mouse anti-nestin Search Results


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  • 94
    Millipore mouse monoclonal anti nestin
    Neurons with action potentials and glial cells differentiated from induced neural stem cells from adult peripheral CD34+ cells. The induced neural stem cells differentiated into βIII- tubulin positive neurons (green, Ai) in neuronal differentiation or GFAP positive astroglia (red, Aii) in astroglial differentiation medium for 1 week. Oligodendrocyte progenitor differentiation was achieved by incubating the neural stem cells with oligodendrocyte differentiation medium. O4 positive cells (green, Aiii, after 2 weeks) and a few of MBP positive cells (red, Aiv, after four weeks) were detected by immunostaining. Spontaneous neuronal differentiation was also observed in low concentration seeded neural stem cell culture (1000 cells/well in a 24 well plate) after incubation in neural stem cell medium for 1 week (Av). Action potentials were recorded from neurons differentiated for 2 weeks using whole cell patch clamp. Action potentials recorded on two separated cells are shown (Avi). Double staining with either VGLUT1 or VGAT with βIII- tubulin showed most βIII- tubulin positive neurons were either glutamatergic or gabaergic neurons while a few of doparminergic neurons were also detected by TH immunostaining after incubation in neuronal differentiation medium for 2 weeks (B). The induced neural stem cells were passaged for 17 passages and <t>immunostained</t> for <t>nestin</t> as a neural stem cell marker. The cells were also cultured in neural differentiation medium for 2 weeks and immunostained for βIII-tubulin to determine their neuronal differentiation capabilities (C).
    Mouse Monoclonal Anti Nestin, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti nestin
    RA regulates RPC proliferation and differentiation through miR-29a. a The results of the CCK-8 analysis showed that RA induced RPC proliferation inhibition, and this trend could be partially rescued by the miR-29a inhibitor. b The qPCR analysis showed that the expression of Ki-67 was upregulated in RPCs treated with both RA and the miR-29a inhibitor compared with those only treated with RA. c The expression levels of RPC differentiation markers (β3-tubulin, rhodopsin and recoverin) were increased RA-treated RPC cultures (compared with control cultures), and miR-29a inhibitor partially reversed RA induced RPC differentiation markers upregulation. d Compared with RPCs only treated with RA, the expression levels of <t>nestin</t> and Pax-6 were upregulated in RPCs treated with both RA and the miR-29a inhibitor, according to the qPCR analysis. e A model of the role of <t>REST</t> mediated by RA in the regulation of RPC proliferation and differentiation. RA could directly degrade REST protein through the proteasome and indirectly suppress REST gene expression through the upregulation of miR-29a. Data are the averages of three independent experiments. Error bars indicate the standard deviation of the mean. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test)
    Mouse Anti Nestin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 557 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Abcam mouse anti nestin
    <t>ADAM17</t> deficiency affected NSCs differentiation and those cells stopped at nIPCs stage as indicated by markers staining. Mouse cortex electroporated at E14.5 with A17-Ri/EYFP were dissected out at E18.5. All sections were cut at 20 mm and processed for IF with antibodies against one of those following markers: <t>Nestin</t> (b), Tuj1 (f), Tbr1 (j) and Map2 n). DAPI staining was used to indicate nuclei and merged images were presented in c-d, g-h, k-l, o-p. The scale bar is 100 mm. Those ADAM17 deficient cells were stained positive with Nestin (c) and Tuj1 (g), but not with Tbr1 (k) or Map2 (o).
    Mouse Anti Nestin, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank mouse anti nestin
    <t>NG2</t> + cell growth is not inhibited by CM from normal peritoneal macrophages (PM) or astrocytes (AST). The numbers and sizes of the spheres ( A ) as well as total cell numbers ( B ) in the presence of the media conditioned by normal PM or AST were similar to the control group (NG2 + cells alone) after 2 weeks culture. There was reduction of sphere formation and total cell numbers when NG2 + cells were cultured with the OX42 + cell-CM. N = 3. *Significantly different from the NG2 + cells in the absence of conditioned medium. C: Representative cells from sphere culture with OX42 + cell-CM. All cells show co-expression of NG2 and <t>nestin.</t> Scale bar = 100 µm.
    Mouse Anti Nestin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mouse anti nestin
    Repression of nNOS negatively regulates neuronal fate commitment. Monolayer-cultured embryonic NSCs differentiated for 4 days and were incubated with L-VNIO (100 μM) or vehicle during the later 2 days of differentiation. (A,B) L-VNIO inhibits neuronal differentiation. (A) Representatives of <t>β-III-Tubulin</t> + neurons. (B) Statistical graph showing the ratio of β-III-Tubulin + neurons. (C,D) L-VNIO has no effect on cell proliferation during NSCs differentiation. BrdU (2.5 μM) was added during the later 2 days of differentiation to label the dividing cells. (C) Representatives of BrdU-labeled cells. (D) Statistical graph showing the ratio of BrdU + cells. (E,F) L-VNIO induces cell apoptosis. Live cultures were stained with PI which stains dead cells and Hoechst 33342 which stains live and dead cells. (E) Representatives of PI + nuclei (arrows). (F) Statistical graph showing the ratio of PI + nuclei. (G) L-VNIO increases the rate of cell death (δ 4 ). (H) L-VNIO decreases the rate of conversion of progenitors to neurons (β 4 ) even if all dead cells are neurons. (I) There are <t>nestin</t> + progenitor cells throughout the differentiation stages. Cells were fixed and stained for nestin at days 1, 2, 3, 4 after differentiation respectively. Data shown are mean ± SEM from three to five independent experiments in parallel cultures; ∗ p
    Mouse Anti Nestin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti nestin
    Cortical spheroids contained CNS cell types, and cells formed laminin-containing 3D networks. Confocal projections of 1, 7, 14, and 21 DIV 8k spheroids revealed the presence of CNS cell types in the cortical spheroids, including neurons (β-III-tubulin, red ), astrocytes (GFAP, green ), oligodendrocytes (O1, yellow ), neural stem/progenitor cells <t>(nestin,</t> cyan ), and microglia (CD11b, magenta ). Nuclei were counterstained with DAPI ( blue ). Cell processes formed 3D networks within the cortical spheroids. Confocal projections showed laminin expression ( gray
    Mouse Anti Nestin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1814 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti nestin monoclonal antibody
    <t>Nestin</t> expression around the central canal. In the cystic spinal cord (A1), 5-bromo-2’-deoxyuridine (BrdU)-labeled cells are seen in the lesion site (B1). The images (C1, D) did not show immunoreactivity for nestin 3 weeks after transplantation. Nestin expression was restricted to ependymal cell layer (star) (C2). In the same region, labeled cells were not observed around the central canal (B2). Magnification at 10 × for A1, B1, C1, D and at 40 × for A2, B2, C2. V: Ventral; D: dorsal; CC: central canal; BF: bright field.
    Mouse Anti Nestin Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal mouse anti nestin
    iPS characterization as pluripotent ES-like cells: Human iPS were derived from HFF cells following retroviral infection with the four reprogramming factors and display morphology similar to that of hES cells: (A) iPS colony, hES (H9.2) colony and HFF morphology. (B) Immunofluorescence staining for pluripotent markers: Oct4, TRA-1–81, TRA-1–60 and SSEA4 for iPS clone 3. DAPI nuclear staining indicated on left panel. (C) Analysis of gene expression by PCR. The transcription of Oct4, Sox2, c-myc, Klf4, Nanog and Rex1 were analysed in three iPS clones C1, C2 and C3 as well in the hESC clone–H9.2 and parental HFF. GAPDH was used as amplification and loading control. (D, E) Spontaneous differentiation of human iPS into EBs and teratomas. Human iPS were induced to spontaneous differentiation in vitro and in vivo. (D) The upper left panel illustrates EBs derived from human iPS clone 3. Upper right and bottom left and right panels show immunofluorescence staining for markers of the three germ layers in human IPS derived EBs. Representative ectoderm marker <t>β-tubulin-III,</t> representative mesoderm marker SMA and representative endoderm marker α-Fetoprotein (E) Immunofluorescence staining for different markers (up) and haematoxylin and eosin staining demonstration of typical tissue morphology (bottom) were performed on teratomas to confirm derivatives of the three germ layers: ectoderm – representative marker <t>Nestin</t> staining and neural rosettes, mesoderm – SMA marker staining and cartilage endoderm – a-Fetoprotein marker staining and gut like epithelium.
    Monoclonal Mouse Anti Nestin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    R&D Systems human nestin antibody
    p38MAPK inhibitor-treated hESC differentiate into all three germ layers in vitro . (A) Immunofluorescence staining of SB203580-treated (p38i) and untreated (Ctl) hEB on day 30 of differentiation demonstrated expression of markers of all three embryonic germ layers, namely <t>nestin</t> and βIII tubulin (ectoderm), <t>α-fetoprotein</t> (AFP; endoderm) and smooth muscle actin (SMA) and cTnT (mesoderm). Bar 10 μm. (B) hEB from SB203580-treated (p38i) and untreated (Ctl) cultures collected prior to treatment, at day 4, and at day 14 were assayed for germ layer-specific gene expression by qPCR. Expression was normalized to GAPDH and calculated relative to proliferating, undifferentiated hESC. There was no significant difference between expression levels of βIII tubulin, SMA or α-fetoprotein in treated and untreated cultures. Data shown are mean percentage ± SEM ( n = 3).
    Human Nestin Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen mouse anti nestin
    Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , <t>NESTIN</t> (E,F) , SOX2, <t>GFAP</t> (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.
    Mouse Anti Nestin, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Becton Dickinson mouse anti rat nestin
    Identification of the core sequence and functional cis -elements in the second intron of the mouse <t>nestin</t> gene. A , the minimal enhancer region was localized to the 3′ 320 bp (1310/1629) region of the second intron. P19EC cells or P19NPCs were transfected with deletion constructs as indicated in the left panel , and the luciferase activity was determined. The minimal enhancer containing 3′ 320 bp is indicated by an asterisk. B , potential cis -elements in the 3′ 320 bp of the second intron. The position of the first nucleotide of the second intron was designated as 1. The consensus sequences of putative cis -elements are shown in boxes. C , alignment of the nucleotide sequence, which contains the binding sites for Sox, POU, HRE, and <t>SF1</t> factors in the 320-bp region with its counterparts in the rat and human nestin genes. D , identification of functional cis -elements. P19EC or P19NPC cells were transfected with site-mutated constructs as indicated in the left panel , and the luciferase activity was determined. The activity of each construct was shown relative to that of the enhancerless pGL3-Px′ vector. Values were normalized for transfection efficiency by co-transfection with a Renilla luciferase expression plasmid, and they are shown as means ± S.D. for three independent experiments.
    Mouse Anti Rat Nestin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc mouse anti nestin
    Time in culture reduces Müller cell reactivity and promotes mature phenotype. A-C. Representative epifluorescence micrographs of primary Müller cells grown for 5, 12, 20 or 30 days in vitro (DIV) in serum-containing media immunolabeled with antibodies against <t>nestin</t> ( A ), vimentin ( B ) and <t>GFAP</t> ( C ) and counterstained with DAPI (blue). Nestin ( A ) and GFAP ( C ) immunolabeling decreased substantially at 20 and 30 DIV. Vimentin immunolabeling also decreased, but not until 30 DIV ( B ). Scale bar = 100μm.
    Mouse Anti Nestin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti nestin antibody
    Tunicamycin inhibits the self-renewal of glioma-initiating cell (GIC) A. Representative images of neurospheres isolated from human GBM samples (T698968, T19002) and tumor xenograft formed by glioma cell line (SHG44). Scale bar represents 100 μm. B. Glioma-initiating cells expressed neural stem cell marker CD133 (red) and <t>Nestin</t> (green), as assessed by immunofluorescence. Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm. C. Glioma-initiating cells expressed core stemness factors Sox2, OCT4 and Nanog, as assessed by western blot. β -actin expression served as loading control. D. Summary of the tumor-initiating capacity of the neurosphere cultures derived from human GBM samples and glioma xenograft. E-I. GICs were plated at 200 cells per well in 96-well plates in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days. Tunicamycin treatment increased expression of ER stress marker <t>CHOP</t> using western blot analysis ( E ). ( F ) Representative photographs of neurospheres formed by SHG44 GICs in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days. Scale bar represents 100 μm. ( G-I ) The numbers of neurospheres formed by SHG44 ( G ), T698968 ( H ) or T19002 GICs ( I ) in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days were determined. Values represent mean ± S.D. (n = 6, *** p
    Mouse Anti Nestin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA mouse anti nestin
    Y3 Counteracts HuD-Induced Neurogenesis (A) Differentiating ESCs cultures assayed for Y3 and HuD expression levels by Northern blot and western blot, respectively. Cultures were immunostained for stage-specific markers: <t>Oct4</t> (ESCs; red), <t>Nestin</t> (NPCs; red), and beta3-tubulin (early neurons; red); the scale bar corresponds to 75 μm. Relative quantification of Y3 and HuD levels are shown (right). (B) Differentiated NSC-34 cells (control or silenced for Y3) immunostained with anti-tubulin antibody (yellow) to detect neurites (left panel); GFP (green) identified transfected cells subjected to high content analysis; the scale bar corresponds to 100 μm. Multiple parameters were analyzed using Operetta HCS device (right panel). (C) Differentiation assay in control Y3 silenced cells, Y3 silenced cells transfected with wild-type HuD or with mutant HuD. A schematic representation of HuD constructs used in the experiment is provided. (D) PC12 cells were co-transfected with HA-tagged HuD and mock or Y3 WT or Y3 “deleted” vectors. Co-transfected cells were immunostained with anti-HA antibody, and the neurites were stained for tubulin. In (A)–(D), data are represented as mean ± SEM t test ∗p
    Mouse Anti Nestin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Neuromics mouse anti nestin
    Phenotypic profiling of astrocytes in the presence and absence of an ECM coating. Representative fluorescent images showing a heterogenous population of cells expressing <t>nestin</t> and <t>GFAP</t> at 15 DIV ( a ) and ~30 DIV ( b ). Scale bar = 50 μm. Bar graph summarizes flow cytometry data highlighting the distribution of astrocytic phenotypes (Nestin−GFAP−, Nestin+GFAP−, Nestin+GFAP+, Nestin−GFAP+) at 15 DIV ( c ) and ~30 DIV ( d ). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats).
    Mouse Anti Nestin, supplied by Neuromics, used in various techniques. Bioz Stars score: 94/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems mouse anti nestin
    Female and male NSPCs were grown as neurospheres without sex hormones for 4 days then allowed to adhere to a laminin substrate and expose to either 10 nM testosterone (T), 17β-estradiol (E 2 ), progesterone (P 4 ) for 20 min before <t>Ki67/nestin</t> double label fluorescent immunocytochemistry was performed (A, female control neurosphere shown). The proportion of DAPI/nestin-labeled cells expressing Ki67 per neurosphere was increased by 20 min of either T, E 2 or P 4 compared to controls in both females (B) and males (C) . Female and male neurospheres maintained without sex hormones were differentiated on a laminin substrate for 72 h ± 10 nM T, E 2 or P 4 when βIII-tubulin (red) immunocytochemistry (D) showed that while T, P 4 and E 2 increased the proportion of neuronal differentiation compared to controls, E 2 was more potent compared to T and P 4 in females (E) and males (F) . Results show mean ± SEM, * p
    Mouse Anti Nestin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse monoclonal anti nestin antibody
    Immunohistochemical staining for <t>DCX</t> in the subventricular zone at different time points (2, 6, 24 and 48 h) after hypoxic-ischemic injury in the control, sham and hypoxic-ischemia groups. DCX, <t>Nestin</t> and doublecortin; MK, MK-801; Ro, Ro25-6981; NVP, NVP-AAM077.
    Mouse Monoclonal Anti Nestin Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson alexa fluor 647 mouse anti nestin
    Immunohistochemical staining for <t>DCX</t> in the subventricular zone at different time points (2, 6, 24 and 48 h) after hypoxic-ischemic injury in the control, sham and hypoxic-ischemia groups. DCX, <t>Nestin</t> and doublecortin; MK, MK-801; Ro, Ro25-6981; NVP, NVP-AAM077.
    Alexa Fluor 647 Mouse Anti Nestin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti human nestin
    Tumor of Mouse-Human NC Chimeras Express Markers of NB. Quantifications of IHC Experiments for the Percentage of Positive Cells Expressing the Typical NB Markers ( A ) Tyrosine hydroxylase (TH), ( B ) <t>Chromogranin</t> A (CgA), ( C ) <t>Nestin</t> (NES), and ( D ) Synaptophysin (SYP), in samples of human NBs from chimeric mice (n=5), subcutaneous xenograft outgrowth of hNCCs (n=4), and NB samples of patients (Data presented as means, error bars represent SD, dots represent fields of view).
    Mouse Anti Human Nestin, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Neurons with action potentials and glial cells differentiated from induced neural stem cells from adult peripheral CD34+ cells. The induced neural stem cells differentiated into βIII- tubulin positive neurons (green, Ai) in neuronal differentiation or GFAP positive astroglia (red, Aii) in astroglial differentiation medium for 1 week. Oligodendrocyte progenitor differentiation was achieved by incubating the neural stem cells with oligodendrocyte differentiation medium. O4 positive cells (green, Aiii, after 2 weeks) and a few of MBP positive cells (red, Aiv, after four weeks) were detected by immunostaining. Spontaneous neuronal differentiation was also observed in low concentration seeded neural stem cell culture (1000 cells/well in a 24 well plate) after incubation in neural stem cell medium for 1 week (Av). Action potentials were recorded from neurons differentiated for 2 weeks using whole cell patch clamp. Action potentials recorded on two separated cells are shown (Avi). Double staining with either VGLUT1 or VGAT with βIII- tubulin showed most βIII- tubulin positive neurons were either glutamatergic or gabaergic neurons while a few of doparminergic neurons were also detected by TH immunostaining after incubation in neuronal differentiation medium for 2 weeks (B). The induced neural stem cells were passaged for 17 passages and immunostained for nestin as a neural stem cell marker. The cells were also cultured in neural differentiation medium for 2 weeks and immunostained for βIII-tubulin to determine their neuronal differentiation capabilities (C).

