twenty μl volumes of rt - pcr products were analyzed by electrophoresis at 1 % agarose gels and visualized by ethidium bromide staining. rna purification and fluorescent dye incorporationfor rna purification of undifferentiated hescs, the latter were mechanically separated from hfcs, collected in a 500 μl volume of vitrohes media, rinsed in pbs buffer and spun down at 300 rcf for 5 min. hesc - derived cells grown atop pa6 cells were harvested using a papain dissociation kit ( worthington biochemical corporation ), rinsed in pbs buffer and spun down as described above. the resulting cell pellets were resuspended in rlt buffer ( qiagen, usa ), passed through the shredder column ( qiagen ) and stored at - 80°c until the rna sample was purified following the rneasy micro kit ( qiagen ) protocol ( without carrier rna ); with dnase i ( quiagen ) treatment incorporated to the latter. rna integrity was tested using both nd - 1000 specrophotometer ( nanodrop, usa ) and rna nano labchip / 2100 bioanalyzer system ( agilent technologies, usa ). fluorescent label ( 24 nmol of the cyanine 3 - ctp ( cy3 ); perkinelmer, usa ) was incorporated to 350 – 500 ng of total rna amplified using low rna input fluorescent linear amplification kit ( agilent technologies ), generally following the kit manufacturer ' s protocol. similarly, 24 nmols of the cyanine 5 - ctp ( cy5; perkinelmer ) fluorescent label were incorporated to 400 ng sample of human universal reference rna ( stratagene, usa ); in addition, dye - swap replicate amplification were performed. amplified fluorescent crna samples were purified using rneasy mini - columns ( quiagen ), and fluorescence of the eluted products was measured using nd - 1000 specrophotometer ( nanodrop ). microarray technologylong oligo nucleot ide probes ( 69 – 71 nucleot ides ) matching gene targets of interest were selected from operon v2 and v3 human aros sets ( operon biotechnologies inc., usa ). arrays were produced by the swegene dna microarray resource centre, department of oncology at lund university ( sweden ) using a microgrid ii 600r arrayer fitted with microspot 10 k pins ( harvard biorobotics, usa ). printing was performed in a temperature - ( 18 – 20°c ) and humidity - ( 44 – 49 % rh ) controlled area on corning ultragaps aminosilane slides ( corning inc., usa ) with 140 μm spot - to - spot centerdistance and 90 – 110 μm average spot size. following printing, arrays were dried for 48 hours andstored in a dessicator until used. microarray slides were uv - cross - linked ( 800 mj / cm2 ), pre - hybri diz edwith fluorescently labeled samples using the pronto ! universal microarray hybri diz ation kit ( corning ) and subsequently hybri diz ed with test ( cy3 - labeled ) / reference ( cy5 - labeled ) rna samples ( or in reverse dye - labeling order ) at 42°c for 17 h using a maui hybri diz ation station ( biomicro systems inc., usa ) and the pronto ! universal microarray hybri diz ation kit, generally following manufacturer ' s instructions, with several minor adaptations [ 31 ]. data acquisition and statistical analysisimmediately following the washing steps, the fluorescence intensities were measured using a confocal laser scanner ( g2505b, agilent technologies ). after image formatting by tiff image channel splitter utility ( agilent technologies ) and grid annotation, a complete set of spots was visually inspected for each slide. using genepix pro ( molecular devices corp. usa ) flags for artifactual spots were annotate d for each spot. median pixel intensity minus the median local background for both dyes was used to obtain a test over reference intensity ratio. data normalization was performed per array subgrid using lowess curve fitting with a smoothing factor of 0. 33 [ 52, 53 ]. all normalizations, filtering, merging of technical replicates and analyses were performed in the bioarray software environment database [ 32 ]. to visualize sample - dependent variation of spot intensities, data was uploaded to the tigr multiexperiment viewer ( mev; [ 34 ] ). backgroundmany novel studies and therapies are possible with the use of human embryonic stem cells ( hes cells ) and their differentiated cell progeny. the hes cell derived cd34 hematopoietic stem cells can be potentially used for many gene therapy applications. here we evaluated the capacity of hes cell derived cd34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for aids. resultsundifferentiated normal and lentiviral vector transduced hes cells were cultured on s17 mouse bone marrow stromal cell layers to derive cd34 hematopoietic progenitor cells. the differentiated cd34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. phenotypic and functional analyses were carried out to compare these with that of fetal liver cd34 cell derived macrophages. as assessed by facs analysis, the hes - cd34 cell derived macrophages displayed characteristic cell surface markers cd14, cd4, ccr5, cxcr4, and hla - dr suggesting a normal phenotype. tests evaluating phagocytosis, upregulation of the costimulatory molecule b7. 1, and cytokine secretion in response to lps stimulation showed that these macrophages are also functionally normal. when infected with hiv - 1, the differentiated macrophages supported productive viral infection. lentiviral vector transduced hes cells expressing the transgene gfp were evaluated similarly like above. the transgenic hes cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. conclusionphenotypically normal and functionally competent macrophages could be derived from hes - cd34 cells. since these cells are susceptible to hiv - 1 infection, they provide a uniform source of macrophages for viral infection studies. based on these results, it is also now feasible to transduce hes - cd34 cells with anti-hiv genes such as inhibitory sirnas and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against hiv - 1 infection. thus, the potential utility of hes derived cd34 hematopoietic cells for hiv - 1 gene therapy can be evaluated. human embryonic stem cells ( hes cells ) show great promise for many novel cellular therapies due to their pluripotent nature [ 1 ]. these cells have the capacity to give rise to mature cells and tissues that arise from all three germ layers during embryonic development [ 2 - 4 ]. several pluripotent hes cell lines have so far been derived from the inner cell mass of human blastocysts and can be cultured indefinitely in an undifferentiated state [ 5 - 7 ]. thus, these cells provide a renewable source of pluripotent stem cells from which many types of differentiated cells could be produced for experimental and therapeutic purposes. cell differentiation protocols currently exist for the derivation of neurons, cardio myoc ytes, endothelial cells, hematopoietic progenitor cells, keratinocytes, osteoblasts, and hepatocytes to name a few [ 2, 3, 8, 9 ]. in addition to providing for potential cellular replacement therapies, opportunities exist in programming hes cells to correct a genetic defect and / or to express a therapeutic transgene of interest. using such approaches, many possibilities exist for treating a number of genetic and immune system disor ders [ 1 ]. many novel applications can be foreseen for hes cells in infectious disease research. aids is a potential disease that can benefit from exploiting hes cells for cell replacement therapy as they have the capacity to differentiate into various hematopoietic cells. hiv continues to be a major global public health problem with infections increasing at an alarming rate [ 10, 11 ].