the surface markers analyzed were cd14, a monocyte / macrophage specific marker, hla - dr ( a class ii mhc molecule found on antigen presenting cells ), cd4, the major receptor for hiv - 1 infection, and ccr5 and cxcr4, chemok ine receptors which are critical coreceptors essential for hiv - 1 entry. egfp expression was also analyzed to determine the levels of transduction and any transgene silencing that may occur during differentiation. fetal liver ( cd34 ), nontransduced ( es ), and vector transduced ( gfp es ) hes cell derived macrophages were all positive for the monocyte / macrophage marker cd14 ( 99. 3 %, 88. 7 %, and 99. 2 %, respectively ) ( fig 2a ). however, the mean fluorescent intensity ( mfi ) was found to be lower on hes cell derived macrophages. surface expression of hla - dr was observed at similar levels between macrophages derived from fetal liver cd34 cells ( 99. 6 % ), nontransduced hes cells ( 92. 8 % ), and transduced hes cells ( 98. 2 % ) ( fig 2a ). cd4 levels were comparable for all stem cell derived macrophages ( 99. 2 %, 83. 3 %, and 88. 7 %, respectively ) ( fig 2a ). ccr5 and cxcr4 cell surface expression was also observed for fetal liver cd34 cell ( 99. 6 % and 99. 3 % ), nontransduced hes cell ( 91. 9 % and 92. 6 % ), and transduced hes cell ( 98. 9 % and 99. 3 % ) derived macrophages ( fig 2a ). as compared to fetal liver cd34 cell derived macrophages, hes cell derived macrophages displayed a higher level of expression of cxcr4. isotype controls for both pe and pecy5 stains are shown in fig 2b. the above phenotypic data are representative of triplic ate experiments. transgenic hes cell derived macrophages are functionally normalthe antigen presenting cell surface specific marker hla - dr ( mhc ii ) on normal macrophages is critical for presenting antigen to cd4 t cells. a second co - stimulatory molecule, b7. 1 is present at low basal levels on resting macrophages and is necessary to activate t cells. its expression is elevated upon activation with certain stimuli such as lps. our results of lps stimulation of respective macrophages have shown upregulation of b7. 1 with values for fetal liver cd34 cell ( cd34 ) ( 27. 9 % to 75. 4 % ) nontransduced ( es ) ( 17. 8 % to 49. 4 % ) and transduced ( gfp es ) ( 35. 6 % to 65. 7 % ) hes cell derived macrophages ( fig 3a ). these values represent a significant upregulation of b7. 1 for all three macrophage populations. figure 3functional analysis of hes cell derived macrophages for b7. 1 costimulatory molecule upregulation and phagocytosis of e. coli particles: a ) mature macrophages were stimulated with lps to determine b7. 1 upregulation. twenty - four hours post - stimulation, macrophages were labeled with a pe - cy5 conjugated anti-b7. 1 antibod y and analyzed by facs. b7. 1 upregulation data are representative of triplic ate experiments. isotype control is shown in the left panel. b ) to assess phagocytic function, e. coli bioparticles® were added directly to the cultured macrophages. twenty four hours post - addition, cells were analyzed by facs. percent positive cells are displayed in the plots for each experiment. these data are representative of triplic ate experiments. another important function of macrophages is their ability to phagocytose foreign material and present antigenic peptides on their cell surface. to evaluate phagocytic function, fluorescently labeled e. coli bioparticles® were added to macrophage cultures followed by facs analysis. nontransduced ( 94. 6 % ) as well as lentiviral vector transduced ( 98. 7 % ) hes cell derived macrophages were found to be capable of phagocytosing the bioparticles® in comparison to fetal liver cd34 cell derived macrophages ( 95. 8 % ) ( fig 3b ). these values are representative of triplic ate experiments. magi - cxcr4 cells with no phagocytic capacity were used as non - phagocytic cell controls and similarly exposed to e. coli bioparticles® ( fig 3b ). no uptake of the bacteria could be seen. thus, uptake of e. coli bioparticles® by macrophages is indicative of active ingestion. macrophages, as effector cells, play a key role in the inflammatory response. activated macrophages secrete various cytokines, two of the major ones being il - 1 and tnf - α. to determine if hes cell derived macrophages have such a capacity, cells were stimulated with lps. on days 1, 2, and 3 post - stimulation, culture supernatants were analyzed by elisa to detect il - 1 and tnf - α. as seen in figure 4a, there were no significant differences in il - 1 secretion between the three sets of macrophages. similarly, nontransduced and transduced hes cell derived macrophages were also capable of tnf - α secretion upon lps stimulation. however, levels of the respective cytokines detected were slightly lower than those from fetal liver cd34 cell derived macrophages ( fig 4b ). the values of cytokine secretion levels represent triplic ate experiments. figure 4cytokine il - 1 and tnfα secretion by stimulated hes cell derived macrophages: macrophages derived from transduced and nontransduced hes and fetal liver cd34 cells were stimulated with 5 μg / ml lps. on days 1, 2, and 3 post - stimulation, supernatants were collected and assayed by elisa for ( a ) il - 1 and ( b ) tnfα. experiments were done in triplic ate. hes cell derived macrophages support productive hiv - 1 infectionthe above data have shown that hes cell derived macrophages are very similar to normal human macrophages based on phenotypic and functional analysis. in addition to being important cells of the immune system, macrophages are among the major target cells for certain viral infections, particularly for hiv - 1. we wanted to determine if hes cell derived macrophages were susceptible to hiv - 1 infection compared to standard macrophages. in these studies, we only used an r5 - tropic strain of hiv - 1 since macrophages are natural targets for this virus. our results from challenge studies of these cells clearly indicated the capacity of hes cell derived macrophages in supporting a productive infection. levels of virus increased up to 15 days similar to non - hes derived macrophages showing that the initial viral input was amplified in productive viral infection. however, the levels of viral yield were found to be slightly lower for the es cell derived macrophages. in the case of gfp - es macrophages, there was a decline in viral titer. this could be due to possible lower numbers of cells present in the initial cultures. as a first step towards the use of hes cells for hematopoietic stem cell and hiv gene therapies, we have shown here that phenotypically and functionally normal macrophages could be derived from hes - cd34 cells. both non transduced and lentiviral vector transduced hes cells were found to be capable of generating cd34 cells that give rise to macrophages which could support productive hiv - 1 infection. current sources of cd34 cells consist of human bone marrow, cytokine mobilized peripheral blood, fetal liver, and cord blood [ 34 ]. however, the number of cells that can be obtained for manipulations is not unlimited. therefore, deriving cd34 cells for therapeutic and investigative purposes from hes cells with unlimited growth potential has the advantage of a consistent and uniform source. the ability to obtain phenotypically normal and functionally competent macrophages from hes cells is important to evaluate their potential therapeutic utilities in the future. additionally, testing of transgenic hes cells derived via lentiviral vector gene transduction is also helpful to determine the stability of the transgene expression and their capacity for differentiation into end stage mature cells such as macrophages. based on these considerations, both non - transduced and lentiviral vector transduced hes cells were evaluated for their capacity to give rise to cd34 progenitor cells.