it was important to point out that while transcriptional expression of these genes did not disting uish these cells from one another, in several instances the reap - predicted as event was able to separate derived nps and hcns - scns. a notable exception was the alternative exon in the slk gene, encoding a serine / threonine kinase protein, which was commonly included in both hescs, i. e., the exon - excluded isoform was not present in hescs compared to nps, as well as in a variety of differentiated tissues. closer inspection of the reap [ + ] - validated as exon in the slk gene revealed strong conservation in the intronic region flanking the exon, a hallmark feature of evolutionarily conserved as exons [ 55, 77, 78 ]. a study analyzing the expression patterns of the slk gene suggested a potential functional role during embryonic development and in the adult central nervous system [ 79 ]; however, to our knowledge, our identification of the slk alternative exon is the first report of a hesc - biased as pattern during neuronal differentiation and across a myriad of differentiated tissues. in agreement, go analysis suggested that genes containing reap [ + ] exons were enriched in serine / threonine kinase activity, of which slk is a family member. future work will be required to study the impact of as in these genes in hescs and nps. we predict it is unlikely that the alternative exon in the slk gene is the only case common across hesc and different from differentiated tissues, but further studies will be necessary to identify other hesc - specific exons. reap [ + ] exons were underrepresented in genes that were differentially transcriptionally regulated in hescs and nps. our results act as a reminder that focusing only on genes that are differentially expressed will overlook rna processing events that may be biologically relevant to the system of interest. finally, we identified potential cis - regulatory intronic elements conserved and enriched proximal to the reap [ + ] exons. in particular, the fox1 / 2 binding site, gcuag, was conserved and enriched in the flanking introns of a subset of reap [ + ] exons. further studies will be required to explore the importance of fox1 family members in early neuronal differentiation. in conclusion, our introduction of reap and its application to identifying as events has revealed new and unanticipated insights into hesc biology and their transition to np cells. collectively, these exons represent a set of molecular changes that are likely to be important for studying human neural differentiation with applications in neuronal regenerative medicine. maintenance and differentiation of hescs and hcns - scns. hesc line cy203 ( cythera ) was cultured as previously described [ 12 ]. to differentiate into neuroepithelial precursor cells, colonies were manually isolated from mouse embryonic fibroblasts ( mefs ) and cut in small pieces. these pieces were transferred to a t75 flask with hescs differentiation media ( same hesc medium but 10 % ksr and no fgf - 2 ). medium was changed the next day by transferring the floating hesc aggregates to a new flask. after culturing for a week, the hesc cell aggregates formed mature embroid bodies ( ebs; ∼10 um round clusters with dark centers ). ebs were plated on a coated 10 - cm dish in hesc differentiation media. the next day, the medium was changed to dmem / f12 supplemented with its and fibronectin. medium was changed every other day for a week or until the cells formed rosette - like columnar structures that were isolated manually. these structures were then transferred to coated dishes in neural induction medium ( dmem / f12 supplemented with n2 and fgf - 2 ) for a week. elongated single cells were separated from leftover aggregates using non - enzymatic dissociation. after one to two passages, the cells formed a monolayer of homogeneous nps ( negative for sox1 immunostaining ). upon confluence, cells will form neurospheres that can also be isolated from the neuroepithelial precursor cells ( positive for sox1 immunostaining ). at any of these two stages, pan - neuronal differentiation can be achieved after three to four weeks. hesc line hues6 was cultured on mef feeders as previously described ( http: / / www. mcb. harvard. edu / melton / hues / ) or on gfr matrigel coated plates. cells grown on matrigel were grown in mef - conditioned medium and fgf - 2 was used at 20 ng / ml instead of 10 ng / ml for cells grown on mefs. to differentiate neuroepithelial precursors, colonies were removed by treatment with collagenase iv ( sigma ) and washed three times in growth media. the pieces of colonies were resuspended in hues growth media without fgf2 in an uncoated bacterial petri dish to form ebs. after one week, ebs were plated on polyornathine / laminin coated plates in dmem / f12 supplemented with n2 and fgf2. rosette structures were manually collected and enzymatically dissociated with tryple ( invitrogen ), plated on polyornathine / laminin coated plates, and grown in dmem / f12 supplemented with n2 and b27 - ra and 20 ng / ml fgf - 2. cells could be grown as a monolayer for up to at least ten passages. cells were sox1 and nestin positive and readily differentiated into neurons upon withdrawal of fgf - 2. human central nervous system stem cell line fbr1664 ( stemcells ) which is referred to as hcns - scns in the main text was cultured as previously described [ 23 ]. the cells were cultured in medium consisting of ex vivo 15 ( biowhittaker ) medium with n2 supplement ( gibco ), fgf2 ( 20 ng / ml ), epidermal growth factor ( 20 ng / ml ), lymphocyte inhibitory factor ( 10 ng / ml ), 0. 2 mg / ml heparin, and 60 ug / ml n - acetylcysteine. cultures were fed weekly and passaged at ∼two to three weeks using collagenases ( roche ). the following antibod ies and corresponding dilutions were utilized for the immunoh istochemical analysis of marker genes in cyt - es and hues6 - es: sox2 ( chemicon, 1: 500 ), oct4 ( santa cruz, 1: 500 ), sox1 ( chemicon, 1: 500 ), nestin ( pharmingen, 1: 250 ); hcns - scns: sox2 ( chemicon, 1: 200 ), nestin ( chemicon, 1: 200 ). rna preparation and array hybri diz ation. total rna from cells was processed as follows. cells were lysed in 1 ml of rna - bee ( teltest ). the rna was isolated by chloroform extraction of the aqueous phase, followed by iso propanol precipitation as per the manufacturer ' s instructions. the precipitated rna was washed in 75 % ethanol and eluted with depc - treated water. five ug of rna was treated with rq1 dnaase ( promega ) according to the manufacturer ' s instructions. one ug of total rna for each sample was processed using the affymetrix genechip whole transcript sense target labeling assay ( affymetrix ). ribosomal rna was reduced with the ribominus kit ( invitrogen ). target material was prepared using commercially available affymetrix genechip wt cdna synthesis kit, wt cdna amplification kit, and wt terminal labeling kit ( affymetrix ) as per manufacturer ' s instructions. hybri diz ation cocktails containing ∼5 ug of fragmented and labeled dna target were prepared and applied to genechip human exon 1. 0 st arrays. hybri diz ation was performed for 16 hours using the fluidics 450 station. arrays were scanned using the affymetrix 3000 7g scanner and genechip operating software version 1. 4 to produce. cel intensity files. detection of as by rt - pcr. cdnas were generated from total rna with superscript iii reverse transcriptase ( invitrogen ). pcr reactions were performed with primer pairs designed for as targets ( annealing at 58 °c and amplification for 30 or 35 cycles ). pcr products were resolved on either 1. 5 % or 3 % agarose gel in tbe. the ethidium bromide - stained gels were scanned with typhoon 8600 scanner ( molecular dynamics ) for quantification. the number of true positives ( tp; false negatives, fn ) was computed as the number of reap [ + ] ( reap [ − ] ) exons that were validated by rt - pcr as as.