    Journal: PLoS ONE

    Article Title: Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

    doi: 10.1371/journal.pone.0081720

    Figure Lengend Snippet: Neurons with action potentials and glial cells differentiated from induced neural stem cells from adult peripheral CD34+ cells. The induced neural stem cells differentiated into βIII- tubulin positive neurons (green, Ai) in neuronal differentiation or GFAP positive astroglia (red, Aii) in astroglial differentiation medium for 1 week. Oligodendrocyte progenitor differentiation was achieved by incubating the neural stem cells with oligodendrocyte differentiation medium. O4 positive cells (green, Aiii, after 2 weeks) and a few of MBP positive cells (red, Aiv, after four weeks) were detected by immunostaining. Spontaneous neuronal differentiation was also observed in low concentration seeded neural stem cell culture (1000 cells/well in a 24 well plate) after incubation in neural stem cell medium for 1 week (Av). Action potentials were recorded from neurons differentiated for 2 weeks using whole cell patch clamp. Action potentials recorded on two separated cells are shown (Avi). Double staining with either VGLUT1 or VGAT with βIII- tubulin showed most βIII- tubulin positive neurons were either glutamatergic or gabaergic neurons while a few of doparminergic neurons were also detected by TH immunostaining after incubation in neuronal differentiation medium for 2 weeks (B). The induced neural stem cells were passaged for 17 passages and immunostained for nestin as a neural stem cell marker. The cells were also cultured in neural differentiation medium for 2 weeks and immunostained for βIII-tubulin to determine their neuronal differentiation capabilities (C).

    Article Snippet: Cells were immunostained with mouse monoclonal anti-Nestin (Clone 10C2, 1:1000; Millipore Billerica, MA), mouse monoclonal anti-β-III-tubulin (1:1000; Promega, Madison, WI), rabbit anti-PAX6 (1:200, Abcam), rabbit anti-vGLUT1 (1:200, Abcam), rabbit anti-vGAT (1:200, Abcam) rabbit anti-TH (1: 1000, Novus) rabbit anti-GFAP (1:1000, Sigma), mouse monoclonal anti-oligodendrocyte marker O4 (1:200, R & D system, Minneapolis, MN), chicken anti-MBP antibody (1:200, Millipore) and ready to use rabbit anti-SOX-2 and anti-OCT4 antibodies from hES/iPS cell characterization kit (Applied Stemcell), followed by corresponding secondary antibodies (anti-mouse Alexa Fluor 488, 1:400; anti-rabbit Alexa Fluor 594, 1:400; anti-chicken Alexa Fluor 594, 1:400; Invitrogen) and DAPI nuclear staining.

    Techniques: Immunostaining, Concentration Assay, Stem Cell Culture, Incubation, Patch Clamp, Double Staining, Marker, Cell Culture

    Generation of neural stem cells from cord blood CD34+ cells. (A) Non adherent cord blood CD34+ cells (Ctrl) were transduced with Sendai virus constructs containing Oct3/4, Sox2, Klf4 and c-Myc and maintained in neural stem cell medium. Adherent cells were observed on day 2 which proliferated rapidly through day 4 post-infection. (B) More than 95% of the generated cells immunostained for nestin (green) and SOX-2 (red), but were OCT4 negative. Cell nuclei were counterstained with DAPI (blue). The cells were subcultured up to passage 7, when a decline in nestin positive cells was first noticed (C). Spontaneously differentiated neuronal like cells were observed when incubated in neural stem cell medium for over a week without changing medium. These cells developed neural sphere like structures from which βIII-tubulin positive cells with long processes grew out (D). When neural stem cells derived from CD34+ cells were placed in neural differentiation medium for two weeks, both βIII-tubulin (green) neurons and GFAP (red) positive astroglia were seen, along with some cell aggregates (E). Immunostaining for NeuN (green) in the nuclei confirms the presence of relatively mature neurons (F).

    Journal: PLoS ONE

    Article Title: Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

    doi: 10.1371/journal.pone.0081720

    Figure Lengend Snippet: Generation of neural stem cells from cord blood CD34+ cells. (A) Non adherent cord blood CD34+ cells (Ctrl) were transduced with Sendai virus constructs containing Oct3/4, Sox2, Klf4 and c-Myc and maintained in neural stem cell medium. Adherent cells were observed on day 2 which proliferated rapidly through day 4 post-infection. (B) More than 95% of the generated cells immunostained for nestin (green) and SOX-2 (red), but were OCT4 negative. Cell nuclei were counterstained with DAPI (blue). The cells were subcultured up to passage 7, when a decline in nestin positive cells was first noticed (C). Spontaneously differentiated neuronal like cells were observed when incubated in neural stem cell medium for over a week without changing medium. These cells developed neural sphere like structures from which βIII-tubulin positive cells with long processes grew out (D). When neural stem cells derived from CD34+ cells were placed in neural differentiation medium for two weeks, both βIII-tubulin (green) neurons and GFAP (red) positive astroglia were seen, along with some cell aggregates (E). Immunostaining for NeuN (green) in the nuclei confirms the presence of relatively mature neurons (F).

    Article Snippet: Cells were immunostained with mouse monoclonal anti-Nestin (Clone 10C2, 1:1000; Millipore Billerica, MA), mouse monoclonal anti-β-III-tubulin (1:1000; Promega, Madison, WI), rabbit anti-PAX6 (1:200, Abcam), rabbit anti-vGLUT1 (1:200, Abcam), rabbit anti-vGAT (1:200, Abcam) rabbit anti-TH (1: 1000, Novus) rabbit anti-GFAP (1:1000, Sigma), mouse monoclonal anti-oligodendrocyte marker O4 (1:200, R & D system, Minneapolis, MN), chicken anti-MBP antibody (1:200, Millipore) and ready to use rabbit anti-SOX-2 and anti-OCT4 antibodies from hES/iPS cell characterization kit (Applied Stemcell), followed by corresponding secondary antibodies (anti-mouse Alexa Fluor 488, 1:400; anti-rabbit Alexa Fluor 594, 1:400; anti-chicken Alexa Fluor 594, 1:400; Invitrogen) and DAPI nuclear staining.

    Techniques: Transduction, Construct, Infection, Generated, Incubation, Derivative Assay, Immunostaining

    RA regulates RPC proliferation and differentiation through miR-29a. a The results of the CCK-8 analysis showed that RA induced RPC proliferation inhibition, and this trend could be partially rescued by the miR-29a inhibitor. b The qPCR analysis showed that the expression of Ki-67 was upregulated in RPCs treated with both RA and the miR-29a inhibitor compared with those only treated with RA. c The expression levels of RPC differentiation markers (β3-tubulin, rhodopsin and recoverin) were increased RA-treated RPC cultures (compared with control cultures), and miR-29a inhibitor partially reversed RA induced RPC differentiation markers upregulation. d Compared with RPCs only treated with RA, the expression levels of nestin and Pax-6 were upregulated in RPCs treated with both RA and the miR-29a inhibitor, according to the qPCR analysis. e A model of the role of REST mediated by RA in the regulation of RPC proliferation and differentiation. RA could directly degrade REST protein through the proteasome and indirectly suppress REST gene expression through the upregulation of miR-29a. Data are the averages of three independent experiments. Error bars indicate the standard deviation of the mean. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test)

    Journal: Cell Death & Disease

    Article Title: REST, regulated by RA through miR-29a and the proteasome pathway, plays a crucial role in RPC proliferation and differentiation

    doi: 10.1038/s41419-018-0473-5

    Figure Lengend Snippet: RA regulates RPC proliferation and differentiation through miR-29a. a The results of the CCK-8 analysis showed that RA induced RPC proliferation inhibition, and this trend could be partially rescued by the miR-29a inhibitor. b The qPCR analysis showed that the expression of Ki-67 was upregulated in RPCs treated with both RA and the miR-29a inhibitor compared with those only treated with RA. c The expression levels of RPC differentiation markers (β3-tubulin, rhodopsin and recoverin) were increased RA-treated RPC cultures (compared with control cultures), and miR-29a inhibitor partially reversed RA induced RPC differentiation markers upregulation. d Compared with RPCs only treated with RA, the expression levels of nestin and Pax-6 were upregulated in RPCs treated with both RA and the miR-29a inhibitor, according to the qPCR analysis. e A model of the role of REST mediated by RA in the regulation of RPC proliferation and differentiation. RA could directly degrade REST protein through the proteasome and indirectly suppress REST gene expression through the upregulation of miR-29a. Data are the averages of three independent experiments. Error bars indicate the standard deviation of the mean. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test)

    Article Snippet: Various antibodies, including rabbit monoclonal anti-REST (Abcam, Cambridge, UK), mouse anti-nestin (BD, San Jose, CA, USA), rabbit monoclonal anti-Pax-6 (Biolegend, San Diego, CA, USA), mouse monoclonal anti-β3-tubulin (Millipore, Billerica, MA, USA), mouse monoclonal anti-rhodopsin, rabbit polyclonal anti-recoverin (Millipore), mouse monoclonal anti-Brn3a (Millipore) and mouse monoclonal anti-Caspase-3 (Santa Cruz, California, USA) were diluted 1:1000, and mouse anti-β-actin (Sigma-Aldrich) was diluted 1:5000.

    Techniques: CCK-8 Assay, Inhibition, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    FMRP is expressed in RGCs at relatively high levels during neocortical development. A rabbit polyclonal FMRP antibody (Sigma) was used for immunoblot and immunohistochemical analysis of the embryonic mouse neocortex. A , FMRP can be detected in neocortical lysates throughout cortical neurogenesis. Neocortices from littermate wild-type (WT) and Fmr1 knock-out (KO) embryos were lysed in radioimmunoprecipitation assay buffer, resolved by SDS-PAGE, and probed for FMRP and β-actin (loading control). B , FMRP is present in perinuclear regions throughout the neocortex but at the highest levels near the pial surface (arrows) and the ventricular surface (arrowheads). Pax6 immunoreactivity labels the ventricular zone. C , D , The FMRP immunoreactivity near the pial surface and the ventricular surface colocalizes with nestin + endfeet of basal ( C , arrows) and apical ( D , arrowheads) processes, respectively. DAPI, 4′,6-Diamidino-2-phenylindole. Scale bars: B , 20 μm; C , D , 5 μm.

    Journal: The Journal of Neuroscience

    Article Title: FMRP Regulates the Transition from Radial Glial Cells to Intermediate Progenitor Cells during Neocortical Development

    doi: 10.1523/JNEUROSCI.4854-10.2011

    Figure Lengend Snippet: FMRP is expressed in RGCs at relatively high levels during neocortical development. A rabbit polyclonal FMRP antibody (Sigma) was used for immunoblot and immunohistochemical analysis of the embryonic mouse neocortex. A , FMRP can be detected in neocortical lysates throughout cortical neurogenesis. Neocortices from littermate wild-type (WT) and Fmr1 knock-out (KO) embryos were lysed in radioimmunoprecipitation assay buffer, resolved by SDS-PAGE, and probed for FMRP and β-actin (loading control). B , FMRP is present in perinuclear regions throughout the neocortex but at the highest levels near the pial surface (arrows) and the ventricular surface (arrowheads). Pax6 immunoreactivity labels the ventricular zone. C , D , The FMRP immunoreactivity near the pial surface and the ventricular surface colocalizes with nestin + endfeet of basal ( C , arrows) and apical ( D , arrowheads) processes, respectively. DAPI, 4′,6-Diamidino-2-phenylindole. Scale bars: B , 20 μm; C , D , 5 μm.

