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. growth, propagation and lentiviral transduction of hes cellsthe nih approved human es h1 cell line was obtained from wicell ( madison, wisconsin ). hes cell colonies were cultured on mouse embryonic fibroblasts ( mef ) ( chemicon, temecula, ca ) in the presence of dmem - f12 ( invitrogen, carlsbad, ca ) supplemented with 20 % knockout serum replacement with 1 mm l-glutamine, 1 % nonessential amino acids, 0. 1 mm β - mercaptoethanol, 0. 5 % penicillin / streptomycin, and 4 ng / ml human basic fibroblast growth factor. culture medium was replaced daily with fresh complete dmem - f12. mature colonies were subcultured weekly by digesting with collagenase iv as previously described [ 5 ]. a vsv - g pseudotyped lentiviral vector ( sinf - ef1a - gfp ) containing a gfp reporter gene ( kindly supplied by r. hawley, george washington university ) was used for hes cell transductions as previously described ( 30, 58 ). generation of the pseudotyped vector in 293t cells and its concentration by ultracentrifugation were described previously [ 30, 48 ]. for vector transduction, the undifferentiated hes cells were prepared into small clumps of 50 – 100 cells with enzyme digestion as done for routine passaging of cells. the cell clumps were incubated with the vector for 2 hrs in the presence of polybrene 6 ug / ml. a secondary cycle of transduction was done by adding fresh vector and incubating for another 2 hrs. the general vector titers were 1 × 107 and the multiplicity of infection was 10. the transduction efficiency was about 50 %. the transduced colonies were cultured on mef like above. derivation and purification of cd34 cells from hes cellsundifferentiated hes cells were cultured on s17 mouse bone marrow stromal cell monolayers to derive cystic bodies containing cd34 + hematopoietic progenitor stem cells. hes cell cultures were treated with collagenase iv ( 1 mg / ml ) for 10 minutes at 37°c and subsequently detached from the plate by gentle scraping of the colonies. the hes cell clusters were then transferred to irradiated ( 35 gy ) s17 cell layers and cultured with rpmi differentiation medium containing 15 % fbs ( hyclone, logan, ut ), 2 mm l-glutamine, 0. 1 mm β - mercaptoethanol, 1 % mem - nonessential amino acids, and 1 % penicillin / streptomycin. media was changed every 2 to 3 days during 14 – 17 days of culture on s17 cells [ 20 ]. after allowing adequate time for differentiation, hes cystic bodies were harvested and processed into a single cell suspension by collagenase iv treatment followed by digestion with trypsin / edta supplemented with 2 % chick serum ( invitrogen, carlsbad, ca ) for 20 minutes at 37°c. cells were washed twice with pbs and filtered through a 70 um cell strainer to obtain a single cell suspension. to assess the levels of cd34 cells in the bulk cell suspension, cells were labeled with pe conjugated anti-cd34 antibod y ( bd biosciences, san jose, ca ) and analyzed by facs. to purify the cd34 cells, direct cd34 progenitor cell isolation kit ( miltenyi biotech, auburn, ca ) was used following the manufacturer ' s protocol. isolated cd34 hematopoietic progenitor stem cells were then analyzed by facs as mentioned above to determine cell purity. for comparative experiments, human cd34 hematopoietic progenitor cells were also purified from fetal liver tissue as described above. derivation of macrophages from hes cell derived and human fetal cd34 cellscd34 cells were cultured initially in semisol id media to derive myelomonocytic colonies followed by liquid culture in cytokine supplemented media as described below. purified cd34 + progenitor cells ( ~ 2. 5 × 105 to 4. 0 × 105 ) were placed directly into methocult semisol id medium ( stem cell technologies, vancouver, bc ), mixed, and cultured in 35 mm plates. myeloid colonies were allowed to develop for 12 – 15 days. upon differentiation and proliferation, myelomonocytic colonies were harvested by the addition of 5 ml dmem containing 10 % fbs, 10 ng / ml each gm - csf and m - csf. cells ( ~ 106 ) were placed in a 35 mm well and allowed to adhere for 48 hours. at two and four days post - harvest, medium was replaced with fresh complete dmem supplemented with 10 ng / ml gm - csf and m - csf. by 4 – 5 days, cells developed into mature macrophages which were used for subsequent phenotypic and functional characterization. phenotypic analysis of hes cell derived macrophagesto determine if nontransduced and lentiviral vector transduced hes cell derived macrophages display normal macrophage surface markers, facs analysis was performed using respective fluorochrome conjugated antibod ies. fetal liver derived cd34 + cells as well as nontransduced and transduced hes cell derived macrophages were evaluated in parallel. cells were scraped from their wells, washed two times with pbs, and stained with the following antibod ies: pe - cd14, pe - hla - dr, pecy5 - cd4, pecy5 - ccr5, pecy5 - cxcr4 ( bd biosciences, san jose, ca ). a blocking step was first performed by incubating the cells with the respective isotype control for 30 minutes at 4c before staining with the respective cell surface marker antibod ies. isotype control staining was used to determine background levels. facs analysis was performed on a beckman - coulter epics ® xl - mcl flow cytometer with data analysis using expo32 adc software ( coulter corporation, miami, fl ). a minimum of 8, 000 cells were analyzed in each facs evaluation. functional analysis of hes cell derived macrophagesphysiological roles of macrophages include phagocytic and immune related functions. to determine if hes cell derived macrophages were functionally normal, a stimulation assay to determine upregulation of the costimulatory molecule b7. 1 was performed. activated macrophages upregulate the expression of b7. 1 upon activation with various stimuli. accordingly, fetal liver cd34, nontransduced hes, and gfp - alone transduced hes cell derived macrophages were stimulated by the addition of lps ( 5 ug / ml ) to the cell culture medium. twenty - four hours post - stimulation, cells were stained with an anti-b7. 1 antibod y labeled with pe - cy5 ( bd biosciences, san jose, ca ) and analyzed by facs. to assess the hes cell derived macrophages ' phagocytic function, 5 ug / ml of fluorescently labeled e. coli bioparticles® ( invitrogen, carlsbad, ca ) were added directly to the cell culture medium. four hours later, macrophages were washed six times with pbs and fresh medium with 10 ng / ml gm - csf and m - csf was added. twenty - four hours later, cells were analyzed by facs for the presence of ingested bioparticles® which can be detected in the pe ( fl2 ) channel. lentiviral vector transduced magi - cxcr4 cells, a hela cell derivative with no phagocytic capacity, were used as non - phagocytic cell controls and similarly exposed to e. coli bioparticles®human es cell derived macrophages were also analyzed for their ability to secrete two major cytokines, il - 1 and tnf - α, upon external stimulation. accordingly, macrophages were stimulated with 5 ug / ml of lps during culture. on days 1, 2, and 3 post - stimulation, cell culture supernatant samples were collected and analyzed by a qu antik ine® elisa kit ( r & d systems, minneapolis, mn ). non - stimulated supernatants were also analyzed for basal levels of cytokine secretion. hiv - 1 infection of hes cell derived macrophagesto determine if hes cell derived macrophages can be infected with hiv - 1 and support viral replication, cells were challenged with a macrophage r5 - tropic bal - 1 strain of hiv - 1.