nevertheless, the cellular mechanisms that underlie the development of neurons in vitro as they are subjected to the specific differentiation protocols are often poorly understood. resultswe have designed a focused dna ( oligo nucleot ide - based ) large - scale microarray platform ( named " neurostem chip " ) and used it to study gene expression patterns in hscs as they differentiate into neurons. we have selected genes that are relevant to cells ( i ) being stem cells, ( ii ) becoming neurons, and ( iii ) being neurons. the neurostem chip has over 1, 300 pre - selected gene targets and multiple controls spotted in quadruplicate s ( ~ 46, 000 spots total ). in this study, we present the neurostem chip in detail and describe the special advantages it offers to the fields of experimental neurology and stem cell biology. to illustrate the utility of neurostem chip platform, we have characterized an undifferentiated population of pluripotent human embryonic stem cells ( hescs, cell line sa02 ). in addition, we have performed a comparative gene expression analysis of those cells versus a heterogeneous population of hesc - derived cells committed towards neuronal / dopaminergic differentiation pathway by co - culturing with pa6 stromal cells for 16 days and containing a few tyrosine hydroxylase - positive dopaminergic neurons. conclusionwe characterized the gene expression profiles of undifferentiated and dopaminergic lineage - committed hesc - derived cells using a highly focused custom microarray platform ( neurostem chip ) that can become an important research tool in human stem cell biology. we propose that the areas of application for neurostem microarray platform could be the following: ( i ) characterization of the expression of established, pre - selected gene targets in 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. modern dna microarrays permit a comprehensive analysis of quantitative and qualitative changes in rna transcript abundance, outlining the cross - sections of gene expression and alterations of these in response to genetic or environmental stimuli. genome - scale microarrays ( cdna - or oligo nucleot ide - based ) are most valuable when screening populations of cells for the novel genes reflecting potential diagnost ic and prognostic markers or for an identification of novel therapeutic targets. on the other hand, custom microarray platforms that focus on specific pre - selected subset of genes relevant to a particular field of investigation can be less costly and more suitable for detection of smaller gene expression changes. microarray technology has added important information on both normal development and pathological changes in neurons. this is well illustrated by multiple studies on substantia nigra dopaminergic neurons, which degenerate in parkinson ' s disease ( pd ) [ 1 - 5 ]. the shortcomings of pharmacological therapies in pd have stimulated a search for alternative treatment strategies. in successful cases, transplants of human embryonic mesencephalic dopaminergic neurons can both restore dopaminergic neurotransmission and provide some symptomatic relief [ 6 - 8 ]. a wider application of neural transplantation in pd is, however, currently not feasible due to the unpredictable and variable outcome, the risks of unwanted side - effects ( dyskinesias ) [ 9, 10 ] and ethical and practical problems associated with using donor cells obtained from aborted embryos and fetuses [ 11, 12 ]. human embryonic stem cells ( hescs ) are considered a promising future source of cells for cell replacement therapy in pd and other neurological conditions [ 13 ]. they could constitute a virtually infinite source of self - renewing cells that can be persuaded to differentiate into specific types of neural cells, including dopaminergic neurons [ 14 - 16 ]. the molecular mechanisms that govern development of cultured hescs into specific types of neural cells are not fully understood. to promote our understanding of such mechanisms, it would be valuable to have tools that readily and reproducibly can help to characterize the cells as they differentiate from pluripotent stem cells into post - mitotic neurons. this important issue was addressed in earlier studies by luo et al. and yang et al., who designed small - to - moderate scale custom microarray platforms ( 281 and 755 gene targets, respectively ) [ 17, 18 ]. in addition superarray bioscience corporation ( frederick, md, usa ) have manufactured a range of small - scale arrays ( 263 gene targets for human array; [ 19 ] ). we sought to create an improved and updated microarray platform for hesc / neuronal differentiation - oriented gene expression studies. therefore, we generated a specialized large - scale dna microarray platform ( the " neurostem chip " ) that has over 1, 300 pre - selected gene targets and multiple controls spotted in quadruplicate s ( ~ 46, 000 spots total ). here we introduce the platform and the advantages it can offers to neuroscientists and stem cell biologists: particularly, in the niche of gene expression - oriented characterization of the samples using an assay of pre - selected, already established gene targets. in the current study, we use the neurostem chip to characterize an undifferentiated population of pluripotent hescs ( cell line sa02, cellartis ab, goteborg, sweden ) and compare the gene expression in those cells with that of a hesc - derived cell population rich in neurons, including tyrosine hydroxylase - positive dopaminergic neurons. stem cells have unique biological characteristics, but only a limited number of genes are currently recognized as established stem cell markers. examples include pou domain, class 5, transcription factor 1 ( oct3 / 4 ), signal transducer and activator of transcription 3 ( stat3 ), teratocarcinoma - derived growth factor ( tdgf1 ), enk - pending ( nanog ), undifferentiated embryonic cell transcription factor 1 ( utf1 ) and dna methyltransferase 3b ( dnmt3b ) [ 20 ]. at the same time, hundreds of genes are suggested as candidate markers for " stemness ", but their coupling to the undifferentiated stem cell state is not yet fully verified [ 21 ]. the concept of " stemness " ( term introduced in 1986 by grossman & levine ) is defined as " core stem cell properties that underlie self - renewal and the ability to generate differentiated progeny " [ 22 ]. considering the complexity of the processes involved, stemness can hardly be ensured by co - operation of just a few genes. nevertheless, three stemness genes ( namely, oct3 / 4, stat3 and nanog ) are considered " master " - genes that control the self - renewing process [ 23, 24 ]. various types of stem cells, such as hematopoietic, mesenchymal and neural ( hscs, mscs and nscs, respectively ), embryonic germ and embryonic carcinoma cells ( egcs and eccs, respectively ) are all characterized by variations in gene expression profiles, and only a few gene markers are associated with all these cell types [ 25, 26 ]. we have aimed to embrace the most comprehensive set of those genes into a solitary array, the neurostem chip. thereby, it is possible to employ it to monitor the relative expression levels of numerous known and candidate stemness genes in a single experiment. similar to the genetic bases underlying stemness, cell differentiation is associated with altered expression levels of certain recognized or candidate genes [ 25 ]. we therefore incorporated gene markers of development and differentiation in general, and that of neuronal and dopaminergic differentiation in particular, into the neurostem chip. examples include markers for the processes of neuronal maturation, axonal branching, neural / neuronal survival, etc.