PMC:1802744 / 24425-34931 JSONTXT

{"project":"CellFinder","denotations":[{"id":"T319","span":{"begin":252,"end":278},"obj":"CellType"},{"id":"T320","span":{"begin":231,"end":243},"obj":"CellType"},{"id":"T321","span":{"begin":252,"end":257},"obj":"Species"},{"id":"T322","span":{"begin":246,"end":250},"obj":"CellType"},{"id":"T323","span":{"begin":309,"end":315},"obj":"CellType"},{"id":"T324","span":{"begin":586,"end":590},"obj":"CellType"},{"id":"T325","span":{"begin":9,"end":14},"obj":"Species"},{"id":"T326","span":{"begin":734,"end":743},"obj":"GeneProtein"},{"id":"T327","span":{"begin":716,"end":721},"obj":"Species"},{"id":"T328","span":{"begin":716,"end":743},"obj":"GeneProtein"},{"id":"T329","span":{"begin":246,"end":247},"obj":"Species"},{"id":"T330","span":{"begin":83,"end":96},"obj":"CellLine"},{"id":"T331","span":{"begin":77,"end":81},"obj":"CellLine"},{"id":"T332","span":{"begin":15,"end":24},"obj":"Anatomy"},{"id":"T333","span":{"begin":224,"end":229},"obj":"Species"},{"id":"T334","span":{"begin":68,"end":73},"obj":"CellType"},{"id":"T335","span":{"begin":309,"end":321},"obj":"CellType"},{"id":"T336","span":{"begin":258,"end":266},"obj":"Anatomy"},{"id":"T337","span":{"begin":267,"end":278},"obj":"CellType"},{"id":"T338","span":{"begin":1509,"end":1513},"obj":"CellType"},{"id":"T339","span":{"begin":745,"end":751},"obj":"GeneProtein"},{"id":"T340","span":{"begin":231,"end":237},"obj":"CellType"},{"id":"T341","span":{"begin":296,"end":306},"obj":"CellLine"},{"id":"T342","span":{"begin":2258,"end":2273},"obj":"GeneProtein"},{"id":"T343","span":{"begin":1784,"end":1790},"obj":"Species"},{"id":"T344","span":{"begin":1774,"end":1779},"obj":"Species"},{"id":"T345","span":{"begin":2736,"end":2740},"obj":"GeneProtein"},{"id":"T346","span":{"begin":2899,"end":2902},"obj":"CellLine"},{"id":"T347","span":{"begin":1910,"end":1919},"obj":"CellType"},{"id":"T348","span":{"begin":1612,"end":1615},"obj":"CellLine"},{"id":"T349","span":{"begin":2439,"end":2442},"obj":"CellLine"},{"id":"T350","span":{"begin":1544,"end":1552},"obj":"Anatomy"},{"id":"T351","span":{"begin":1758,"end":1767},"obj":"CellType"},{"id":"T352","span":{"begin":2084,"end":2087},"obj":"CellLine"},{"id":"T353","span":{"begin":1544,"end":1552},"obj":"CellType"},{"id":"T354","span":{"begin":1861,"end":1865},"obj":"GeneProtein"},{"id":"T355","span":{"begin":1709,"end":1714},"obj":"Species"},{"id":"T356","span":{"begin":2439,"end":2448},"obj":"CellType"},{"id":"T357","span":{"begin":1910,"end":1913},"obj":"CellLine"},{"id":"T358","span":{"begin":1715,"end":1723},"obj":"Anatomy"},{"id":"T359","span":{"begin":1761,"end":1768},"obj":"CellType"},{"id":"T360","span":{"begin":1826,"end":1859},"obj":"GeneProtein"},{"id":"T361","span":{"begin":2042,"end":2047},"obj":"CellType"},{"id":"T362","span":{"begin":1530,"end":1535},"obj":"CellType"},{"id":"T363","span":{"begin":2231,"end":2234},"obj":"CellLine"},{"id":"T364","span":{"begin":2231,"end":2240},"obj":"CellType"},{"id":"T365","span":{"begin":1791,"end":1795},"obj":"CellType"},{"id":"T366","span":{"begin":6451,"end":6456},"obj":"GeneProtein"},{"id":"T367","span":{"begin":6458,"end":6462},"obj":"GeneProtein"},{"id":"T368","span