    Article Snippet: The following primary antibodies were also used: mouse monoclonal anti-β-actin (GenScript, 1:5000 for Western blot), mouse monoclonal anti-Pax6 (Developmental Studies Hybridoma Bank, 1:100 for immunostaining), mouse monoclonal anti-Ser10 phosphorylated histone H3 (Abcam, 1:200 for immunostaining), mouse monoclonal anti-nestin (BD Biosciences, 1:1000 for immunostaining), mouse monoclonal Tuj1 (Covance, 1:1000 for immunostaining), rabbit polyclonal anti-Tbr2 (Abcam, 1:1000 for immunostaining), rabbit polyclonal anti-Pfn1 (Abcam, 1:200 for immunostaining), rabbit polyclonal anti-activated caspase 3 (Millipore Bioscience Research Reagents, 1:200 for immunostaining), and chicken polyclonal anti-GFP (Aves Labs, 1:500 for immunostaining).

    Techniques: Immunohistochemistry, Knock-Out, Radio Immunoprecipitation, SDS Page

    Immunocytochemical analysis of sphere colonies from the peripheral stroma on day 7. Bright-field images and immunostaining of spheres are shown. The spheres were stained for vimentin (a mesenchymal cell marker), \alpha-smooth muscle actin (α-SMA, a mesenchymal cell marker), cytokeratin 3 (a differentiated epithelial cell marker), nestin (a neural stem cell marker), microtubule-associated protein 2 (MAP2, a differentiated neural cell marker), neuron-specific enolase (NSE, a differentiated neural cell marker, and CD34 (a stem cell marker). Each colony is also labeled by BrdU. As a negative control, IgG was used instead of the primary antibody. Scale bar=100 μm.

    Journal: Molecular Vision

    Article Title: Isolation and distribution of rabbit keratocyte precursors

    doi:

    Figure Lengend Snippet: Immunocytochemical analysis of sphere colonies from the peripheral stroma on day 7. Bright-field images and immunostaining of spheres are shown. The spheres were stained for vimentin (a mesenchymal cell marker), \alpha-smooth muscle actin (α-SMA, a mesenchymal cell marker), cytokeratin 3 (a differentiated epithelial cell marker), nestin (a neural stem cell marker), microtubule-associated protein 2 (MAP2, a differentiated neural cell marker), neuron-specific enolase (NSE, a differentiated neural cell marker, and CD34 (a stem cell marker). Each colony is also labeled by BrdU. As a negative control, IgG was used instead of the primary antibody. Scale bar=100 μm.

    Article Snippet: Next, the cells were incubated for 2 h at room temperature with the following primary antibodies diluted in BSA/PBST: mouse anti-cytokeratin 3 monoclonal antibody (mAb, AE-5, Progen Biotechnik GMBH, Heidelberg, Germany), mouse anti-\alpha-smooth muscle actin (α-SMA) mAb (1:400; Sigma-Aldrich), mouse anti-vimentin mAb (1:400; Dako, Glostrup, Denmark), mouse anti-nestin mAb (1:400; BD Biosciences), mouse anti-microtubule–associated protein (MAP)-2 mAb (1:400; Chemicon, Temecula, CA), mouse anti-neuron–specific enolase mAb (NSE, 1:400; Dako), mouse anti-CD34 mAb (NCL-END 1:100; Novocastra Laboratories Ltd., Newcastle upon Tyne, UK), and FITC-conjugated mouse anti-5-bromo2’-deoxyuridine (BrdU)/fluorescence mAb (1:100; Roche Diagnostics, Basel, Switzerland).

    Techniques: Immunostaining, Staining, Marker, Labeling, Negative Control

    Reverse-transcription polymerase chain reaction analysis of corneal stromal tissue, spheres, and sphere progeny. G3PDH gene expression can be detected in all samples except those processed without reverse-transcriptase (RT). Vimentin is expressed by the corneal stromal tissues and the spheres derived from the peripheral or central regions and their progeny but is not detected by PCR of total RNA without RT. Expression of nestin by the progeny is lower than by the spheres from both the peripheral and central regions of the cornea. No expression of keratin 3 or 12 is detected in any of these samples.

    Journal: Molecular Vision

    Article Title: Isolation and distribution of rabbit keratocyte precursors

    doi:

    Figure Lengend Snippet: Reverse-transcription polymerase chain reaction analysis of corneal stromal tissue, spheres, and sphere progeny. G3PDH gene expression can be detected in all samples except those processed without reverse-transcriptase (RT). Vimentin is expressed by the corneal stromal tissues and the spheres derived from the peripheral or central regions and their progeny but is not detected by PCR of total RNA without RT. Expression of nestin by the progeny is lower than by the spheres from both the peripheral and central regions of the cornea. No expression of keratin 3 or 12 is detected in any of these samples.

    Article Snippet: Next, the cells were incubated for 2 h at room temperature with the following primary antibodies diluted in BSA/PBST: mouse anti-cytokeratin 3 monoclonal antibody (mAb, AE-5, Progen Biotechnik GMBH, Heidelberg, Germany), mouse anti-\alpha-smooth muscle actin (α-SMA) mAb (1:400; Sigma-Aldrich), mouse anti-vimentin mAb (1:400; Dako, Glostrup, Denmark), mouse anti-nestin mAb (1:400; BD Biosciences), mouse anti-microtubule–associated protein (MAP)-2 mAb (1:400; Chemicon, Temecula, CA), mouse anti-neuron–specific enolase mAb (NSE, 1:400; Dako), mouse anti-CD34 mAb (NCL-END 1:100; Novocastra Laboratories Ltd., Newcastle upon Tyne, UK), and FITC-conjugated mouse anti-5-bromo2’-deoxyuridine (BrdU)/fluorescence mAb (1:100; Roche Diagnostics, Basel, Switzerland).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Polymerase Chain Reaction

    ADAM17 deficiency affected NSCs differentiation and those cells stopped at nIPCs stage as indicated by markers staining. Mouse cortex electroporated at E14.5 with A17-Ri/EYFP were dissected out at E18.5. All sections were cut at 20 mm and processed for IF with antibodies against one of those following markers: Nestin (b), Tuj1 (f), Tbr1 (j) and Map2 n). DAPI staining was used to indicate nuclei and merged images were presented in c-d, g-h, k-l, o-p. The scale bar is 100 mm. Those ADAM17 deficient cells were stained positive with Nestin (c) and Tuj1 (g), but not with Tbr1 (k) or Map2 (o).

    Journal: PLoS ONE

    Article Title: ADAM17 Is Critical for Multipolar Exit and Radial Migration of Neuronal Intermediate Progenitor Cells in Mice Cerebral Cortex

    doi: 10.1371/journal.pone.0065703

    Figure Lengend Snippet: ADAM17 deficiency affected NSCs differentiation and those cells stopped at nIPCs stage as indicated by markers staining. Mouse cortex electroporated at E14.5 with A17-Ri/EYFP were dissected out at E18.5. All sections were cut at 20 mm and processed for IF with antibodies against one of those following markers: Nestin (b), Tuj1 (f), Tbr1 (j) and Map2 n). DAPI staining was used to indicate nuclei and merged images were presented in c-d, g-h, k-l, o-p. The scale bar is 100 mm. Those ADAM17 deficient cells were stained positive with Nestin (c) and Tuj1 (g), but not with Tbr1 (k) or Map2 (o).

    Article Snippet: The following primary antibodies were used: goat anti-ADAM17 (Abcam, ab13535); rabbit anti-Tbr1 (Abcam, 31940), anti-Tbr-2 (Abcam, ab23345), anti-GFP (Beyotime); mouse anti-Nestin (Abcam, ab6142), anti-Tuj1 (class III beta-tubulin, Abcam, ab78078), anti-Map2 (Abcam, ab11268); and rat anti-L1-CAM (Millipore, MAB5272) antibodies.

    Techniques: Staining

    Knocking down ADAM17 expression affected the radial glia fiber at E16.5 in cerebral cortex. Mouse embryos were electroporated at E14.5 and dissected at E16.5 for confocal imaging with Nestin or Tuj1 staining. All sections were cut at 20 µm. Each group was shown as the label on top. DAPI staining was used to indicate nuclei. All scale bars are 100 µm. All images presented the VZ/SVZ to IZ layers with the superficial layer on top. The radial glia fibers in the control group and overexpressing group were smoother and better aligned as indicated by the white arrow heads in (a, c). The radial glia fibers in the ADAM17 knockdown group appeared with more branches and not as well aligned, indicated by white arrows in (b).

    Journal: PLoS ONE

    Article Title: ADAM17 Is Critical for Multipolar Exit and Radial Migration of Neuronal Intermediate Progenitor Cells in Mice Cerebral Cortex

    doi: 10.1371/journal.pone.0065703

    Figure Lengend Snippet: Knocking down ADAM17 expression affected the radial glia fiber at E16.5 in cerebral cortex. Mouse embryos were electroporated at E14.5 and dissected at E16.5 for confocal imaging with Nestin or Tuj1 staining. All sections were cut at 20 µm. Each group was shown as the label on top. DAPI staining was used to indicate nuclei. All scale bars are 100 µm. All images presented the VZ/SVZ to IZ layers with the superficial layer on top. The radial glia fibers in the control group and overexpressing group were smoother and better aligned as indicated by the white arrow heads in (a, c). The radial glia fibers in the ADAM17 knockdown group appeared with more branches and not as well aligned, indicated by white arrows in (b).

    Article Snippet: The following primary antibodies were used: goat anti-ADAM17 (Abcam, ab13535); rabbit anti-Tbr1 (Abcam, 31940), anti-Tbr-2 (Abcam, ab23345), anti-GFP (Beyotime); mouse anti-Nestin (Abcam, ab6142), anti-Tuj1 (class III beta-tubulin, Abcam, ab78078), anti-Map2 (Abcam, ab11268); and rat anti-L1-CAM (Millipore, MAB5272) antibodies.

    Techniques: Expressing, Imaging, Staining

    NG2 + cell growth is not inhibited by CM from normal peritoneal macrophages (PM) or astrocytes (AST). The numbers and sizes of the spheres ( A ) as well as total cell numbers ( B ) in the presence of the media conditioned by normal PM or AST were similar to the control group (NG2 + cells alone) after 2 weeks culture. There was reduction of sphere formation and total cell numbers when NG2 + cells were cultured with the OX42 + cell-CM. N = 3. *Significantly different from the NG2 + cells in the absence of conditioned medium. C: Representative cells from sphere culture with OX42 + cell-CM. All cells show co-expression of NG2 and nestin. Scale bar = 100 µm.

    Journal: Glia

    Article Title: Interaction of NG2+ glial progenitors and microglia/macrophages from the injured spinal cord

    doi: 10.1002/glia.20932

    Figure Lengend Snippet: NG2 + cell growth is not inhibited by CM from normal peritoneal macrophages (PM) or astrocytes (AST). The numbers and sizes of the spheres ( A ) as well as total cell numbers ( B ) in the presence of the media conditioned by normal PM or AST were similar to the control group (NG2 + cells alone) after 2 weeks culture. There was reduction of sphere formation and total cell numbers when NG2 + cells were cultured with the OX42 + cell-CM. N = 3. *Significantly different from the NG2 + cells in the absence of conditioned medium. C: Representative cells from sphere culture with OX42 + cell-CM. All cells show co-expression of NG2 and nestin. Scale bar = 100 µm.

    Article Snippet: After 2 h in such adherent culture conditions, the coverslips were fixed and processed for immunocytochemistry with rabbit anti-NG2 (1:400, Chemicon) and a mouse anti-nestin (rat-401, 1:50, Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: AST Assay, Cell Culture, Expressing

    Repression of nNOS negatively regulates neuronal fate commitment. Monolayer-cultured embryonic NSCs differentiated for 4 days and were incubated with L-VNIO (100 μM) or vehicle during the later 2 days of differentiation. (A,B) L-VNIO inhibits neuronal differentiation. (A) Representatives of β-III-Tubulin + neurons. (B) Statistical graph showing the ratio of β-III-Tubulin + neurons. (C,D) L-VNIO has no effect on cell proliferation during NSCs differentiation. BrdU (2.5 μM) was added during the later 2 days of differentiation to label the dividing cells. (C) Representatives of BrdU-labeled cells. (D) Statistical graph showing the ratio of BrdU + cells. (E,F) L-VNIO induces cell apoptosis. Live cultures were stained with PI which stains dead cells and Hoechst 33342 which stains live and dead cells. (E) Representatives of PI + nuclei (arrows). (F) Statistical graph showing the ratio of PI + nuclei. (G) L-VNIO increases the rate of cell death (δ 4 ). (H) L-VNIO decreases the rate of conversion of progenitors to neurons (β 4 ) even if all dead cells are neurons. (I) There are nestin + progenitor cells throughout the differentiation stages. Cells were fixed and stained for nestin at days 1, 2, 3, 4 after differentiation respectively. Data shown are mean ± SEM from three to five independent experiments in parallel cultures; ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Neuronal Nitric Oxide Synthase in Neural Stem Cells Induces Neuronal Fate Commitment via the Inhibition of Histone Deacetylase 2

    doi: 10.3389/fncel.2017.00066

    Figure Lengend Snippet: Repression of nNOS negatively regulates neuronal fate commitment. Monolayer-cultured embryonic NSCs differentiated for 4 days and were incubated with L-VNIO (100 μM) or vehicle during the later 2 days of differentiation. (A,B) L-VNIO inhibits neuronal differentiation. (A) Representatives of β-III-Tubulin + neurons. (B) Statistical graph showing the ratio of β-III-Tubulin + neurons. (C,D) L-VNIO has no effect on cell proliferation during NSCs differentiation. BrdU (2.5 μM) was added during the later 2 days of differentiation to label the dividing cells. (C) Representatives of BrdU-labeled cells. (D) Statistical graph showing the ratio of BrdU + cells. (E,F) L-VNIO induces cell apoptosis. Live cultures were stained with PI which stains dead cells and Hoechst 33342 which stains live and dead cells. (E) Representatives of PI + nuclei (arrows). (F) Statistical graph showing the ratio of PI + nuclei. (G) L-VNIO increases the rate of cell death (δ 4 ). (H) L-VNIO decreases the rate of conversion of progenitors to neurons (β 4 ) even if all dead cells are neurons. (I) There are nestin + progenitor cells throughout the differentiation stages. Cells were fixed and stained for nestin at days 1, 2, 3, 4 after differentiation respectively. Data shown are mean ± SEM from three to five independent experiments in parallel cultures; ∗ p

    Article Snippet: The primary antibodies used were as follows: mouse anti-nestin (1:100; sc-33677; Santa Cruz Biotechnology), mouse anti-β-III-Tubulin (1:200; MAB1637; Millipore Bioscience Research Reagents) or mouse anti-glial fibrillary acidic protein (GFAP; 1:1000; MAB360; Millipore Bioscience Research Reagents).

