of 0. 01 in the presence of 4 ug / ml polybrene was used. at different days post - infection, culture supernatants were collected and assayed for p24 antigen by elisa. to quantify viral p24 levels, a coulter - p24 kit ( beckman coulter, fullerton, ca ) was used. human embryonic stem cells ( hescs ) and neural progenitor ( np ) cells are excellent models for recapitulating early neuronal development in vitro, and are key to establishing strategies for the treatment of degenerative disor ders. while much effort had been undertaken to analyze transcriptional and epigenetic differences during the transition of hesc to np, very little work has been performed to understand post - transcriptional changes during neuronal differentiation. alternative rna splicing ( as ), a major form of post - transcriptional gene regulation, is important in mammalian development and neuronal function. human esc, hesc - derived np, and human central nervous system stem cells were compared using affymetrix exon arrays. we introduced an outlier detection approach, reap ( regression - based exon array protocol ), to identify 1, 737 internal exons that are predicted to undergo as in np compared to hesc. experimental validation of reap - predicted as events indicated a threshold - dependent sensitivity ranging from 56 % to 69 %, at a specificity of 77 % to 96 %. reap predictions significantly overlapped sets of alternative events identified using expressed sequence tags and evolutionarily conserved as events. our results also reveal that focusing on differentially expressed genes between hesc and np will overlook 14 % of potential as genes. in addition, we found that reap predictions are enriched in genes encoding serine / threonine kinase and helicase activities. an example is a reap - predicted alternative exon in the slk ( serine / threonine kinase 2 ) gene that is differentially included in hesc, but skipped in np as well as in other differentiated tissues. lastly, comparative sequence analysis revealed conserved intronic cis - regulatory elements such as the fox1 / 2 binding site gcaug as being proximal to candidate as exons, suggesting that fox1 / 2 may participate in the regulation of as in np and hesc. in summary, a new methodology for exon array analysis was introduced, leading to new insights into the complexity of as in human embryonic stem cells and their transition to neural stem cells. author summaryderiving neural progenitors ( np ) from human embryonic stem cells ( hesc ) is the first step in creating homogeneous populations of cells that will differentiate into myriad neuronal subtypes necessary to form a human brain. during alternative rna splicing ( as ), noncoding sequences ( introns ) in a pre - mrna are differentially removed in different cell types and tissues, and the remaining sequences ( exons ) are joined to form multiple forms of mature rna, playing an important role in cellular diversity. the authors utilized affymetrix exon arrays with probes targeting hundreds of thousands of exons to study as comparing human es to np. to accomplish this, a novel computational method, reap ( regression - based exon array protocol ), is introduced to analyze the exon array data. the authors showed that reap candidates are consistent with other types of methods for discovering alternative exons. in addition, reap candidate alternative exons are enriched in genes encoding serine / theronine kinases and helicase activities. an example is the alternative exon in the slk ( serine / threonine kinase 2 ) gene that is included in hesc, but excluded in np as well as in other differentiated tissues. finally, by comparing genomic sequences across multiple mammals, the authors identified dozens of conserved candidate binding sites that were enriched proximal to reap candidate exons. the human central nervous system is compose d of thousands of neuronal subtypes originating from neural stem cells ( nscs ) that migrate from the developing neural tube. such neuronal complexity is generated by a vast repertoire of molecular, genetic, and epigenetic mechanisms, such as the active retrotransposition of transposable elements [ 1 ], alternative promoter usage, alternative rna splicing ( as ), alternative polyadenylation, rna editing, post - translational modifications, and epigenetic modulation [ 2 ]. understanding the processes that generate neuronal diversity is key to gaining insights into neuronal development and paving new avenues for biomedical research. human embryonic stem cells ( hescs ) are pluripotent cells that propagate perpetually in culture as undifferentiated cells and can be induced to differentiate into a multitude of cell types both in vitro and in vivo [ 3 ]. as hescs can theoretically generate all cell types that make up an organism, they serve as an important model for understanding early human embryonic development. in addition, the hescs are a nearly infinite source for generating specialized cells such as neurons and glia for potential therapeutic purposes [ 4, 5 ]. in recent years, methods have been introduced to induce hescs to differentiate into neural progenitors ( nps ) [ 6, 7 ] and neuronal and glial subtypes [ 8 – 12 ]. the therapeutic interest in understanding the molecular basis of pluripotency and differentiation has led to many studies comparing transcriptional profiles in different hesc lines and the study of expression changes during the differentiation of hescs to various lineages [ 13 – 17 ]. nscs and progenitor cells ( nps ) are present throughout development and persist into adulthood [ 18 – 20 ]. they are critical for both basic research and developing approaches to treat neurological disor ders, such as parkinson disease and amyot rophic lateral sclerosis ( als ), and stroke or head injuries [ 21, 22 ]. nscs and npcs can be isolated from human fetal brain tissue [ 23 – 26 ], as well as from several regions of the adult human brain, such as the cortex, hippocampus, and the subventricular zone ( svz ) of the lateral ventricles [ 26 – 35 ]. several studies have explored expression patterns of npcs. for example, wright et al. identified “ expressed ” and “ not expressed ” genes in npcs isolated from the human embryonic cortex [ 24 ]; cai et al. used the massively parallel signature sequencing profiling ( mpss ) technique to analyze expression of fetal npcs in comparison to hescs and astrocyte precursors [ 27 ]; maisel et al. used affymetrix gene chip arrays to compare adult and fetal npcs propagated in neurospheres [ 35 ]. however, as with hescs, the focus thus far has been primarily on transcriptional differences, which ignores differential rna processing such as as, polyadenylation, degradation, or promoter usage. as is frequently used to regulate gene expression and to generate tissue - specific mrna and protein isoforms [ 36 – 39 ]. recent studies using splicing - sensitive microarrays suggested that up to 75 % of human genes undergo as, where multiple isoforms are derived from the same genetic loci [ 40 ]. this functional complexity underscores the challenge and importance of elucidating as regulation. as appears to play a dominant role in regulating neuronal gene expression and function [ 41, 42 ]. examples of splicing regulators that are enriched and function specifically in neuronal cells include the brain - specific splicing factor nova [ 43, 44 ] and neural - specific polypyrimidine tract binding protein ( nptb ), which antagonizes its paralogous ptb to regulate exon exclusion in neuronal cells [ 45 – 47 ]. finally, an early report estimating that 15 % of point mutations disrup t splicing underscores the importance of splicing in human disease [ 48 ]. indeed, the disrup tion of specific as events has been implicated in several human genetic diseases, such as frontotemporal dementia and parkinsonism, frasier syndrome, and atypical cystic fibrosis [ 49 ].