":{"begin":3267,"end":3276},"obj":"CellType"},{"id":"T369","span":{"begin":4213,"end":4215},"obj":"GeneProtein"},{"id":"T370","span":{"begin":6819,"end":6824},"obj":"CellType"},{"id":"T371","span":{"begin":7011,"end":7014},"obj":"CellLine"},{"id":"T372","span":{"begin":3267,"end":3270},"obj":"CellLine"},{"id":"T373","span":{"begin":6366,"end":6370},"obj":"GeneProtein"},{"id":"T374","span":{"begin":4201,"end":4207},"obj":"Species"},{"id":"T375","span":{"begin":6355,"end":6359},"obj":"GeneProtein"},{"id":"T376","span":{"begin":3197,"end":3202},"obj":"CellType"},{"id":"T377","span":{"begin":7994,"end":7999},"obj":"Species"},{"id":"T378","span":{"begin":4158,"end":4164},"obj":"Species"},{"id":"T379","span":{"begin":4811,"end":4822},"obj":"GeneProtein"},{"id":"T380","span":{"begin":5946,"end":5950},"obj":"CellComponent"},{"id":"T381","span":{"begin":7730,"end":7733},"obj":"CellComponent"},{"id":"T382","span":{"begin":7044,"end":7050},"obj":"GeneProtein"},{"id":"T383","span":{"begin":8020,"end":8023},"obj":"CellComponent"},{"id":"T384","span":{"begin":8127,"end":8131},"obj":"CellComponent"},{"id":"T385","span":{"begin":7754,"end":7757},"obj":"CellComponent"},{"id":"T386","span":{"begin":6516,"end":6521},"obj":"GeneProtein"},{"id":"T387","span":{"begin":6503,"end":6505},"obj":"GeneProtein"},{"id":"T388","span":{"begin":6282,"end":6285},"obj":"GeneProtein"},{"id":"T389","span":{"begin":6529,"end":6533},"obj":"GeneProtein"},{"id":"T390","span":{"begin":6782,"end":6785},"obj":"CellComponent"},{"id":"T391","span":{"begin":6523,"end":6527},"obj":"GeneProtein"},{"id":"T392","span":{"begin":6870,"end":6874},"obj":"CellType"},{"id":"T393","span":{"begin":4100,"end":4117},"obj":"GeneProtein"},{"id":"T394","span":{"begin":4111,"end":4117},"obj":"GeneProtein"},{"id":"T395","span":{"begin":3720,"end":3726},"obj":"Species"},{"id":"T396","span":{"begin":3732,"end":3738},"obj":"Species"},{"id":"T397","span":{"begin":5240,"end":5244},"obj":"GeneProtein"},{"id":"T398","span":{"begin":5434,"end":5440},"obj":"GeneProtein"},{"id":"T399","span":{"begin":5246,"end":5269},"obj":"GeneProtein"},{"id":"T400","span":{"begin":6494,"end":6501},"obj":"GeneProtein"},{"id":"T401","span":{"begin":5521,"end":5528},"obj":"GeneProtein"},{"id":"T402","span":{"begin":5123,"end":5165},"obj":"GeneProtein"},{"id":"T403","span":{"begin":5114,"end":5121},"obj":"GeneProtein"},{"id":"T404","span":{"begin":5353,"end":5362},"obj":"GeneProtein"},{"id":"T405","span":{"begin":5347,"end":5351},"obj":"GeneProtein"},{"id":"T406","span":{"begin":5515,"end":5519},"obj":"GeneProtein"},{"id":"T407","span":{"begin":5696,"end":5700},"obj":"GeneProtein"},{"id":"T408","span":{"begin":5702,"end":5748},"obj":"GeneProtein"},{"id":"T409","span":{"begin":3739,"end":3742},"obj":"GeneProtein"},{"id":"T410","span":{"begin":3720,"end":3742},"obj":"GeneProtein"},{"id":"T411","span":{"begin":3772,"end":3777},"obj":"Species"},{"id":"T412","span":{"begin":4100,"end":4105},"obj":"Species"},{"id":"T413","span":{"begin":6308,"end":6312},"obj":"GeneProtein"},{"id":"T414","span":{"begin":4722,"end":4726},"obj":"GeneProtein"},{"id":"T415","span":{"begin":3429,"end":3438},"obj":"CellType"},{"id":"T416","span":{"