    Techniques: Cell Culture, Incubation, Labeling, Staining

    HDAC2 mediates the role of nNOS in regulating the fate of adult NSCs. (A) Identification of cultured adult NSCs. Single-cell suspensions were seeded on polyornithine/laminin-coated coverslips, cultured as a monolayer for 24 h, and then fixed for nestin staining. At least 92% cells were nestin + NSCs. In addition, cells were monolayer-cultured in the presence of 10 μM BrdU for 24 h and fixed for stain, and most cells were BrdU + labeled. These cells could differentiate into β-III-Tubulin + neurons and GFAP + astrocytes after differentiation for 4 days. (B) Monolayer-cultured adult NSCs treated with 100 μM L-VNIO during the later 2 days of 4-day differentiation exhibit a marked decrease of neuronal differentiation. Immunoblots showing HDAC2 levels (C) and bar graph showing HDAC2 activity (D) in cultures treated with 100 μM L-VNIO or vehicle for the first 24 h during differentiation. (E,F) HDAC2 down-regulation rescues L-VNIO-induced neuronal differentiation reduction. 100 μM L-VNIO or vehicle was treated for the later 2 days during the 4-day differentiation of LV-HDAC2 shRNA- or LV-Control shRNA-infected adult NSCs. (E) Bar graph showing the percentage of newborn neurons. (F) Representatives of β-III-Tubulin + neurons. Scale bars = 50 μm. Data are mean ± SEM ( n = 3); ∗ p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Neuronal Nitric Oxide Synthase in Neural Stem Cells Induces Neuronal Fate Commitment via the Inhibition of Histone Deacetylase 2

    doi: 10.3389/fncel.2017.00066

    Figure Lengend Snippet: HDAC2 mediates the role of nNOS in regulating the fate of adult NSCs. (A) Identification of cultured adult NSCs. Single-cell suspensions were seeded on polyornithine/laminin-coated coverslips, cultured as a monolayer for 24 h, and then fixed for nestin staining. At least 92% cells were nestin + NSCs. In addition, cells were monolayer-cultured in the presence of 10 μM BrdU for 24 h and fixed for stain, and most cells were BrdU + labeled. These cells could differentiate into β-III-Tubulin + neurons and GFAP + astrocytes after differentiation for 4 days. (B) Monolayer-cultured adult NSCs treated with 100 μM L-VNIO during the later 2 days of 4-day differentiation exhibit a marked decrease of neuronal differentiation. Immunoblots showing HDAC2 levels (C) and bar graph showing HDAC2 activity (D) in cultures treated with 100 μM L-VNIO or vehicle for the first 24 h during differentiation. (E,F) HDAC2 down-regulation rescues L-VNIO-induced neuronal differentiation reduction. 100 μM L-VNIO or vehicle was treated for the later 2 days during the 4-day differentiation of LV-HDAC2 shRNA- or LV-Control shRNA-infected adult NSCs. (E) Bar graph showing the percentage of newborn neurons. (F) Representatives of β-III-Tubulin + neurons. Scale bars = 50 μm. Data are mean ± SEM ( n = 3); ∗ p

    Article Snippet: The primary antibodies used were as follows: mouse anti-nestin (1:100; sc-33677; Santa Cruz Biotechnology), mouse anti-β-III-Tubulin (1:200; MAB1637; Millipore Bioscience Research Reagents) or mouse anti-glial fibrillary acidic protein (GFAP; 1:1000; MAB360; Millipore Bioscience Research Reagents).

    Techniques: Cell Culture, Staining, Labeling, Western Blot, Activity Assay, shRNA, Infection

    The effects of maxadilan on the chemical neural induction of hADSC s. ( A ) hADSC s treated without or with maxadilan were induced in chemical neural induction medium (group‐C and group‐D) or in hADSC culture medium (group‐A and group‐B) for 1 or 3 days. ( B ) Comparison of the relative gene expression levels of Nestin, MAP ‐2, NF ‐M, NSE and Tuj‐III in different groups on day 3 using qPCR analysis. ( C ) Assessment of protein expressions of NSE , MAP ‐2, Nestin and NF ‐M in different groups on day 3 using Western blot assays. ( D ) Quantification of protein expression levels of Western blot. ( E ) Detection of protein expressions of Vimentin, MAP ‐2, NF ‐M and NSE of hADSC s in different groups on day 3 using immunofluorescence assays. Differences with * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: PAC1R agonist maxadilan enhances hADSC viability and neural differentiation potential

    doi: 10.1111/jcmm.12772

    Figure Lengend Snippet: The effects of maxadilan on the chemical neural induction of hADSC s. ( A ) hADSC s treated without or with maxadilan were induced in chemical neural induction medium (group‐C and group‐D) or in hADSC culture medium (group‐A and group‐B) for 1 or 3 days. ( B ) Comparison of the relative gene expression levels of Nestin, MAP ‐2, NF ‐M, NSE and Tuj‐III in different groups on day 3 using qPCR analysis. ( C ) Assessment of protein expressions of NSE , MAP ‐2, Nestin and NF ‐M in different groups on day 3 using Western blot assays. ( D ) Quantification of protein expression levels of Western blot. ( E ) Detection of protein expressions of Vimentin, MAP ‐2, NF ‐M and NSE of hADSC s in different groups on day 3 using immunofluorescence assays. Differences with * P

    Article Snippet: Then, the membranes were incubated overnight at 4°C with primary antibodies as follow: rabbit polyclonal anti‐PAC1R antibody (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti‐Cleaved Caspase 3 (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti‐Cleaved Caspase 9 (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti‐MAP‐2 (1:5000; Sigma‐Aldrich), mouse polyclonal anti‐NF‐M (1:5000; Abcam, Cambridge, MA, USA), chicken polyclonal anti‐NSE (1:5000; Merck Millipore, Billerica, MA, USA), mouse monoclonal anti‐Nestin (1:3000; Santa Cruz Biotechnology), rabbit polyclonal anti‐Tuj‐III (1:3000; Abcam), rabbit polyclonal‐β‐catenin (1:5000; Santa Cruz Biotechnology), rabbit polyclonal‐Cyclin D1 (1:3000; Santa Cruz Biotechnology), rabbit polyclonal‐c‐myc (1:3000; Santa Cruz Biotechnology), rabbit polyclonal anti‐survivin (1:3000; Santa Cruz Biotechnology), mouse monoclonal anti‐β‐actin (1:1000; Santa Cruz Biotechnology) and rabbit polyclonal anti‐GADPH (1:10,000; Bioworld, Minneapolis, MN, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

    The potential molecular mechanisms underlying the promotion of hADSC viability and neural differentiation potential effects induced by maxadilan. ( A ) Domain structure analysis of maxadilan using PredictProtein. ( B ) The 3D structures of PACAP and maxadilan using SWISSMODEL . ( C ) Assessment of protein expression levels of β‐catenin, Cyclin D1, c‐myc and survivin in different groups using Western blot assays. ( D ) Detection of the Bcl‐2 levels in different groups using ELISA . ( E ) Comparison of the Caspase 3 activity in different groups using Caspase 3 activity assays. ( F ) Assessment of protein expression levels of NSE , Nestin, NF ‐M and Tuj‐III in different groups using Western blot assays. ( G ) Quantification of the cAMP levels in different groups using ELISA . ( H ) Diagram of the potential molecular mechanisms underlying the promotion of hADSC viability and neural differentiation potential effects induced by maxadilan. Differences with * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: PAC1R agonist maxadilan enhances hADSC viability and neural differentiation potential

    doi: 10.1111/jcmm.12772

    Figure Lengend Snippet: The potential molecular mechanisms underlying the promotion of hADSC viability and neural differentiation potential effects induced by maxadilan. ( A ) Domain structure analysis of maxadilan using PredictProtein. ( B ) The 3D structures of PACAP and maxadilan using SWISSMODEL . ( C ) Assessment of protein expression levels of β‐catenin, Cyclin D1, c‐myc and survivin in different groups using Western blot assays. ( D ) Detection of the Bcl‐2 levels in different groups using ELISA . ( E ) Comparison of the Caspase 3 activity in different groups using Caspase 3 activity assays. ( F ) Assessment of protein expression levels of NSE , Nestin, NF ‐M and Tuj‐III in different groups using Western blot assays. ( G ) Quantification of the cAMP levels in different groups using ELISA . ( H ) Diagram of the potential molecular mechanisms underlying the promotion of hADSC viability and neural differentiation potential effects induced by maxadilan. Differences with * P

    Article Snippet: Then, the membranes were incubated overnight at 4°C with primary antibodies as follow: rabbit polyclonal anti‐PAC1R antibody (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti‐Cleaved Caspase 3 (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti‐Cleaved Caspase 9 (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti‐MAP‐2 (1:5000; Sigma‐Aldrich), mouse polyclonal anti‐NF‐M (1:5000; Abcam, Cambridge, MA, USA), chicken polyclonal anti‐NSE (1:5000; Merck Millipore, Billerica, MA, USA), mouse monoclonal anti‐Nestin (1:3000; Santa Cruz Biotechnology), rabbit polyclonal anti‐Tuj‐III (1:3000; Abcam), rabbit polyclonal‐β‐catenin (1:5000; Santa Cruz Biotechnology), rabbit polyclonal‐Cyclin D1 (1:3000; Santa Cruz Biotechnology), rabbit polyclonal‐c‐myc (1:3000; Santa Cruz Biotechnology), rabbit polyclonal anti‐survivin (1:3000; Santa Cruz Biotechnology), mouse monoclonal anti‐β‐actin (1:1000; Santa Cruz Biotechnology) and rabbit polyclonal anti‐GADPH (1:10,000; Bioworld, Minneapolis, MN, USA).

    Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay

    Electrophysiological analyses of cytokine neural redifferentiated hADSC s in different groups. ( A ) The holding potential was −60 mV and depolarizing steps were ranged from −60 to +40 mV at pulse of 2 msec. Representative traces of sodium currents and I–V curves demonstrated the voltage dependence of sodium currents in different groups ( n = 20). ( B ) Sodium currents were blocked using 1 μM TTX and voltage‐dependent sodium currents were activated from a depolarizing step in mV intervals from −100 to +40 mV at pulse of 50 msec. Representative traces of potassium currents and I–V curves demonstrated the voltage dependence of sodium currents in different groups ( n = 25). ( C ) Step current injection protocols were used from −80 to +40 pA at pulse of 100 msec. Representative traces showed repetitive action potentials in group‐H ( n = 6) after cytokine neural redifferentiation for 2 weeks. ( D ) Typical recording of single whole‐cell patch‐clamp mode. ( E ) Detection of protein expressions of NF ‐M, Tuj‐III, MAP ‐2, NSE , Nestin and Vimentin in group‐H using immunofluorescence assays.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: PAC1R agonist maxadilan enhances hADSC viability and neural differentiation potential

    doi: 10.1111/jcmm.12772

    Figure Lengend Snippet: Electrophysiological analyses of cytokine neural redifferentiated hADSC s in different groups. ( A ) The holding potential was −60 mV and depolarizing steps were ranged from −60 to +40 mV at pulse of 2 msec. Representative traces of sodium currents and I–V curves demonstrated the voltage dependence of sodium currents in different groups ( n = 20). ( B ) Sodium currents were blocked using 1 μM TTX and voltage‐dependent sodium currents were activated from a depolarizing step in mV intervals from −100 to +40 mV at pulse of 50 msec. Representative traces of potassium currents and I–V curves demonstrated the voltage dependence of sodium currents in different groups ( n = 25). ( C ) Step current injection protocols were used from −80 to +40 pA at pulse of 100 msec. Representative traces showed repetitive action potentials in group‐H ( n = 6) after cytokine neural redifferentiation for 2 weeks. ( D ) Typical recording of single whole‐cell patch‐clamp mode. ( E ) Detection of protein expressions of NF ‐M, Tuj‐III, MAP ‐2, NSE , Nestin and Vimentin in group‐H using immunofluorescence assays.

    Article Snippet: Then, the membranes were incubated overnight at 4°C with primary antibodies as follow: rabbit polyclonal anti‐PAC1R antibody (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti‐Cleaved Caspase 3 (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti‐Cleaved Caspase 9 (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti‐MAP‐2 (1:5000; Sigma‐Aldrich), mouse polyclonal anti‐NF‐M (1:5000; Abcam, Cambridge, MA, USA), chicken polyclonal anti‐NSE (1:5000; Merck Millipore, Billerica, MA, USA), mouse monoclonal anti‐Nestin (1:3000; Santa Cruz Biotechnology), rabbit polyclonal anti‐Tuj‐III (1:3000; Abcam), rabbit polyclonal‐β‐catenin (1:5000; Santa Cruz Biotechnology), rabbit polyclonal‐Cyclin D1 (1:3000; Santa Cruz Biotechnology), rabbit polyclonal‐c‐myc (1:3000; Santa Cruz Biotechnology), rabbit polyclonal anti‐survivin (1:3000; Santa Cruz Biotechnology), mouse monoclonal anti‐β‐actin (1:1000; Santa Cruz Biotechnology) and rabbit polyclonal anti‐GADPH (1:10,000; Bioworld, Minneapolis, MN, USA).

    Techniques: Injection, Patch Clamp, Immunofluorescence

    Cortical spheroids contained CNS cell types, and cells formed laminin-containing 3D networks. Confocal projections of 1, 7, 14, and 21 DIV 8k spheroids revealed the presence of CNS cell types in the cortical spheroids, including neurons (β-III-tubulin, red ), astrocytes (GFAP, green ), oligodendrocytes (O1, yellow ), neural stem/progenitor cells (nestin, cyan ), and microglia (CD11b, magenta ). Nuclei were counterstained with DAPI ( blue ). Cell processes formed 3D networks within the cortical spheroids. Confocal projections showed laminin expression ( gray

    Journal: Tissue Engineering. Part C, Methods

    Article Title: Three-Dimensional Neural Spheroid Culture: An In Vitro Model for Cortical Studies

    doi: 10.1089/ten.tec.2015.0135

    Figure Lengend Snippet: Cortical spheroids contained CNS cell types, and cells formed laminin-containing 3D networks. Confocal projections of 1, 7, 14, and 21 DIV 8k spheroids revealed the presence of CNS cell types in the cortical spheroids, including neurons (β-III-tubulin, red ), astrocytes (GFAP, green ), oligodendrocytes (O1, yellow ), neural stem/progenitor cells (nestin, cyan ), and microglia (CD11b, magenta ). Nuclei were counterstained with DAPI ( blue ). Cell processes formed 3D networks within the cortical spheroids. Confocal projections showed laminin expression ( gray

    Article Snippet: The following antibodies were used: mouse anti-β-III-tubulin (Covance MMS-435P, 1:50), rabbit anti-glial fibrillary acidic protein (GFAP, DAKO Z0334, 1:200), rabbit anti-laminin (BTI BT594, 1:100), mouse anti-nestin (Millipore MAB353, 1:200), mouse anti-CD11b (Millipore CBL1512, 1:25), mouse anti-O1 (Millipore MAB344, 1:50), Cy3 goat anti-mouse (Jackson 115-165-068, 1:500), and Alexa488 goat anti-rabbit (Jackson 115-545-146, 1:500).

