while insights into the regulation of as have come predominantly from the molecular dissection of individual genes [ 36, 49 ], it is becoming clear that molecular rules can be identified from large - scale studies of both constitutive splicing and as [ 40 ]. most systematic global analyses on as have focused on comparisons across differentiated human tissues [ 50 – 52 ]. only one study, utilizing expressed sequence tag ( est ) collections from stem cells, has attempted to find as differences between embryonic and hematopoietic stem cells [ 53 ]. however, utilizing ests to identify as has intrinsic problems, as ests tend to be biased for the 3 ′ ends of genes, and full coverage of the genome by ests is severely limited by sequencing costs. the commercial availability of affymetrix exon arrays provides an alternative approach to interrogate the expression of every known and predicted exon in the human genome. the affymetrix genechip human exon 1. 0 st array contains ∼5. 4 million features used to interrogate ∼1 million exon clusters ( collections of overlapping ) of known and predicted exons with more than 1. 4 million probesets, with an average of four probes per exon. our goal was to identify and characterize as events that disting uish pluripotent hescs from multipotent nps, paving the way for future candidate gene approaches to study the impact of as in hescs and nps. however, as different hesc lines were established under different culture conditions from embryos with unique genetic backgrounds, we expected that hescs and their derived nps might have distinc t epigenetic and molecular signatures [ 54 ]. as both common and cell - line specific alternatively spliced exons are likely to be important in regenerative research, in our study two separate hesc lines were used, with independent protocols for differentiating the hescs into nps positive for sox1, an early neuroectodermal marker. as an endogenously occurring population of nps, human central nervous system stem cells grown as neurospheres ( hcns - scns ) were utilized as a natural benchmark for derived nps. we developed an approach called reap ( regression - based exon array protocol ), which is based on robust regression that analyzed signal estimates from affymetrix exon array data to identify as exons. experimental validation revealed alternative exons that disting uish hescs from nps; some of them also disting uish hescs from a variety of differentiated tissues. a comparison of reap - predicted alternative events with independent methods, such as using publicly available transcripts ( ests and mrnas ) and computational predictions based on genomic sequence information alone [ 55 ], showed a strong concordance of reap - identified as exons with as events identified from these orthogonal methods. finally, using analysis of the sequences flanking reap - identified alternative exons, we were able to discover known and novel cis - regulatory elements that potentially regulate these as events. derivation of neural progenitors from embryonic stem cellsnps were independently derived from two hesc lines, and rna extracted from the cell lines was processed and hybri diz ed onto affymetrix human 1. 0 st exon arrays. immunoh istochemical and reverse - transcriptase polymerase chain reaction ( rt - pcr ) analyses demonstrated that the hescs expressed pluripotent marker genes, and the derived nps expressed multipotent and neurogenic markers similar to hcns - scns. undifferentiated cythera ( cyt - es ) and hues6 ( hues6 - es ) hesc lines were maintained in culture as previously described [ 12, 23, 56 ]. utilizing specific antibod ies, we observed that undifferentiated cyt - es and hues6 - es cells were positive for the pluripotent markers oct4, ssea - 4, and tra - 1 – 80 ( unpublished data ). nps were derived from the hesc cell lines using protocols optimized for each line ( see materials and methods ). greater than 90 % of derived np cells ( cyt - np from cyt - es and hues6 - np from hues6 - es ) were positive for sox1, an early neuroectodermal marker, and nestin ( figure 1a ), and negative for oct4 ( unpublished data ). as a natural benchmark for the derived nps, we utilized hcns - scns, which were previously isolated from fresh human fetal brain tissues using antibod ies to cell - surface markers and fluorescence - activated cell sorting [ 12, 23 ]. the hcns - scns form neurospheres in culture which are greater than 90 % nestin and sox1 positive, and differentiate into both neurons and glial cells in vitro [ 12, 23 ]. immunoh istochemical analysis confirmed that hcns - scns were negative for oct4 ( unpublished data ) and positive for sox1 and nestin ( figure 1a ). figure 1molecular characterization of human embryonic stem cell lines and neuronal progenitors ( a ) immunoh istochemical analysis of markers in nps derived from the hesc lines ( cyt - np from cyt - es; and hues6 - np from hues6 - es ) and in hcns - scns. cyt - np, hues6 - np, and hcns - scns cells were nestin and sox1 positive. nuclei stained positive for dapi. white horizontal bar indicated 15 μm. ( b ) gene - level signal estimates of marker genes ( gapdh, oct4, nanog, nestin, notch1, dner, and sox1 ) from affymetrix exon array analysis. vertical bars indicated average log2 normalized signal estimates, and error bars represented standard deviations from three independent replicate experiments per cell type. ( c ) rt - pcr of marker genes ( gapdh, oct4, nanog, nestin, notch1, dner, and sox1 ). here, known molecular markers were subjected to rt - pcr measurements, which were compared to gene - level signal estimates generated from the exon array data. total rna was extracted, and labeled cdna targets were generated from three independent preparations of each cell type, namely cyt - es, hues6 - es, cyt - np, hues6 - np, and hcns - scns. to facilitate downstream analyses, instead of utilizing the meta - gene sets available from the manufacturers, we generated our own gene models by clustering alignments of ests and mrnas to annotate d known genes from the university of california santa cruz ( ucsc ) genome browser database. after hybri diz ation, scanning, and extraction of signal estimates for each probeset on the exon arrays, gene - level estimates were computed based on our gene models using available normalization and signal estimation software from affymetrix. for every gene, a t - statistic and corresponding p - value were computed representing the relative enrichment of the expression of the gene in hesc versus np, such as in cyt - es versus cyt - np. after correcting for multiple hypothesis testing using the benjamini - hochberg method, a p - value cutoff of 0. 01 was used to identify enriched genes. close inspection of all pairs of hesc - np comparisons revealed a generally significant overlap from 31 % to 85 % of the smaller of two compared sets of enriched genes ( see figure s1 ). thus for the purpose of identifying overall pluripotent and neural lineage - specific genes, the collective set of nps ( cyt - np, hues6 - np, and hcns - scns ) was compared to the collective set of hescs ( cyt - es and hues6 - es ). oct4 and nanog, which are important in maintaining the pluripotent state of embryonic stem cells ( escs ), were highly expressed in hescs but were significantly lower in nps ( figure 1b ). rt - pcr of oct4 and nanog mrna levels ac curate ly reflected the signal estimates from the array ( figure 1c ). interestingly, nestin was not significantly higher in nps as compared to the hesc from the gene - level estimates ( p - value 0. 065 ), which was further confirmed by rt - pcr ( figure 1c ). notch was recently identified to be important in promoting the neural lineage entry in mouse escs [ 57 ] and was shown to regulate stem cell proliferation in somatic mouse and hesc [ 58 ].