begin":3546,"end":3552},"obj":"Species"},{"id":"T417","span":{"begin":4201,"end":4215},"obj":"GeneProtein"},{"id":"T418","span":{"begin":4158,"end":4174},"obj":"GeneProtein"},{"id":"T419","span":{"begin":4728,"end":4737},"obj":"GeneProtein"},{"id":"T420","span":{"begin":3429,"end":3432},"obj":"CellLine"},{"id":"T421","span":{"begin":4170,"end":4174},"obj":"GeneProtein"},{"id":"T422","span":{"begin":6445,"end":6449},"obj":"GeneProtein"},{"id":"T423","span":{"begin":6431,"end":6436},"obj":"GeneProtein"},{"id":"T424","span":{"begin":5825,"end":5871},"obj":"GeneProtein"},{"id":"T425","span":{"begin":5818,"end":5823},"obj":"GeneProtein"},{"id":"T426","span":{"begin":5986,"end":5989},"obj":"CellComponent"},{"id":"T427","span":{"begin":4990,"end":4995},"obj":"GeneProtein"},{"id":"T428","span":{"begin":4997,"end":5038},"obj":"GeneProtein"},{"id":"T429","span":{"begin":6379,"end":6384},"obj":"GeneProtein"},{"id":"T430","span":{"begin":3112,"end":3117},"obj":"CellType"},{"id":"T431","span":{"begin":3248,"end":3253},"obj":"CellType"},{"id":"T432","span":{"begin":3214,"end":3218},"obj":"CellType"},{"id":"T433","span":{"begin":6322,"end":6324},"obj":"GeneProtein"},{"id":"T434","span":{"begin":6294,"end":6301},"obj":"GeneProtein"},{"id":"T435","span":{"begin":6337,"end":6341},"obj":"GeneProtein"},{"id":"T436","span":{"begin":6314,"end":6320},"obj":"GeneProtein"},{"id":"T437","span":{"begin":7410,"end":7417},"obj":"GeneProtein"},{"id":"T438","span":{"begin":4908,"end":4913},"obj":"GeneProtein"},{"id":"T439","span":{"begin":6479,"end":6483},"obj":"GeneProtein"},{"id":"T440","span":{"begin":6287,"end":6292},"obj":"GeneProtein"},{"id":"T441","span":{"begin":6464,"end":6470},"obj":"GeneProtein"},{"id":"T442","span":{"begin":5608,"end":5628},"obj":"GeneProtein"},{"id":"T443","span":{"begin":6326,"end":6330},"obj":"GeneProtein"},{"id":"T444","span":{"begin":6343,"end":6348},"obj":"GeneProtein"},{"id":"T445","span":{"begin":5604,"end":5606},"obj":"GeneProtein"},{"id":"T446","span":{"begin":6426,"end":6429},"obj":"GeneProtein"},{"id":"T447","span":{"begin":7466,"end":7469},"obj":"CellComponent"},{"id":"T448","span":{"begin":4806,"end":4809},"obj":"GeneProtein"},{"id":"T449","span":{"begin":7011,"end":7020},"obj":"CellType"}],"text":"Methods\n\nHuman embryonic stem cell (hESC) cultures\nUndifferentiated hESCs of SA02 (Sahlgrenska 2) line (Cellartis AB, Göteborg, Sweden; see NIH Human Embryonic Stem Cell Registry at [46]) were maintained over a monolayer of human \"feeder cells\" (hFCs; human foreskin fibroblasts, ATCC; cell line CCD-1112Sk). Feeder cells were grown in hFC medium (Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% heat-inactivated FCS (Stem Cell Technologies, USA) and 0.5% Penicillin/Streptomycin mix) for 11 passages. One day prior to hESC plating, hFC medium was washed away from the hFCs, the latter were resuspended in a hESC proliferation medium (VitroHES media (Vitrolife AB, Sweden) supplemented with 4 ng/ml human recombinant basic FGF (hrbFGF, Biosource International, USA) and plated in a central ring of gelatinized in vitro fertilization (IVF) dishes with a cell density of 120,000 cells/dish. The outer rings of the IVF dishes