    Techniques: Expressing

    Nestin expression around the central canal. In the cystic spinal cord (A1), 5-bromo-2’-deoxyuridine (BrdU)-labeled cells are seen in the lesion site (B1). The images (C1, D) did not show immunoreactivity for nestin 3 weeks after transplantation. Nestin expression was restricted to ependymal cell layer (star) (C2). In the same region, labeled cells were not observed around the central canal (B2). Magnification at 10 × for A1, B1, C1, D and at 40 × for A2, B2, C2. V: Ventral; D: dorsal; CC: central canal; BF: bright field.

    Journal: Neural Regeneration Research

    Article Title: Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury

    doi: 10.3969/j.issn.1673-5374.2013.36.001

    Figure Lengend Snippet: Nestin expression around the central canal. In the cystic spinal cord (A1), 5-bromo-2’-deoxyuridine (BrdU)-labeled cells are seen in the lesion site (B1). The images (C1, D) did not show immunoreactivity for nestin 3 weeks after transplantation. Nestin expression was restricted to ependymal cell layer (star) (C2). In the same region, labeled cells were not observed around the central canal (B2). Magnification at 10 × for A1, B1, C1, D and at 40 × for A2, B2, C2. V: Ventral; D: dorsal; CC: central canal; BF: bright field.

    Article Snippet: The following primary antibodies were used during BrdU/βIII-tubulin and BrdU/RIP double-label staining: sheep anti-BrdU polyclonal antibody (1:100; AB1893, Abcam, Cambridge, MA, USA); mouse anti-nestin monoclonal antibody (1:100, MAB353; Millipore, Billerica, MA, USA); mouse anti-βIII-tubulin antibody (1:200; Sigma-Aldrich); mouse anti-RIP monoclonal antibody (1:50 000; MAB1580; Millipore).

    Techniques: Expressing, Labeling, Transplantation Assay

    Accumulation of the DNA demethylation intermediate 5hmC increases DSBs in Polβ-deficient neural progenitors. A , Immunoblot analysis shows 5mC global levels in genomic DNA from E14.5 Pol β fl/fl and Emx1-Cre/Pol β fl/fl mouse cortex. B , Immunoblot analysis shows global 5hmC levels in genomic DNA. Dissociated cell cultures from E14.5 cortices were incubated with or without vitamin C for 24 h. Immunocytochemistry was performed with anti-γH2AX ( C, F, G, J, K, N, O, R ) and anti-Nestin ( D, F, H, J, L, N, P, R ) antibodies in dissociated cells from E14.5 Pol β fl/fl ( C–J ) and Emx1-Cre/Pol β fl/fl mouse cortex ( K–R ). Nuclei were stained with DAPI ( E, F, I, J, M, N, Q, R ). The cells were untreated ( C–F, K–N ) or treated with 100 μg/ml vitamin C ( G–J, O–R ). Scale bar, 5 μm. S , Histogram showing the number of γH2AX foci in individual Nestin-positive cells. Values indicate the mean ± SEM from 24 Pol β fl/fl or Emx1-Cre/Pol β fl/fl cells. Asterisks indicate a significant difference (* p

    Journal: The Journal of Neuroscience

    Article Title: Genome Stability by DNA Polymerase β in Neural Progenitors Contributes to Neuronal Differentiation in Cortical Development

    doi: 10.1523/JNEUROSCI.0665-17.2017

    Figure Lengend Snippet: Accumulation of the DNA demethylation intermediate 5hmC increases DSBs in Polβ-deficient neural progenitors. A , Immunoblot analysis shows 5mC global levels in genomic DNA from E14.5 Pol β fl/fl and Emx1-Cre/Pol β fl/fl mouse cortex. B , Immunoblot analysis shows global 5hmC levels in genomic DNA. Dissociated cell cultures from E14.5 cortices were incubated with or without vitamin C for 24 h. Immunocytochemistry was performed with anti-γH2AX ( C, F, G, J, K, N, O, R ) and anti-Nestin ( D, F, H, J, L, N, P, R ) antibodies in dissociated cells from E14.5 Pol β fl/fl ( C–J ) and Emx1-Cre/Pol β fl/fl mouse cortex ( K–R ). Nuclei were stained with DAPI ( E, F, I, J, M, N, Q, R ). The cells were untreated ( C–F, K–N ) or treated with 100 μg/ml vitamin C ( G–J, O–R ). Scale bar, 5 μm. S , Histogram showing the number of γH2AX foci in individual Nestin-positive cells. Values indicate the mean ± SEM from 24 Pol β fl/fl or Emx1-Cre/Pol β fl/fl cells. Asterisks indicate a significant difference (* p

    Article Snippet: They were incubated with rabbit polyclonal anti-γH2AX antibody at 1:200, rabbit polyclonal anti-53BP1 antibody at 1:100, or mouse monoclonal anti-nestin antibody (MAB353, Millipore) at 1:200 in buffer G overnight at 4°C.

    Techniques: Incubation, Immunocytochemistry, Staining

    Floxed TW mouse glioma model ( A ) Scheme used to investigate TW function in TWflox:mTmG transgenic mice. ( B ) Immunophentyping of NPCs prior to transformation showing expression of neural stem and progenitor markers Sox2, Olig2 and nestin. Scale bar, 30 μm. ( C ) NPCs are multi-potent expressing astrocytic (GFAP) and neuronal (Map2) markers after exposure to differentiating conditions. Scale bar, 20 μm. ( D ) Conversion of mTmG Cre reporter after exposure to Cre recombinase. Scale bar, 10 μm.

    Journal: Oncotarget

    Article Title: Twist1 mediated regulation of glioma tumorigenicity is dependent on mode of mouse neural progenitor transformation

    doi: 10.18632/oncotarget.22593

    Figure Lengend Snippet: Floxed TW mouse glioma model ( A ) Scheme used to investigate TW function in TWflox:mTmG transgenic mice. ( B ) Immunophentyping of NPCs prior to transformation showing expression of neural stem and progenitor markers Sox2, Olig2 and nestin. Scale bar, 30 μm. ( C ) NPCs are multi-potent expressing astrocytic (GFAP) and neuronal (Map2) markers after exposure to differentiating conditions. Scale bar, 20 μm. ( D ) Conversion of mTmG Cre reporter after exposure to Cre recombinase. Scale bar, 10 μm.

    Article Snippet: Fixed cells were then subject to overnight incubation at 4°C with primary antibodies 10% problock, 0.1% Triton-X in PBS including anti-Nestin mouse monoclonal (Chemicon MAB353, 1:500, Millipore), anti-SOX2 goat polyclonal (SC17320, 1:250, Santa Cruz), and anti-Olig2 rabbit polyclonal antibody (gift from H. Yokoo [ , ].

    Techniques: Transgenic Assay, Mouse Assay, Transformation Assay, Expressing

    iPS characterization as pluripotent ES-like cells: Human iPS were derived from HFF cells following retroviral infection with the four reprogramming factors and display morphology similar to that of hES cells: (A) iPS colony, hES (H9.2) colony and HFF morphology. (B) Immunofluorescence staining for pluripotent markers: Oct4, TRA-1–81, TRA-1–60 and SSEA4 for iPS clone 3. DAPI nuclear staining indicated on left panel. (C) Analysis of gene expression by PCR. The transcription of Oct4, Sox2, c-myc, Klf4, Nanog and Rex1 were analysed in three iPS clones C1, C2 and C3 as well in the hESC clone–H9.2 and parental HFF. GAPDH was used as amplification and loading control. (D, E) Spontaneous differentiation of human iPS into EBs and teratomas. Human iPS were induced to spontaneous differentiation in vitro and in vivo. (D) The upper left panel illustrates EBs derived from human iPS clone 3. Upper right and bottom left and right panels show immunofluorescence staining for markers of the three germ layers in human IPS derived EBs. Representative ectoderm marker β-tubulin-III, representative mesoderm marker SMA and representative endoderm marker α-Fetoprotein (E) Immunofluorescence staining for different markers (up) and haematoxylin and eosin staining demonstration of typical tissue morphology (bottom) were performed on teratomas to confirm derivatives of the three germ layers: ectoderm – representative marker Nestin staining and neural rosettes, mesoderm – SMA marker staining and cartilage endoderm – a-Fetoprotein marker staining and gut like epithelium.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells

    doi: 10.1111/j.1582-4934.2009.00996.x

    Figure Lengend Snippet: iPS characterization as pluripotent ES-like cells: Human iPS were derived from HFF cells following retroviral infection with the four reprogramming factors and display morphology similar to that of hES cells: (A) iPS colony, hES (H9.2) colony and HFF morphology. (B) Immunofluorescence staining for pluripotent markers: Oct4, TRA-1–81, TRA-1–60 and SSEA4 for iPS clone 3. DAPI nuclear staining indicated on left panel. (C) Analysis of gene expression by PCR. The transcription of Oct4, Sox2, c-myc, Klf4, Nanog and Rex1 were analysed in three iPS clones C1, C2 and C3 as well in the hESC clone–H9.2 and parental HFF. GAPDH was used as amplification and loading control. (D, E) Spontaneous differentiation of human iPS into EBs and teratomas. Human iPS were induced to spontaneous differentiation in vitro and in vivo. (D) The upper left panel illustrates EBs derived from human iPS clone 3. Upper right and bottom left and right panels show immunofluorescence staining for markers of the three germ layers in human IPS derived EBs. Representative ectoderm marker β-tubulin-III, representative mesoderm marker SMA and representative endoderm marker α-Fetoprotein (E) Immunofluorescence staining for different markers (up) and haematoxylin and eosin staining demonstration of typical tissue morphology (bottom) were performed on teratomas to confirm derivatives of the three germ layers: ectoderm – representative marker Nestin staining and neural rosettes, mesoderm – SMA marker staining and cartilage endoderm – a-Fetoprotein marker staining and gut like epithelium.

    Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-α Fetaprotein (Dako), mouse monoclonal anti-smooth muscle actin (SMA) antibody (1:50, Dako), polyclonal rabbit anti-β-tubulin III (1:1000, Covance, Princeton, NJ, USA) and monoclonal mouse anti-Nestin (1:200, Chemicon).

    Techniques: Derivative Assay, Infection, Immunofluorescence, Staining, Expressing, Polymerase Chain Reaction, Clone Assay, Amplification, In Vitro, In Vivo, Marker

    p38MAPK inhibitor-treated hESC differentiate into all three germ layers in vitro . (A) Immunofluorescence staining of SB203580-treated (p38i) and untreated (Ctl) hEB on day 30 of differentiation demonstrated expression of markers of all three embryonic germ layers, namely nestin and βIII tubulin (ectoderm), α-fetoprotein (AFP; endoderm) and smooth muscle actin (SMA) and cTnT (mesoderm). Bar 10 μm. (B) hEB from SB203580-treated (p38i) and untreated (Ctl) cultures collected prior to treatment, at day 4, and at day 14 were assayed for germ layer-specific gene expression by qPCR. Expression was normalized to GAPDH and calculated relative to proliferating, undifferentiated hESC. There was no significant difference between expression levels of βIII tubulin, SMA or α-fetoprotein in treated and untreated cultures. Data shown are mean percentage ± SEM ( n = 3).

    Journal: Cytotherapy

    Article Title: Timed inhibition of p38MAPK directs accelerated differentiation of human embryonic stem cells into cardiomyocytes

    doi: 10.3109/14653249.2010.491821

    Figure Lengend Snippet: p38MAPK inhibitor-treated hESC differentiate into all three germ layers in vitro . (A) Immunofluorescence staining of SB203580-treated (p38i) and untreated (Ctl) hEB on day 30 of differentiation demonstrated expression of markers of all three embryonic germ layers, namely nestin and βIII tubulin (ectoderm), α-fetoprotein (AFP; endoderm) and smooth muscle actin (SMA) and cTnT (mesoderm). Bar 10 μm. (B) hEB from SB203580-treated (p38i) and untreated (Ctl) cultures collected prior to treatment, at day 4, and at day 14 were assayed for germ layer-specific gene expression by qPCR. Expression was normalized to GAPDH and calculated relative to proliferating, undifferentiated hESC. There was no significant difference between expression levels of βIII tubulin, SMA or α-fetoprotein in treated and untreated cultures. Data shown are mean percentage ± SEM ( n = 3).

    Article Snippet: Primary antibodies used were mouse anti-human α-fetoprotein (Sigma-Aldrich, St. Louis, MO, USA, A8452), mouse anti-human nestin (R & D Systems MAB1259), mouse anti-human βIII tubulin (R & DSystemsMAB1195), mouse anti-humans mooth muscle actin (R & D Systems MAB1420) and mouse anti-human cardiac troponin T (cTnT; Lab Vision Corporation, Fremont, CA, USA, MS-295).

    Techniques: In Vitro, Immunofluorescence, Staining, CTL Assay, Expressing, Real-time Polymerase Chain Reaction

    Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

    doi: 10.3389/fnmol.2018.00415

    Figure Lengend Snippet: Long-term marking of hippocampal NSCs by LV PGK-GFP. LV PGK-GFP was unilaterally injected into the hippocampal DG; brain sections were analyzed 15 days (A) and 6 months (B) later. GFP expression was evident in the DG at both time points. A higher magnification view is displayed in insets (A,B) . GFP-expressing cells co-labeled with NSCs markers such as BLBP (C,D) , NESTIN (E,F) , SOX2, GFAP (G,H) , and MUSASHI-1 (I,J) (arrows). Note that some GFP-positive cells stained for SOX2 showed co-localization with radial glial cell markers such as GFAP in their processes (G,H) . DG, dentate gyrus; SGZ is marked with dotted lines.

    Article Snippet: Cells were washed three times with PBS 0,1% Triton X-100 (PBS-T) and blocked for 2 h with PBS-T containing 5% normal goat serum (Vector laboratories), followed by overnight incubation with primary antibodies: rabbit anti- GFAP (1:1000; Dako); mouse anti-Nestin (1:500; Pharmingen); rabbit anti-Sox2 (1:200; Chemicon); anti-TUJ-1 (1:1,000; Chemicon).

