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia.

Deregulation of Jak-Stat Signaling in EVI1 Leukemia Global biological function analysis using all significant EVI1 binding gene targets revealed the Pathways in cancer and Jak-Stat signaling pathways were most aberrant. Given a surprising 88% of the EVI1 binding sites contained an ETS-like AGGAAG binding motif, we repeated the analysis using only EVI1 gene targets with the motif. This revealed the Jak-Stat signaling was the most significantly enriched KEGG pathway. We found EVI1 signifi- cantly binds to the promoter region of a remarkable 50 gene targets involved in the Jak-Stat signaling pathway (Figure 10). Of these 50 genes, expression levels of 10 were significantly aberrant. Jak-Stat signaling is one of the principal mechanism by which extracellular signals, specifically cytokines and growth factors, are translated into intracellular responses [65]. Various ligands such as erythropoietin, growth hormones, interferons and interleukins bind their cognate receptors which are associated with JAK tyrosine kinases (JAK1, JAK2, JAK 3 and TYK2) [66,67]. Upon ligand binding, JAKs are transphosphorylated and subsequently phosphorylate latent STAT transcription factors in the cytoplasm. Phosphorylated STATs enter the nucleus and activate or repress gene targets critical for cellular differentiation, proliferation and death [68]. STAT transcription factors are regulated through various inhibitory factors, including the suppressor of cytokine signaling (SOCS) proteins [69].
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Looking at tricellulin expression in the brain

Looking at tricellulin expression in the brain

particularly in undifferentiated state, whereas Shimizu et al. (2008) found TJ molecules in pericytes. Furthermore, it was reported occludin expression by neuron and glial cells in human brain (Romanitan et al., 2007). Moreover, TJ not only provide structural integrity but also function as landmarks, spatially confining signaling molecules and polarity cues, as well as serving as docking sites for vesicles (Nelson, 2003). Given these facts, we hypothesized that TRIC might also be expressed in neurons and analyzed rat neurons in primary culture at 3 DIV, which correspond to immature neurons (Falcão et al., 2006), for TRIC expression. The observation of TRIC mRNA expression, but not of protein expression, as assessed by western blot and immunofluorescence, may result from low expression levels of the protein, whereas the mRNA levels point to the capacity of neurons to retain, or even regain, some of their neuroepithelial characteristics throughout cultivation (Bauer et al. 1999). It is also conceivable that TRIC protein expression increases in neuropathological conditions, in an attempt to contribute to the barrier function. In fact, Brightman and Reese (1969) reported that, in a few instances, TJ are found between neuronal processes. Furthermore, occludin and claudins increase in Alzheimer‟s disease and vascular dementia‟s brains (Romanitan et al., 2007; Romanitan et al., 2010), pathologies associated with a disruption of the BBB (Zlokovic, 2008).
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Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.

Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.

Human induced pluripotent stem cells (hiPSCs) generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs), as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as ‘‘embryonic stem cell-like’’, these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the ‘‘distance’’ between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.
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Acinetobacter baumannii outer membrane vesicles elicit a potent innate immune response via membrane proteins.

Acinetobacter baumannii outer membrane vesicles elicit a potent innate immune response via membrane proteins.

To determine which OMV components are responsible for pro- inflammatory cytokine response, HEp-2 cells were treated with proteinase K-treated A. baumannii OMVs and gene expression of pro-inflammatory cytokines was measured. Treatment with proteinase K resulted in alteration of the protein profile of OMVs (Fig 4A). All proteins in the OMVs were not degraded, but many high molecular weight bands disappeared, suggesting degradation of membrane proteins and protection of luminal proteins. Up- regulation of pro-inflammatory cytokine genes was not observed in response to proteinase K-treated A. baumannii OMVs (Fig. 4B). Next, in order to determine whether lysed OMVs stimulated pro- inflammatory response like that of intact OMVs, A. baumannii OMVs were pre-treated with EDTA in order to disintegrate the OMV membrane, resulting in lysis of OMVs, and HEp-2 cells were treated with lysed OMVs for 24 h. Treatment with EDTA did not result in alteration of the protein profile of OMVs (Fig 4A). The lysed A. baumannii OMVs induced up-regulation of IL-1b, IL- 6, IL-8, and MIP-1a genes, but not induce MCP-1 gene (Fig. 4B). Intact A. baumannii OMVs elicited greater pro-inflammatory cytokine gene expression than the response to the lysed A. baumannii OMVs (Fig 4B). To determine whether AbOmpA in A. baumannii OMVs was responsible for pro-inflammatory cytokine response, HEp-2 cells were treated with OMVs purified from A. baumannii ATCC 19606 T and its isogenic AbOmpA-deficient
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Characterization of human adipose-derived stem cells

Characterization of human adipose-derived stem cells

ibrous material and visible blood vessels, adipose tissue samples were cut into small pieces ( 10 to 20 mm) and digested in 10 mM Phosphate- Buffered Saline (PBS) (Sigma Aldrich, St. Louis, Missouri, USA) containing 200 U/ml crude collagenase II (Sigma Aldrich, St. Louis, Missouri, USA) for 40 minutes in a shaking water bath at 37ºC. The dispersed material was iltered through a nylon mesh with a pore size of 150 μm. After centrifugation (300g), the mature adipocytes remained on the supernatant surface and the ASCs formed a pellet at the bottom of the tube. Ficoll- hypaque (Histopaque ® ) was used as a centrifugation gradient for
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Dental Press J. Orthod.  vol.16 número6 en a17v16n6

Dental Press J. Orthod. vol.16 número6 en a17v16n6

constant irrigation, and the pulp tissue removed with the aid of curettes and endodontic files. Only a small amount of pulp tissue was obtained. One of the four deciduous teeth extracted no longer contained pulp tissue and the others had a small amount of pulp. Therefore, few cells were availed to begin cultivation. Once collected, the tissue was immediately placed in culture bottles containing DMEM supplemented with 10% fetal bovine se- rum (SBF, Cultilab, Campinas, Brazil) and stored at 37 °C and 5% CO 2 for cell proliferation and adherence to the bottle. The medium was com- pletely replaced every three days during a period of approximately 10 days, when culture reached about 80-90% confluence. The culture was moni- tored by means of an inverted optical microscope. After adherence to the plastic surface, SCs ini- tially exhibited an ovoid shape that evolved early during the first 24 hours to a fibroblastoid form, which remained until confluence (Fig 1). As the culture was replaced, the adherent mesenchymal SCs were selected while cells suspended in cul- ture medium were gradually discarded.
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