were filled with Dulbecco's modified Eagle medium (DMEM) supplemented with 0.5% Penicillin/Streptomycin mix. One half of the culture medium was replaced every other day. The cells were maintained at 37°C, 5% CO2, 95% humidity settings. Every 6 days, fragments of the hESC colonies (around 10–14 colonies per dish, measuring around 0.015 × 0.015 mm) that had an unaltered morphology (indicating lack of spontaneous differentiation) were mechanically cut from dishes using stem cell knives/transfer pipettes (SweMed Lab International AB, Sweden) and then plated on fresh hFCs.\n\nCommitment of hESCs towards neuronal/dopaminergic differentiation pathway\nCo-culturing with the PA6 stromal cell line (MC3T3-G2/Pa6, from RIKEN Cell Bank Japan (RCB 1127), derived from newborn mouse calvaria rapidly generates high numbers of DA neurons from mouse and monkey ESCs by an unknown mechanism named stromal-derived inducing activity (SDIA; [28,29]). For differentiation experiments, PA6 cells were plated on gelatine-coated T25 flasks at 16 × 103 cells/cm2 (400,000 cells/flask) density 2 days prior to introducing hESCs into the co-culture and cultured at PA6 culturing media (containing minimum essential medium alpha (α-MEM) supplemented with 10% FCS and 0.5% Penicillin/Streptomycin). Alternatively, PA6 cells were plated over Type I collagen-coated glass cover-slips placed in wells of 4-well-plates (50,000 cells/well, for immunocytochemical (ICC) analysis). Three hours prior to initiation of co-culture, PA6 cells were rinsed 3 times with PBS and media was replaced with co-culture media (Glasgow's modified Eagle's media (G-MEM) supplemented with 8% knock-out serum replacement (KSR), 2 mM glutamine, 0.1 mM non-essential amino-acids (NEAA), 1 mM pyruvate, 0.1 mM β-mercaptoethanol (βME) and 4 ng/μl bFGF). Fragments of hESC colonies (80–90 per flask; 4–5 per well of 4-well-plate) presenting undifferentiated morphology were manually passaged onto the confluent PA6 monolayer and cell co-cultures were maintained at 37°C, 5% CO2, 95% humidity settings. One half of the co-culture medium was replaced every other day for first 10 days, and daily onwards.\n\nCharacterization of hESCs and hESC-derived cells by immunocytochemistry (ICC) and RT-PCR\nIVF dishes with hESCs grown atop hFCs and 4-well plate dishes with hESCs growing atop PA6 cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at the day of passage/harvest (Day 6 of hESC/hFC co-culturing) and Day 16 of co-culturing with PA6 cells, respectively. Cells were pre-incubated with blocking solution containing PBS, 0.5% Triton X-100 and 5% of donkey serum. They were then incubated with primary antibodies in blocking solution overnight at room temperature. After three washes with PBS, cells were incubated with the donkey anti-rabbit IgG conjugated with FITC or anti-mouse Cy3 (1:200, Jackson ImmunoResearch Laboratories). Cells were then washed once with PBS, incubated with 1:1000 DAPI in PBS for 10 minutes, followed by another wash with PBS. Coverslips were mounted onto glass slides with PVA mounting medium containing anti-fading reagent DABCO. The following primary antibodies were used: mouse anti-Oct3/4 (1:500, Santa Cruz Biotechnology Inc.); rabbit anti-Ki67 (1:200, Novocastra Ltd.); rabbit anti-TH (1:500, Chemicon). Immunostained cell cultures were visualized using a Zeiss fluorescent microscope attached to a Nikon digital camera.