    Techniques: Injection, Expressing, Labeling, Staining

    Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

    doi: 10.3389/fnmol.2018.00415

    Figure Lengend Snippet: Long-term maintenance of NSCs in the adult hippocampus. Fate mapping of GFP + identified NSCs that proliferate and produce neurons (A) and astrocytes (B) . Some NSCs underwent cell proliferation proliferated twice in a one-month interval (C) . GFP-labeled cells in vivo gave rise to in vitro NSCs. In vitro , GFP + NSCs expressed NSC markers such as NESTIN and Sox2 (D) and differentiated into neurons (TUJ1) and astrocytes (GFAP) (E) . GFP + derived-neurons and astrocytes at day 7 of differentiation (F) .

    Article Snippet: Cells were washed three times with PBS 0,1% Triton X-100 (PBS-T) and blocked for 2 h with PBS-T containing 5% normal goat serum (Vector laboratories), followed by overnight incubation with primary antibodies: rabbit anti- GFAP (1:1000; Dako); mouse anti-Nestin (1:500; Pharmingen); rabbit anti-Sox2 (1:200; Chemicon); anti-TUJ-1 (1:1,000; Chemicon).

    Techniques: Labeling, In Vivo, In Vitro, Derivative Assay

    Identification of the core sequence and functional cis -elements in the second intron of the mouse nestin gene. A , the minimal enhancer region was localized to the 3′ 320 bp (1310/1629) region of the second intron. P19EC cells or P19NPCs were transfected with deletion constructs as indicated in the left panel , and the luciferase activity was determined. The minimal enhancer containing 3′ 320 bp is indicated by an asterisk. B , potential cis -elements in the 3′ 320 bp of the second intron. The position of the first nucleotide of the second intron was designated as 1. The consensus sequences of putative cis -elements are shown in boxes. C , alignment of the nucleotide sequence, which contains the binding sites for Sox, POU, HRE, and SF1 factors in the 320-bp region with its counterparts in the rat and human nestin genes. D , identification of functional cis -elements. P19EC or P19NPC cells were transfected with site-mutated constructs as indicated in the left panel , and the luciferase activity was determined. The activity of each construct was shown relative to that of the enhancerless pGL3-Px′ vector. Values were normalized for transfection efficiency by co-transfection with a Renilla luciferase expression plasmid, and they are shown as means ± S.D. for three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Different Transcription Factors Regulate nestin Gene Expression during P19 Cell Neural Differentiation and Central Nervous System Development

    doi: 10.1074/jbc.M805632200

    Figure Lengend Snippet: Identification of the core sequence and functional cis -elements in the second intron of the mouse nestin gene. A , the minimal enhancer region was localized to the 3′ 320 bp (1310/1629) region of the second intron. P19EC cells or P19NPCs were transfected with deletion constructs as indicated in the left panel , and the luciferase activity was determined. The minimal enhancer containing 3′ 320 bp is indicated by an asterisk. B , potential cis -elements in the 3′ 320 bp of the second intron. The position of the first nucleotide of the second intron was designated as 1. The consensus sequences of putative cis -elements are shown in boxes. C , alignment of the nucleotide sequence, which contains the binding sites for Sox, POU, HRE, and SF1 factors in the 320-bp region with its counterparts in the rat and human nestin genes. D , identification of functional cis -elements. P19EC or P19NPC cells were transfected with site-mutated constructs as indicated in the left panel , and the luciferase activity was determined. The activity of each construct was shown relative to that of the enhancerless pGL3-Px′ vector. Values were normalized for transfection efficiency by co-transfection with a Renilla luciferase expression plasmid, and they are shown as means ± S.D. for three independent experiments.

    Article Snippet: The primary antibodies and their final concentrations were as follows: rabbit anti-human Oct1 (1:1000, Santa Cruz Biotechnology), mouse anti-human Oct4 (1:200, Santa Cruz Biotechnology), goat anti-human Brn1 (1:1000, Santa Cruz Biotechnology), goat anti-human Brn2 (1:1000, Santa Cruz Biotechnology), rabbit anti-mouse Sox2 (1:200) , rabbit anti-mouse SF1 (1:1000) , rabbit anti-mouse nestin (1:200) , mouse anti-rat nestin (1:200, BD Biosciences, San Jose, CA).

    Techniques: Sequencing, Functional Assay, Transfection, Construct, Luciferase, Activity Assay, Binding Assay, Plasmid Preparation, Cotransfection, Expressing

    In vivo characterization of the functional cis -elements in the second intron of the mouse nestin gene. The pxtkEGFP vector containing a TK promoter and GFP coding sequence was used as a negative control. The second intron of the mouse nestin gene with wild-type or mutated cis -elements was placed upstream of the TK promoter to generate a series of GFP reporter gene constructs. The GFP constructs were electroporated into chick neural tubes together with the tracer plasmid, pHcRed1-N1, at HH stage 10. For G and H , a GFP construct and the tracer plasmid, pHcRed1-N1, were also injected and electroporated into the surrounding somite. The expressions of GFP and HcRed were detected 24 h later (at HH stage 17). Neural tubes expressing HcRed were selected and cut from chick embryos for cryosection, and the expression of GFP in the thoracic spinal cord was examined under a confocal microscope. The numbers of independent electroporated embryos that expressed GFP and HcRed for different constructs were as follows: 0/10 for the vector pxtkEGFP ( A-C ), 9/9 for the full-length second intron construct pxtkEGFP2In32/1628 ( D-F ), 30/31 for the 320-bp minimal enhancer construct pxtkEGFP2In1310/1629 ( G-I ), 5/21 for the Sox-mutated construct pxtkEGFP2InSOXm ( J-L ), 0/7 for the POU-mutated construct pxtkEGFP2InPOUm ( M-O ), 5/19 for the HRE-mutated construct pxtkEGFP2InHREm ( P-R ), and 13/13 for the SF1-mutated construct pxtkEGFP2InSF1m ( S-U ). Bar , 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Different Transcription Factors Regulate nestin Gene Expression during P19 Cell Neural Differentiation and Central Nervous System Development

    doi: 10.1074/jbc.M805632200

    Figure Lengend Snippet: In vivo characterization of the functional cis -elements in the second intron of the mouse nestin gene. The pxtkEGFP vector containing a TK promoter and GFP coding sequence was used as a negative control. The second intron of the mouse nestin gene with wild-type or mutated cis -elements was placed upstream of the TK promoter to generate a series of GFP reporter gene constructs. The GFP constructs were electroporated into chick neural tubes together with the tracer plasmid, pHcRed1-N1, at HH stage 10. For G and H , a GFP construct and the tracer plasmid, pHcRed1-N1, were also injected and electroporated into the surrounding somite. The expressions of GFP and HcRed were detected 24 h later (at HH stage 17). Neural tubes expressing HcRed were selected and cut from chick embryos for cryosection, and the expression of GFP in the thoracic spinal cord was examined under a confocal microscope. The numbers of independent electroporated embryos that expressed GFP and HcRed for different constructs were as follows: 0/10 for the vector pxtkEGFP ( A-C ), 9/9 for the full-length second intron construct pxtkEGFP2In32/1628 ( D-F ), 30/31 for the 320-bp minimal enhancer construct pxtkEGFP2In1310/1629 ( G-I ), 5/21 for the Sox-mutated construct pxtkEGFP2InSOXm ( J-L ), 0/7 for the POU-mutated construct pxtkEGFP2InPOUm ( M-O ), 5/19 for the HRE-mutated construct pxtkEGFP2InHREm ( P-R ), and 13/13 for the SF1-mutated construct pxtkEGFP2InSF1m ( S-U ). Bar , 50 μm.

    Article Snippet: The primary antibodies and their final concentrations were as follows: rabbit anti-human Oct1 (1:1000, Santa Cruz Biotechnology), mouse anti-human Oct4 (1:200, Santa Cruz Biotechnology), goat anti-human Brn1 (1:1000, Santa Cruz Biotechnology), goat anti-human Brn2 (1:1000, Santa Cruz Biotechnology), rabbit anti-mouse Sox2 (1:200) , rabbit anti-mouse SF1 (1:1000) , rabbit anti-mouse nestin (1:200) , mouse anti-rat nestin (1:200, BD Biosciences, San Jose, CA).

    Techniques: In Vivo, Functional Assay, Plasmid Preparation, Sequencing, Negative Control, Construct, Injection, Expressing, Microscopy

    Different transcription factors are recruited to the nestin enhancer during neural differentiation of P19 cells. A , a 32 P-labeled SoxPOU probe (1360/1395) was incubated with NE from P19 cells. EMSA was performed with the same amount of NE from P19 cells (5 μg). Specific gel-shifted bands ( 1-4 ) formed in P19EC cells and P19NPCs are indicated by arrows. B , 32 P-labeled SoxPOU probe was incubated with NE from P19EC cells ( lanes 1-7 ) or P19NPCs ( lanes 8-14 ). The competition experiment was performed with 100-fold excess of unlabeled non-relevant probe CIE ( lanes 2 and 9 ), SoxPOU probe ( lanes 3 and 10 ), POU1(Sox2) probe ( lanes 4 and 11 ), Sox-mutated probe SoxmPOU ( lanes 5 and 12 ), POU-mutated probe SoxPOUm ( lanes 6 and 13 ), or a probe with both the Sox and POU sites mutated probe SoxmPOUm ( lane 7 and 14 ). C , a 32 P-labeled SoxPOU probe was incubated with NE from P19EC cells ( lanes 1-4 ) or P19NPCs ( lanes 5-7 ). The supershift assay was performed with an antibody against Oct1 ( lane 2 ), Oct2 ( lane 3 ), Oct4 ( lane 4 ), Brn1 ( lane 6 ), or Brn2 ( lane 7 ). Note that there were supershifted bands in lanes 2 and 7. D , 32 P-labeled HRESF1 (1409/1441) probe was incubated with NE from P19 cells. EMSA was performed with the same amount of NE from P19 cells (5 μg). Specific gel-shifted bands ( 5-7 ) formed in P19EC cells and P19NPCs are indicated by arrows. E , a 32 P-labeled HRESF1 probe was incubated with NE from P19EC cells. The competition experiment was performed with 100-fold excess of unlabeled probe HRESF1 ( lane 2 ) or the non-relevant probe CIE ( lane 3 ). The supershift assay was performed with an antibody against SF1 ( lane 4 ). Note that there were supershifted bands in lanes 4. F, in vivo occupancy of the nestin enhancer by different transcription factors. In ChIP assays, antibodies against Brn1, Brn2, Sox2, or SF1 were used to immunoprecipitate cross-linked chromatin fragments prepared from P19EC cells or P19NPCs. Immunoprecipitates were analyzed for the abundance of the nestin enhancer by quantitative PCR. The data were normalized relative to nestin enhancer occupancy by pre-immune IgG. Ss , supershift.

    Journal: The Journal of Biological Chemistry

    Article Title: Different Transcription Factors Regulate nestin Gene Expression during P19 Cell Neural Differentiation and Central Nervous System Development

    doi: 10.1074/jbc.M805632200

    Figure Lengend Snippet: Different transcription factors are recruited to the nestin enhancer during neural differentiation of P19 cells. A , a 32 P-labeled SoxPOU probe (1360/1395) was incubated with NE from P19 cells. EMSA was performed with the same amount of NE from P19 cells (5 μg). Specific gel-shifted bands ( 1-4 ) formed in P19EC cells and P19NPCs are indicated by arrows. B , 32 P-labeled SoxPOU probe was incubated with NE from P19EC cells ( lanes 1-7 ) or P19NPCs ( lanes 8-14 ). The competition experiment was performed with 100-fold excess of unlabeled non-relevant probe CIE ( lanes 2 and 9 ), SoxPOU probe ( lanes 3 and 10 ), POU1(Sox2) probe ( lanes 4 and 11 ), Sox-mutated probe SoxmPOU ( lanes 5 and 12 ), POU-mutated probe SoxPOUm ( lanes 6 and 13 ), or a probe with both the Sox and POU sites mutated probe SoxmPOUm ( lane 7 and 14 ). C , a 32 P-labeled SoxPOU probe was incubated with NE from P19EC cells ( lanes 1-4 ) or P19NPCs ( lanes 5-7 ). The supershift assay was performed with an antibody against Oct1 ( lane 2 ), Oct2 ( lane 3 ), Oct4 ( lane 4 ), Brn1 ( lane 6 ), or Brn2 ( lane 7 ). Note that there were supershifted bands in lanes 2 and 7. D , 32 P-labeled HRESF1 (1409/1441) probe was incubated with NE from P19 cells. EMSA was performed with the same amount of NE from P19 cells (5 μg). Specific gel-shifted bands ( 5-7 ) formed in P19EC cells and P19NPCs are indicated by arrows. E , a 32 P-labeled HRESF1 probe was incubated with NE from P19EC cells. The competition experiment was performed with 100-fold excess of unlabeled probe HRESF1 ( lane 2 ) or the non-relevant probe CIE ( lane 3 ). The supershift assay was performed with an antibody against SF1 ( lane 4 ). Note that there were supershifted bands in lanes 4. F, in vivo occupancy of the nestin enhancer by different transcription factors. In ChIP assays, antibodies against Brn1, Brn2, Sox2, or SF1 were used to immunoprecipitate cross-linked chromatin fragments prepared from P19EC cells or P19NPCs. Immunoprecipitates were analyzed for the abundance of the nestin enhancer by quantitative PCR. The data were normalized relative to nestin enhancer occupancy by pre-immune IgG. Ss , supershift.

    Article Snippet: The primary antibodies and their final concentrations were as follows: rabbit anti-human Oct1 (1:1000, Santa Cruz Biotechnology), mouse anti-human Oct4 (1:200, Santa Cruz Biotechnology), goat anti-human Brn1 (1:1000, Santa Cruz Biotechnology), goat anti-human Brn2 (1:1000, Santa Cruz Biotechnology), rabbit anti-mouse Sox2 (1:200) , rabbit anti-mouse SF1 (1:1000) , rabbit anti-mouse nestin (1:200) , mouse anti-rat nestin (1:200, BD Biosciences, San Jose, CA).

    Techniques: Labeling, Incubation, In Vivo, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Time in culture reduces Müller cell reactivity and promotes mature phenotype. A-C. Representative epifluorescence micrographs of primary Müller cells grown for 5, 12, 20 or 30 days in vitro (DIV) in serum-containing media immunolabeled with antibodies against nestin ( A ), vimentin ( B ) and GFAP ( C ) and counterstained with DAPI (blue). Nestin ( A ) and GFAP ( C ) immunolabeling decreased substantially at 20 and 30 DIV. Vimentin immunolabeling also decreased, but not until 30 DIV ( B ). Scale bar = 100μm.

