given the present lack of effective vaccines and the ineffectiveness of drug based therapies for a complete cure, new and innovative approaches are essential. gene therapy through intracellular immunization offers a promising alternative approach and possible supplement to current haart therapy [ 12 - 14 ]. hiv mainly targets cells of the hematopoietic system, namely, t cells, macrophages, and dendritic cells [ 15 ]. as infection progresses, the immune system is rendered defenseless against other invading pathogens and succumbs to opportunistic infections. there is a great deal of progress in the area of stem cell gene therapy for aids [ 12 ]. a primary goal of many ongoing studies is to introduce an effective anti-hiv gene into hematopoietic stem cells [ 16 - 18 ]. as these cells possess the ability to self renew, they have the potential to continually produce hiv resistant t cells and macrophages in the body thus providing long term immune reconstitution. these approaches use cd34 hematopoietic stem cells for anti-hiv gene transduction via integrating viral vectors such as lentiviral vectors [ 16 - 18 ]. lentiviral vectors have several advantages over conventional retroviral vectors since higher transduction efficiencies can be obtained and there is less gene silencing. the cd34 cells currently used for many therapies are primarily obtained from bone marrow or mobilized peripheral blood [ 1, 19 ]. thus, cd34 progenitor cells are an essential ingre dien t for hiv gene therapy. in view of the need for cd34 cells for hiv gene therapy as well as for other hematopoietic disor ders, if one can produce these cells in unlimited quantities from a renewable source, it will overcome the limitations of securing large numbers of cd34 cells for therapeutic purposes. in this regard, progress has been made in deriving cd34 cells from hes cells ( hes - cd34 ). different methods currently exist to derive cd34 cells from hes cells with varying efficiencies [ 20 - 27 ]. recent reports have indicated the capacity of hes cell derived cd34 cells to give rise to lymphoid and myeloid lineages thus paving the way for utilization of these cells for hematopoietic cell therapy [ 20, 27 - 29 ]. for the effective utilization of hes - cd34 cells for hiv gene therapy, a number of parameters need to be examined. first, one has to demonstrate that hes - cd34 cells can give rise to macrophages and helper t cells which are the main cells that need to be protected against hiv infection. recent evidence has shown that hes - cd34 cells can give rise to myelomonocytic cells [ 21 ]. however, thorough phenotypic or functional characterization of these cells is lacking. it is also not clear if these cells are susceptible to hiv infection. similarly, although the hes - cd34 cells were shown to have lymphoid progenitor capacity, only b cell and natural killer ( nk ) cell differentiation has been examined so far [ 21, 28 ]. the capacity to generate t cells remains to be evaluated. with this background, as a first step, our primary goal in these studies is to examine the capacity of hes - cd34 cells to give rise to phenotypically and functionally normal macrophages and whether such cells are susceptible to productive hiv infection. since lentiviral vectors have been shown to successfully transduce hes cells [ 30 - 33 ], we further investigated the ability of transduced hes cells to differentiate into transgenic macrophages that can support hiv - 1 infection. demonstration of hiv - 1 productive infection in these cells will permit future efficacy evaluations of anti-hiv genes in this system. here we show that normal and lentiviral vector transduced hes - cd34 cells can give rise to phenotypically and functionally normal macrophages that support hiv infection thus paving the way for many novel approaches to evaluate their potential for hiv gene therapy. derivation of macrophages from hes cellsundifferentiated hes cell colonies grown in media supplemented with 4 ng / ml bfgf displayed normal morphology of pluripotent human embryonic stem cells with tight and discreet borders on the mef feeder layers ( fig 1a ). similarly, lentiviral vector transduced hes cell colonies, also displayed normal morphology and growth characteristics ( fig 1a ). as expected, the vector transduced colonies displayed green fluorescence due to the presence of the gfp reporter gene. when cultured on irradiated s17 mouse bone marrow stromal cells, both nontransduced and transduced hes cells developed into embryonic cystic bodies ( fig 1a ). facs analysis of single cell suspensions of the cystic bodies showed levels of cd34 cells which ranged from 7 – 15 %. figure 1b displays a representative facs profile of hes - cd34 cells. purified cd34 cells were later cultured in semi - solid methylcellulose medium to derive myeloid colonies. both nontransduced ( denoted as es in figures ) and vector transduced ( denoted as gfp es in figures ) hes cell derived cd34 cells gave rise to normal myelomonocytic colonies similar to human fetal liver derived cd34 cells ( denoted as cd34 in figures ) ( fig 1a ). when pooled colonies were cultured further in liquid cytokine media for 12 – 15 days for differentiation, the cells developed into morphologically distinc t macrophages ( fig 1a ). when compared, the morphology of macrophages derived from all stem cell progenitor populations appeared similar. these results were found to be consistent in replicative experiments. the transgene gfp expression was also maintained during the differentiation of hes cells into mature macrophages. gfp expression in cystic body derived cd34 cells was around 80 % ( data not shown ) with similar levels seen in differentiated macrophages ( fig 2 ). figure 1derivation of macrophages from lentiviral vector transduced and normal hes cells. a ) transduced and non - transduced h1 hes cells were cultured on mouse s17 bone marrow stromal cell layers to derive cystic bodies. cystic body derived cd34 cells were purified by positive selection with antibod y conjugated magnetic beads and placed in methocult media to obtain myelomonocytic colonies. pooled colonies were cultured in liquid cytokine media supplemented with gm - csf and m - csf to promote macrophage growth. for comparison, fetal liver derived cd34 cells were cultured similarly to derive macrophages. representative es cell colonies, cystic bodies, methocult colonies, and derivative macrophages are shown with gfp expressing cells fluorescing green under uv illumination. b ) representative facs profile of hes cell derived cd34 cells stained with pe conjugated antibod ies. percent positive cd34 cells are shown with isotype control shown in the left panel. figure 2phenotypic facs analysis of hes cell derived macrophages. a ) macrophages derived from transduced and nontransduced hes cd34 and fetal liver cd34 cells were stained with antibod ies to cd14, hla - dr, cd4, ccr5, and cxcr4 and the expression of these surface markers was analyzed by facs. b ) isotype controls for pe and pe - cy5 antibod ies. percent positive cells are displayed in the plots for each respective cell surface marker staining. dot plots are representative of triplic ate experiments. hes cell derived macrophages display a normal phenotypic profilemacrophages play a critical role in immune system function and are also major target cells for many viral infections including hiv - 1. distinc t surface phenotypic markers exist on these cells and, thus far, there has been no thorough evaluation of hes cell derived macrophages. therefore we analyzed hes cell derived macrophages for the presence of characteristic cell surface markers and compared these to the phenotypic profile displayed on fetal cd34 cell derived macrophages.