\nUsing RT-PCR, all RNA samples used in this study were tested negative for the presence of gDNA (data not shown). The intron-spanning primers for RT-PCR were selected from published works or designed using Oligo 4.0 software (Molecular Biology Insight) or Clone Manager Suite 7.1 (Sci Ed Software, NC, USA) and ordered from TAG Copenhagen A/S, Denmark, as the following: Sox2, SRY-box 2: 5'-TAC CTC TTC CTC CC CTC CA-3', 5'-ACT CTC CTC TTT TGC ACC CC-3'; En1, Engrailed 1: 5'-AAG GGA CGA AAC TGC GAA CTC C-3', 5'-GAC ACG AAA GGA AAC ACA CAC TCT CG-3' [47]; Nanog: 5'-TGC TTA TTC AGG ACA GCC T-3', 5'-TCT GGT CTT CTG TTT CTT GAC T-3' [48]; Gapdh, glyceraldehydes-3-phosphate dehydrogenase: 5'-GGA AGG TGA AGG TCG GAG TCA A-3', 5'-GAT CTC GCT CCT GGA AGA TGG T-3'; Aldh1A1, aldehyde dehydrogenase 1 family, member A1: 5'-GGG CAG CCA TTT CTT CTC AC-3', 5'-CTT CTT AGC CCG CTC AAC AC-3' [49]; Sdha, succinate dehydrogenase: 5'-TGG GAA CAA GAG GGC ATC TG-3', 5'-CCA CCA CTG CAT CAA ATT CAT G-3' [50]; Tubb, β-tubulin: 5'-CTC ACA AGT ACG TGC CTC GAG-3', 5'-GCA CGA CGC TGA AGG TGT TCA-3'; Nestin: 5'-AGA GGG GAA TTC CTG CT GAG-3', 5'-CTG AGG ACC AGG ACT CTC TA-3' [47]; Actb, β-actin: 5'-CAT CGA GCA CGG CAT CGT CA-3', 5'-TAG CAC AGC CTG GAT AGC AAC-3' [51]; Th, Tyrosine hydroxylase: 5'-CGA GCT GTG AAG GTG TTT G-3', 5'-TTG GTG ACC AGG TGA TGA C-3'; Msx1, homolog of Drosophila muscle segment homolog 1: 5'-CTC AAG CTG CCA GAA GAT GC-3', 5'-TCC AGC TCT GCC TCT TGT AG-3'; Pitx2, paired-like homeodomain transcription factor 2: 5'-ACC TTA CGG AAG CCC GAG TC-3', 5'-TGG ATA GGG AGG CGG ATG TA-3' [49]. cDNA was synthesized from 1 mg of total RNA using SuperScript II (Invitrogen), and RT-PCR amplifications were performed using the MiniOpticon system (Bio-Rad) with REDTaq Polymerase (Sigma-Aldrich) essentially as described by the manufacturer. Following initial denaturation for 5 min at 95°C, DNA amplifications were performed for 35 (En1, Nanog, Aldh1a1), 33 (Sox2, Nestin, Th, Msx1), 32 (Tubb, Pitx2), 27 (Sdha), 25 (Actb) or 22 (Gapdh) cycles of 1 min at 95°C, 1 min at 55°C (En1, Pitx2), 57°C (Sox2, Nanog, Sdha, Nestin), 58°C (Tubb), 58.5°C (Aldh1a1, Th) or 59°C (Gapdh, Actb, Msx1), and 1 min at 72°C. The final extension was 5 min at 72°C. Twenty μl volumes of RT-PCR products were analyzed by electrophoresis at 1% agarose gels and visualized by ethidium bromide staining.\nRNA purification and fluorescent dye incorporationFor RNA purification of undifferentiated hESCs, the latter were mechanically separated from hFCs, collected in a 500 μl volume of VitroHES media, rinsed in PBS buffer and spun down at 300 rcf for 5 min. hESC-derived cells grown atop PA6 cells were harvested using a papain dissociation kit (Worthington Biochemical Corporation), rinsed in PBS buffer and spun down as described above. The resulting cell pellets were resuspended in RLT buffer (Qiagen, USA), passed through the shredder column (Qiagen) and stored at -80°C until the RNA sample was purified following the RNeasy Micro Kit (Qiagen) protocol (without carrier RNA); with DNase I (Quiagen) treatment incorporated to the latter. RNA integrity was tested using both ND-1000 specrophotometer (NanoDrop, USA) and RNA Nano LabChip/2100 Bioanalyzer system (Agilent Technologies, USA).