some suggested areas of application for neurostem microarray platform could be the following: ( i ) characterization of the expression of established, pre - selected gene targets in human stem cell ( hsc ) lines, including newly derived ones, ( ii ) longitudinal quality control for maintained hsc populations, ( iii ) following gene expression changes during differentiation under defined cell culture conditions, and ( iv ) confirming the success of differentiation into specific neuronal subtypes. in addition, the neurostem chip can be used to characterize gene changes in intracerebral grafts of human cells, even when they are transplanted into experimental animals. we specifically wish to stress that we are about to make the neurostem chip available at a non - profit cost to the research community. we believe it has the potential to become an important screening tool in the expanding field of hsc studies in application to neurological / neurodegenerative disor ders. human embryonic stem cell ( hesc ) culturesundifferentiated hescs of sa02 ( sahlgrenska 2 ) line ( cellartis ab, goteborg, 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. commitment of hescs towards neuronal / dopaminergic differentiation pathwayco - 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. characterization of hescs and hesc - derived cells by immunocytochemistry ( icc ) and rt - pcrivf 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 % trit on x - 100 and 5 % of donkey serum. they were then incubated with primary antibod ies 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 immunorese arch 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 antibod ies 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. using 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.