    Journal: Experimental eye research

    Article Title: Phenotypes of Primary Retinal Macroglia: Implications for Purification and Culture Conditions

    doi: 10.1016/j.exer.2019.03.008

    Figure Lengend Snippet: Time in culture reduces Müller cell reactivity and promotes mature phenotype. A-C. Representative epifluorescence micrographs of primary Müller cells grown for 5, 12, 20 or 30 days in vitro (DIV) in serum-containing media immunolabeled with antibodies against nestin ( A ), vimentin ( B ) and GFAP ( C ) and counterstained with DAPI (blue). Nestin ( A ) and GFAP ( C ) immunolabeling decreased substantially at 20 and 30 DIV. Vimentin immunolabeling also decreased, but not until 30 DIV ( B ). Scale bar = 100μm.

    Article Snippet: Cells were labeled with rabbit anti-vimentin (Cell Signaling Technology CST, Danvers, MA; 1:100), mouse anti-GFAP (CST, 1:300), rabbit anti-GFAP (Dako/Agilent, Santa Clara, CA; 1:1000), mouse anti-Nestin (CST, 1:300), or rabbit anti-PAX2 (BioLegend, San Diego, CA; 1:200).

    Techniques: In Vitro, Immunolabeling

    Time in culture does not alter intermediate filament phenotypes of primary astrocytes. A,B. Representative epifluorescence micrographs of primary astrocytes grown for 5, 12, 20 or 30 days in vitro (DIV) in serum and immunolabeled with antibodies against nestin (green) and counterstained with DAPI (blue; A ) or co-immunolabeled with antibodies against vimentin (red) and GFAP (green; B ). Nestin immunolabeling decreased over time ( A ), while vimentin immunolabeling remained consistent across all DIV ( B ). GFAP was minimally detected at 20DIV and 30DIV, but appeared perinuclear rather than filamentous ( B ). Scale bar = 100μm.

    Journal: Experimental eye research

    Article Title: Phenotypes of Primary Retinal Macroglia: Implications for Purification and Culture Conditions

    doi: 10.1016/j.exer.2019.03.008

    Figure Lengend Snippet: Time in culture does not alter intermediate filament phenotypes of primary astrocytes. A,B. Representative epifluorescence micrographs of primary astrocytes grown for 5, 12, 20 or 30 days in vitro (DIV) in serum and immunolabeled with antibodies against nestin (green) and counterstained with DAPI (blue; A ) or co-immunolabeled with antibodies against vimentin (red) and GFAP (green; B ). Nestin immunolabeling decreased over time ( A ), while vimentin immunolabeling remained consistent across all DIV ( B ). GFAP was minimally detected at 20DIV and 30DIV, but appeared perinuclear rather than filamentous ( B ). Scale bar = 100μm.

    Article Snippet: Cells were labeled with rabbit anti-vimentin (Cell Signaling Technology CST, Danvers, MA; 1:100), mouse anti-GFAP (CST, 1:300), rabbit anti-GFAP (Dako/Agilent, Santa Clara, CA; 1:1000), mouse anti-Nestin (CST, 1:300), or rabbit anti-PAX2 (BioLegend, San Diego, CA; 1:200).

    Techniques: In Vitro, Immunolabeling

    Primary astrocytes and Müller cells exhibit an immature phenotype. A,B. Representative epifluorescence micrographs of primary astrocytes ( A ) and primary Müller cells ( B ) labeled with DAPI (blue) and immunolabeled with antibodies against Pax2 (left) and nestin (right). Both primary astrocytes ( A ) and Müller cells ( B ) were nestin-positive and Pax2-negative. Scale bar = 100μm.

    Journal: Experimental eye research

    Article Title: Phenotypes of Primary Retinal Macroglia: Implications for Purification and Culture Conditions

    doi: 10.1016/j.exer.2019.03.008

    Figure Lengend Snippet: Primary astrocytes and Müller cells exhibit an immature phenotype. A,B. Representative epifluorescence micrographs of primary astrocytes ( A ) and primary Müller cells ( B ) labeled with DAPI (blue) and immunolabeled with antibodies against Pax2 (left) and nestin (right). Both primary astrocytes ( A ) and Müller cells ( B ) were nestin-positive and Pax2-negative. Scale bar = 100μm.

    Article Snippet: Cells were labeled with rabbit anti-vimentin (Cell Signaling Technology CST, Danvers, MA; 1:100), mouse anti-GFAP (CST, 1:300), rabbit anti-GFAP (Dako/Agilent, Santa Clara, CA; 1:1000), mouse anti-Nestin (CST, 1:300), or rabbit anti-PAX2 (BioLegend, San Diego, CA; 1:200).

    Techniques: Labeling, Immunolabeling

    Tunicamycin inhibits the self-renewal of glioma-initiating cell (GIC) A. Representative images of neurospheres isolated from human GBM samples (T698968, T19002) and tumor xenograft formed by glioma cell line (SHG44). Scale bar represents 100 μm. B. Glioma-initiating cells expressed neural stem cell marker CD133 (red) and Nestin (green), as assessed by immunofluorescence. Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm. C. Glioma-initiating cells expressed core stemness factors Sox2, OCT4 and Nanog, as assessed by western blot. β -actin expression served as loading control. D. Summary of the tumor-initiating capacity of the neurosphere cultures derived from human GBM samples and glioma xenograft. E-I. GICs were plated at 200 cells per well in 96-well plates in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days. Tunicamycin treatment increased expression of ER stress marker CHOP using western blot analysis ( E ). ( F ) Representative photographs of neurospheres formed by SHG44 GICs in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days. Scale bar represents 100 μm. ( G-I ) The numbers of neurospheres formed by SHG44 ( G ), T698968 ( H ) or T19002 GICs ( I ) in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days were determined. Values represent mean ± S.D. (n = 6, *** p

    Journal: Oncotarget

    Article Title: ER stress inducer tunicamycin suppresses the self-renewal of glioma-initiating cell partly through inhibiting Sox2 translation

    doi: 10.18632/oncotarget.8954

    Figure Lengend Snippet: Tunicamycin inhibits the self-renewal of glioma-initiating cell (GIC) A. Representative images of neurospheres isolated from human GBM samples (T698968, T19002) and tumor xenograft formed by glioma cell line (SHG44). Scale bar represents 100 μm. B. Glioma-initiating cells expressed neural stem cell marker CD133 (red) and Nestin (green), as assessed by immunofluorescence. Nuclei were stained with Hoechst 33258 (blue). Scale bar represents 10 μm. C. Glioma-initiating cells expressed core stemness factors Sox2, OCT4 and Nanog, as assessed by western blot. β -actin expression served as loading control. D. Summary of the tumor-initiating capacity of the neurosphere cultures derived from human GBM samples and glioma xenograft. E-I. GICs were plated at 200 cells per well in 96-well plates in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days. Tunicamycin treatment increased expression of ER stress marker CHOP using western blot analysis ( E ). ( F ) Representative photographs of neurospheres formed by SHG44 GICs in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days. Scale bar represents 100 μm. ( G-I ) The numbers of neurospheres formed by SHG44 ( G ), T698968 ( H ) or T19002 GICs ( I ) in the presence of DMSO or 2.5 μM tunicamycin (TM) for seven days were determined. Values represent mean ± S.D. (n = 6, *** p

    Article Snippet: Antibodies The antibodies used were as follows: mouse anti-β-catenin antibody, mouse anti-Stat3 antibody and mouse anti-BrdU antibody were from BD Biosciences; mouse anti-Bmi-1 antibody and rabbit anti-OLIG2 and rabbit anti-POU3F2 was from Abcam; mouse anti-PTEN antibody and rabbit anti-CHOP antibody were from Cell Signaling Technology; mouse anti-Nestin antibody and rabbit anti-Sox2 antibody were from Millipore, mouse monoclonal anti-CD133 (W6B3C1 clone) was from Miltenyi Biotec.

    Techniques: Isolation, Marker, Immunofluorescence, Staining, Western Blot, Expressing, Derivative Assay

    Y3 Counteracts HuD-Induced Neurogenesis (A) Differentiating ESCs cultures assayed for Y3 and HuD expression levels by Northern blot and western blot, respectively. Cultures were immunostained for stage-specific markers: Oct4 (ESCs; red), Nestin (NPCs; red), and beta3-tubulin (early neurons; red); the scale bar corresponds to 75 μm. Relative quantification of Y3 and HuD levels are shown (right). (B) Differentiated NSC-34 cells (control or silenced for Y3) immunostained with anti-tubulin antibody (yellow) to detect neurites (left panel); GFP (green) identified transfected cells subjected to high content analysis; the scale bar corresponds to 100 μm. Multiple parameters were analyzed using Operetta HCS device (right panel). (C) Differentiation assay in control Y3 silenced cells, Y3 silenced cells transfected with wild-type HuD or with mutant HuD. A schematic representation of HuD constructs used in the experiment is provided. (D) PC12 cells were co-transfected with HA-tagged HuD and mock or Y3 WT or Y3 “deleted” vectors. Co-transfected cells were immunostained with anti-HA antibody, and the neurites were stained for tubulin. In (A)–(D), data are represented as mean ± SEM t test ∗p

    Journal: Molecular Cell

    Article Title: HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA

    doi: 10.1016/j.molcel.2018.06.032

    Figure Lengend Snippet: Y3 Counteracts HuD-Induced Neurogenesis (A) Differentiating ESCs cultures assayed for Y3 and HuD expression levels by Northern blot and western blot, respectively. Cultures were immunostained for stage-specific markers: Oct4 (ESCs; red), Nestin (NPCs; red), and beta3-tubulin (early neurons; red); the scale bar corresponds to 75 μm. Relative quantification of Y3 and HuD levels are shown (right). (B) Differentiated NSC-34 cells (control or silenced for Y3) immunostained with anti-tubulin antibody (yellow) to detect neurites (left panel); GFP (green) identified transfected cells subjected to high content analysis; the scale bar corresponds to 100 μm. Multiple parameters were analyzed using Operetta HCS device (right panel). (C) Differentiation assay in control Y3 silenced cells, Y3 silenced cells transfected with wild-type HuD or with mutant HuD. A schematic representation of HuD constructs used in the experiment is provided. (D) PC12 cells were co-transfected with HA-tagged HuD and mock or Y3 WT or Y3 “deleted” vectors. Co-transfected cells were immunostained with anti-HA antibody, and the neurites were stained for tubulin. In (A)–(D), data are represented as mean ± SEM t test ∗p

    Article Snippet: The following primary antibodies were used: mouse anti-MAP2 1:300 (M4403, Sigma-Aldrich), rabbit anti-Tau 1:300 (314 002, SynapticSystem), anti-SMI32 (200 KDa neurofilament) 1:300 (Ab7795, Abcam), rabbit anti-MNX1 1:100 (ABN174, Millipore), mouse anti-HuD 1:200 (sc-28299, Santa Cruz), mouse anti-beta III Tubulin (T8578, Sigma Aldrich), mouse anti-eEF1A1 (05235, Millipore), rabbit anti-eIF4A2 (31218, Abcam), rabbit anti-PABP 1:500 (Ab21060, Abcam), mouse anti-DCP1A 1:200 (Ab57654, Abcam), goat anti-TIA1 1:100 (sc-166247, Santa Cruz), mouse anti-Oct4 1:400 (sc-5279, Santa Cruz), mouse anti Nestin 1:400 (MAB353, Merck-Millipore), mouse anti beta3-Tubulin 1:1000 (G712A, Promega).

    Techniques: Expressing, Northern Blot, Western Blot, Transfection, High Content Screening, Differentiation Assay, Mutagenesis, Construct, Staining

    Phenotypic profiling of astrocytes in the presence and absence of an ECM coating. Representative fluorescent images showing a heterogenous population of cells expressing nestin and GFAP at 15 DIV ( a ) and ~30 DIV ( b ). Scale bar = 50 μm. Bar graph summarizes flow cytometry data highlighting the distribution of astrocytic phenotypes (Nestin−GFAP−, Nestin+GFAP−, Nestin+GFAP+, Nestin−GFAP+) at 15 DIV ( c ) and ~30 DIV ( d ). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats).

    Journal: Scientific Reports

    Article Title: Tissue-specific extracellular matrix accelerates the formation of neural networks and communities in a neuron-glia co-culture on a multi-electrode array

    doi: 10.1038/s41598-019-40128-1

    Figure Lengend Snippet: Phenotypic profiling of astrocytes in the presence and absence of an ECM coating. Representative fluorescent images showing a heterogenous population of cells expressing nestin and GFAP at 15 DIV ( a ) and ~30 DIV ( b ). Scale bar = 50 μm. Bar graph summarizes flow cytometry data highlighting the distribution of astrocytic phenotypes (Nestin−GFAP−, Nestin+GFAP−, Nestin+GFAP+, Nestin−GFAP+) at 15 DIV ( c ) and ~30 DIV ( d ). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats).

    Article Snippet: Neurons were characterized using Alexa Fluor 647 mouse anti-Tuj1 (1:25), and astrocytes with Alexa Fluor 488 mouse anti-GFAP (1:100) and mouse anti-Nestin (1:50, Neuromics), conjugated to PE-Cy7 using the Lightning-Link R-PE-Cy7 Kit (Novus Biologicals, Littleton, CO) according to manufacturer’s instructions.