\nFluorescent label (24 nmol of the Cyanine 3-CTP (Cy3); PerkinElmer, USA) was incorporated to 350–500 ng of total RNA amplified using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies), generally following the kit manufacturer's protocol. Similarly, 24 nmols of the Cyanine 5-CTP (Cy5; PerkinElmer) fluorescent label were incorporated to 400 ng sample of Human Universal Reference RNA (Stratagene, USA); in addition, dye-swap replicate amplification were performed. Amplified fluorescent cRNA samples were purified using RNeasy mini-columns (Quiagen), and fluorescence of the eluted products was measured using ND-1000 specrophotometer (NanoDrop).\n\nMicroarray technology\nLong oligonucleotide probes (69–71 nucleotides) matching gene targets of interest were selected from Operon V2 and V3 human AROS sets (Operon Biotechnologies Inc., USA). Arrays were produced by the SweGene DNA Microarray Resource Centre, Department of Oncology at Lund University (Sweden) using a MicroGrid II 600R arrayer fitted with MicroSpot 10 K pins (Harvard BioRobotics, USA). Printing was performed in a temperature- (18–20°C) and humidity- (44–49% RH) controlled area on Corning UltraGAPS aminosilane slides (Corning Inc., USA) with 140 μm spot-to-spot centerdistance and 90–110 μm average spot size. Following printing, arrays were dried for 48 hours andstored in a dessicator until used. Microarray slides were UV-cross-linked (800 mJ/cm2), pre-hybridizedwith fluorescently labeled samples using the Pronto! Universal Microarray Hybridization Kit (Corning) and subsequently hybridized with test (Cy3-labeled)/reference (Cy5-labeled) RNA samples (or in reverse dye-labeling order) at 42°C for 17 h using a MAUI hybridization station (BioMicro Systems Inc., USA) and the Pronto! Universal Microarray Hybridization Kit, generally following manufacturer's instructions, with several minor adaptations [31].\n\nData acquisition and statistical analysis\nImmediately following the washing steps, the fluorescence intensities were measured using a confocal laser scanner (G2505B, Agilent Technologies). After image formatting by Tiff Image Channel Splitter Utility (Agilent Technologies) and grid annotation, a complete set of spots was visually inspected for each slide. Using GenePix Pro (Molecular Devices Corp. USA) flags for artifactual spots were annotated for each spot. Median pixel intensity minus the median local background for both dyes was used to obtain a test over reference intensity ratio. Data normalization was performed per array subgrid using LOWESS curve fitting with a smoothing factor of 0.33 [52,53]. All normalizations, filtering, merging of technical replicates and analyses were performed in the BioArray Software Environment database [32]. To visualize sample-dependent variation of spot intensities, data was uploaded to the TIGR MultiExperiment Viewer (MEV; [34]).\n"}