    Techniques: Expressing, Flow Cytometry, Cytometry

    Characterization of cellular composition. Flow cytometry analysis of primary neuron and glial cells co-cultured in the presence or absence of an ECM coating. ( a ) Representative flow cytometry plots illustrate the gating strategy for the co-culture system: single cell population (top left) was gated based on forward-scatter characteristics, Zombie viability dye was used to exclude dead cells (high fluorescence) (top right), and Tuj1 staining was used to identify neuronal (Tuj1+) and glia (Tuj1−) subpopulation of cells (bottom left). The glial subpopulation (Tuj1− cells) was further gated to determine the phenotype of the astrocytic population based on nestin and GFAP expression (bottom right). Scatter plots summarizing the % of Tuj1+ ( b ) and % Tuj1− cells ( c ) from PDL, MaxGel, and bECM culture conditions across time (n = 2–3 cultures from 2 biological repeats). ( d ) Representative fluorescent images of DAPI-stained nuclei (blue) and those positive for the Ki67 proliferative marker (red). Scale bar = 50 μm. ( e ) Bar graph summarizes the total nuclei count (top) and % of Ki67 + nuclei (bottom). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats) indicated at the base of each bar and were analyzed using two-way ANOVA with Tukey’s post hoc test. Statistical significances between time points are at a level of ** p

    Journal: Scientific Reports

    Article Title: Tissue-specific extracellular matrix accelerates the formation of neural networks and communities in a neuron-glia co-culture on a multi-electrode array

    doi: 10.1038/s41598-019-40128-1

    Figure Lengend Snippet: Characterization of cellular composition. Flow cytometry analysis of primary neuron and glial cells co-cultured in the presence or absence of an ECM coating. ( a ) Representative flow cytometry plots illustrate the gating strategy for the co-culture system: single cell population (top left) was gated based on forward-scatter characteristics, Zombie viability dye was used to exclude dead cells (high fluorescence) (top right), and Tuj1 staining was used to identify neuronal (Tuj1+) and glia (Tuj1−) subpopulation of cells (bottom left). The glial subpopulation (Tuj1− cells) was further gated to determine the phenotype of the astrocytic population based on nestin and GFAP expression (bottom right). Scatter plots summarizing the % of Tuj1+ ( b ) and % Tuj1− cells ( c ) from PDL, MaxGel, and bECM culture conditions across time (n = 2–3 cultures from 2 biological repeats). ( d ) Representative fluorescent images of DAPI-stained nuclei (blue) and those positive for the Ki67 proliferative marker (red). Scale bar = 50 μm. ( e ) Bar graph summarizes the total nuclei count (top) and % of Ki67 + nuclei (bottom). Data are mean ± SEM for the number of cultures (n = 3–4 biological repeats) indicated at the base of each bar and were analyzed using two-way ANOVA with Tukey’s post hoc test. Statistical significances between time points are at a level of ** p

    Article Snippet: Neurons were characterized using Alexa Fluor 647 mouse anti-Tuj1 (1:25), and astrocytes with Alexa Fluor 488 mouse anti-GFAP (1:100) and mouse anti-Nestin (1:50, Neuromics), conjugated to PE-Cy7 using the Lightning-Link R-PE-Cy7 Kit (Novus Biologicals, Littleton, CO) according to manufacturer’s instructions.

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Co-Culture Assay, Fluorescence, Staining, Expressing, Marker

    Female and male NSPCs were grown as neurospheres without sex hormones for 4 days then allowed to adhere to a laminin substrate and expose to either 10 nM testosterone (T), 17β-estradiol (E 2 ), progesterone (P 4 ) for 20 min before Ki67/nestin double label fluorescent immunocytochemistry was performed (A, female control neurosphere shown). The proportion of DAPI/nestin-labeled cells expressing Ki67 per neurosphere was increased by 20 min of either T, E 2 or P 4 compared to controls in both females (B) and males (C) . Female and male neurospheres maintained without sex hormones were differentiated on a laminin substrate for 72 h ± 10 nM T, E 2 or P 4 when βIII-tubulin (red) immunocytochemistry (D) showed that while T, P 4 and E 2 increased the proportion of neuronal differentiation compared to controls, E 2 was more potent compared to T and P 4 in females (E) and males (F) . Results show mean ± SEM, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression

    doi: 10.3389/fncel.2015.00253

    Figure Lengend Snippet: Female and male NSPCs were grown as neurospheres without sex hormones for 4 days then allowed to adhere to a laminin substrate and expose to either 10 nM testosterone (T), 17β-estradiol (E 2 ), progesterone (P 4 ) for 20 min before Ki67/nestin double label fluorescent immunocytochemistry was performed (A, female control neurosphere shown). The proportion of DAPI/nestin-labeled cells expressing Ki67 per neurosphere was increased by 20 min of either T, E 2 or P 4 compared to controls in both females (B) and males (C) . Female and male neurospheres maintained without sex hormones were differentiated on a laminin substrate for 72 h ± 10 nM T, E 2 or P 4 when βIII-tubulin (red) immunocytochemistry (D) showed that while T, P 4 and E 2 increased the proportion of neuronal differentiation compared to controls, E 2 was more potent compared to T and P 4 in females (E) and males (F) . Results show mean ± SEM, * p

    Article Snippet: Cells were then washed in ice-cold 0.1 M phosphate buffered saline (PBS) then fixed in 4% paraformaldehyde (PFA) on ice for 20 min. Immunocytochemistry was then performed; cells were blocked in 0.1 M PBS containing 5% normal donkey serum (Millipore) and 0.1% triton-X100 (Sigma-Aldrich) for 60 min at 20°C then incubated with primary antibodies, rabbit anti-Ki67 (1:300, DKSH Australia Pty Ltd., VIC, Australia), mouse anti-nestin (1:300, R and D Systems, MN, USA) for 3 h, washed in PBS then incubated in secondary antibodies, Alexa fluor 488-conjugated donkey anti-rabbit (1:400, Molecular Probes, Life Technologies) and Alexa fluor 555-conjugated donkey anti-mouse (1:400, Molecular Probes) then counterstained with 4′,6-Diamidino-2-Phenylindole (1:2000, DAPI, Sigma-Aldrich) for 10 min and coverslipped in Fluoromount (DAKO, North Sydney, NSW, Australia).

    Techniques: Immunocytochemistry, Labeling, Expressing

    Immunohistochemical staining for DCX in the subventricular zone at different time points (2, 6, 24 and 48 h) after hypoxic-ischemic injury in the control, sham and hypoxic-ischemia groups. DCX, Nestin and doublecortin; MK, MK-801; Ro, Ro25-6981; NVP, NVP-AAM077.

    Journal: Molecular Medicine Reports

    Article Title: NMDA receptors promote neurogenesis in the neonatal rat subventricular zone following hypoxic-ischemic injury

    doi: 10.3892/mmr.2015.4501

    Figure Lengend Snippet: Immunohistochemical staining for DCX in the subventricular zone at different time points (2, 6, 24 and 48 h) after hypoxic-ischemic injury in the control, sham and hypoxic-ischemia groups. DCX, Nestin and doublecortin; MK, MK-801; Ro, Ro25-6981; NVP, NVP-AAM077.

    Article Snippet: Mouse monoclonal anti-Nestin antibody (ab11306), rabbit polycloncal anti-DCX antibody (ab18723), rabbit polycloncal anti-NR2A antibody (ab84181), and rabbit polyclonal anti-NR2B antibody (ab65875) were purchased from Abcam (Hong Kong, China).

    Techniques: Immunohistochemistry, Staining

    Morphine injections for four days decreased the number of later stage progenitors and immature neurons. (A-E) Fluorescent images of EGFP labeled cells and multiple neurogenesis markers: Nestin, Sox2, NeuroD, DCX, TUJ-1. There were no or very little Nestin or Sox2 positive neurons that were also EGFP positive (

    Journal: PLoS ONE

    Article Title: Morphine Modulates Adult Neurogenesis and Contextual Memory by Impeding the Maturation of Neural Progenitors

    doi: 10.1371/journal.pone.0153628

    Figure Lengend Snippet: Morphine injections for four days decreased the number of later stage progenitors and immature neurons. (A-E) Fluorescent images of EGFP labeled cells and multiple neurogenesis markers: Nestin, Sox2, NeuroD, DCX, TUJ-1. There were no or very little Nestin or Sox2 positive neurons that were also EGFP positive (

    Article Snippet: The following primary antibodies were used in our studies: Mouse monoclonal anti-GFP (1:2000; Life technologies, Grand Island, NY), Rat anti-BrdU (1:800; Abcam, Cambridge, MA), Mouse monoclonal anti-nestin (1:500; Abcam), Rabbit anti-Sox2 (1:500; Abcam), Rabbit DCX antibodies (1:1000; Abcam), Goat NeuroD antibodies (1:400; Santa Cruz Biotechnology, Santa Cruz, CA), Mouse monoclonal anti-Tuj1 IgG (1:1000; Covance, Princeton, NJ), Rabbit anti-GFAP (1:1000, Dako, Carpinteria, CA), Rabbit Caspase-3 antibodies (1:1000, Cell Signaling, Danvers, MA); Rabbit MOR antibodies (1:1000; Genentech, Customer ordered; San Francisco, CA) For the TUNEL Assay, we use Click-iT® Plus TUNEL Assay for in situ apoptosis detection with the Alexa Fluor® dyes kit (Life technologies, Grand Island, NY; Catalog number: C10618) following the manufacturer’s instructions.

    Techniques: Labeling

    Tumor of Mouse-Human NC Chimeras Express Markers of NB. Quantifications of IHC Experiments for the Percentage of Positive Cells Expressing the Typical NB Markers ( A ) Tyrosine hydroxylase (TH), ( B ) Chromogranin A (CgA), ( C ) Nestin (NES), and ( D ) Synaptophysin (SYP), in samples of human NBs from chimeric mice (n=5), subcutaneous xenograft outgrowth of hNCCs (n=4), and NB samples of patients (Data presented as means, error bars represent SD, dots represent fields of view).

    Journal: bioRxiv

    Article Title: Development of Human Neuroblastomas in Mouse-Human Neural Crest Chimeras

    doi: 10.1101/523795

    Figure Lengend Snippet: Tumor of Mouse-Human NC Chimeras Express Markers of NB. Quantifications of IHC Experiments for the Percentage of Positive Cells Expressing the Typical NB Markers ( A ) Tyrosine hydroxylase (TH), ( B ) Chromogranin A (CgA), ( C ) Nestin (NES), and ( D ) Synaptophysin (SYP), in samples of human NBs from chimeric mice (n=5), subcutaneous xenograft outgrowth of hNCCs (n=4), and NB samples of patients (Data presented as means, error bars represent SD, dots represent fields of view).

    Article Snippet: For immunostainings, samples were blocked with 2% BSA and incubated with primary antibodies including Rabbit anti-ALK (1:200, Cell Signaling), Rabbit anti-MYCN (1:50, Cell Signaling), Rabbit anti-CD3 (1:300, Thermo Fisher Scientific), Rabbit anti-CD8a (1:300, Cell Signaling), Rabbit anti-FoxP3 (1:75, R & D systems), Mouse anti-Il2ra (CD25, 1:100, Novus), Rabbit anti-human-PD-L1 (1:300, Cell Signaling), Sheep anti-human-CD47 (1:100, R & D systems), Rat anti-mouse F4/80 (1:100, Thermo Fisher Scientific), Rabbit anti-γH2AX (1:300, Abcam), Rabbit anti-human-Ki67 (1:20, Thermo Fisher Scientific), Rabbit anti-Chromogranin A (1:500, Novus), Rabbit anti-Synaptophysin (1:200, Cell Signaling), Mouse anti-human-Nestin (1:300, Abcam), Rabbit anti-TH (1:500, PelFreez), Rabbit anti-Peripherin (1:500, Abcam), Rabbit anti-human neurofilament (1:100, 160kD, Abcam) and anti-eGFP (1:1000, Aves Labs) overnight at 4°C followed by appropriate secondary antibody incubation for 1-2h (Thermo Fisher Scientific).

    Techniques: Immunohistochemistry, Expressing, Mouse Assay

    Tumor of Mouse-Human Neural Crest Chimeras Present Phenotypes of Human NB in Vivo. ( A ) H E and IHC comparing assay show that human NB tumors of chimeric mice express the typical NB markers Synaptophysin (SYP), Nestin (NES), Tyrosine hydroxylase (TH) and Chromogranin A (CgA), similarly to their expression found in NB samples of patient. hNCCS, which expressed oncogenes and were subcutaneously injected into immunocompromised mice to form xenograft tumors (left column) did not express these NB markers. (See IHC quantifications in Supplementary Figure 4 ; scale bars =100µm). ( B ) RNA-Seq of CHNB tumor samples (n=4) were separated in-silico into human and mouse reads to separate the tumor and hosts’-environment compartments (See material and methods). The analysis of RNA-Seq of the human-gene expression profile revealed that the human tumors in chimeric mice expressed a set of key genes normally associated with NB tumors ( ABCC1, BIRC5, CAMTA1, CCND1, DDX1, ENOS, IGF1R, KIF1B, KRAS, MAX, NES, NGFR, NME1, NRAS, PHOX2B, RAF1, SNW1, TH, TP53 and VEGFA ) with a significant correlation to expression in NB cell lines (Kelly and SHSY-5Y). linear regression p -value =0.011.

    Journal: bioRxiv

    Article Title: Development of Human Neuroblastomas in Mouse-Human Neural Crest Chimeras

    doi: 10.1101/523795

    Figure Lengend Snippet: Tumor of Mouse-Human Neural Crest Chimeras Present Phenotypes of Human NB in Vivo. ( A ) H E and IHC comparing assay show that human NB tumors of chimeric mice express the typical NB markers Synaptophysin (SYP), Nestin (NES), Tyrosine hydroxylase (TH) and Chromogranin A (CgA), similarly to their expression found in NB samples of patient. hNCCS, which expressed oncogenes and were subcutaneously injected into immunocompromised mice to form xenograft tumors (left column) did not express these NB markers. (See IHC quantifications in Supplementary Figure 4 ; scale bars =100µm). ( B ) RNA-Seq of CHNB tumor samples (n=4) were separated in-silico into human and mouse reads to separate the tumor and hosts’-environment compartments (See material and methods). The analysis of RNA-Seq of the human-gene expression profile revealed that the human tumors in chimeric mice expressed a set of key genes normally associated with NB tumors ( ABCC1, BIRC5, CAMTA1, CCND1, DDX1, ENOS, IGF1R, KIF1B, KRAS, MAX, NES, NGFR, NME1, NRAS, PHOX2B, RAF1, SNW1, TH, TP53 and VEGFA ) with a significant correlation to expression in NB cell lines (Kelly and SHSY-5Y). linear regression p -value =0.011.

    Article Snippet: For immunostainings, samples were blocked with 2% BSA and incubated with primary antibodies including Rabbit anti-ALK (1:200, Cell Signaling), Rabbit anti-MYCN (1:50, Cell Signaling), Rabbit anti-CD3 (1:300, Thermo Fisher Scientific), Rabbit anti-CD8a (1:300, Cell Signaling), Rabbit anti-FoxP3 (1:75, R & D systems), Mouse anti-Il2ra (CD25, 1:100, Novus), Rabbit anti-human-PD-L1 (1:300, Cell Signaling), Sheep anti-human-CD47 (1:100, R & D systems), Rat anti-mouse F4/80 (1:100, Thermo Fisher Scientific), Rabbit anti-γH2AX (1:300, Abcam), Rabbit anti-human-Ki67 (1:20, Thermo Fisher Scientific), Rabbit anti-Chromogranin A (1:500, Novus), Rabbit anti-Synaptophysin (1:200, Cell Signaling), Mouse anti-human-Nestin (1:300, Abcam), Rabbit anti-TH (1:500, PelFreez), Rabbit anti-Peripherin (1:500, Abcam), Rabbit anti-human neurofilament (1:100, 160kD, Abcam) and anti-eGFP (1:1000, Aves Labs) overnight at 4°C followed by appropriate secondary antibody incubation for 1-2h (Thermo Fisher Scientific).

    Techniques: In Vivo, Immunohistochemistry, Mouse Assay, Expressing, Injection, RNA Sequencing Assay, In Silico