in colony forming assays using semisol id methylcellulose medium, the morphology of myelomonocytic colonies derived from hes cd34 cells appeared similar to that of fetal liver cd34 cells. when subsequently cultured in cytokine media that promotes macrophage differentiation, morphologically normal macrophages were obtained with hes - cd34 cells similar to that of fetal liver cd34 cells. at higher magnification, the macrophages displayed flat projecting cellular borders with fried egg appearance with distinc t refractory lyso somal granules in the cytoplasm ( data not shown ). lentiviral vector transduced hes cells also did not display any abnormal growth or differentiation characteristics as compared to nontransduced hes - cd34 cells indicating no adverse effects due to vector integration and expression. transduced cells gave rise to cystic bodies with similar cd34 cell content and profiles upon development. the transduced hes - cd34 cells also gave rise to apparently normal macrophages that expressed the transgene as shown by gfp expression. these results are consistent with those of others that showed normal differentiation of hes cells to other cell types following lentiviral transduction [ 32 ]. a requirement for successful cellular and hiv - 1 gene therapy is that mature end stage cells derived from cd34 progenitor cells be phenotypically and functionally normal to maintain and restore the body ' s immunological function. accordingly, hes cell derived macrophages were evaluated to determine if they met these criteria. macrophages display distinc t cell surface markers upon end stage differentiation. to determine whether hes cell derived macrophages display these surface markers, facs analysis was performed to detect the presence of cd14, hla - dr ( mhcii ), cd4, ccr5, and cxcr4. as observed in fig 2a, both nontransduced and transduced hes cell derived macrophages expressed all of these markers with some differences in their levels of expression. hla - dr, cd4, and ccr5 expression profiles were comparable between all cell types analyzed. even though all cell types analyzed stained positive for cd14, relative expression of cd14 was slightly lower on hes cell derived macrophages compared to fetal liver cd34 cell derived macrophages. on the contrary, the levels of cxcr4, a chemok ine receptor involved in cellular homing, were found to be higher on hes - cd34 cell derived macrophages. this may be due to inherent differences in the cell types and / or due to their physiological state at the time of harvest [ 35 ]. additional hes cell lines need to be evaluated in the future to establish if these differences are consistent. a major functional role of macrophages in vivo is their ability to serve as professional antigen presenting cells. during this process macrophages present antigen peptide fragments complexed with both classes of mhc molecules and deliver a costimulatory signal through the expression of b7 molecules. upon stimulation with lps, hes - cd34 cell derived macrophages had shown upregulation of the costimulatory molecule b7. 1 similar to cells derived from fetal liver. furthermore, the hes - cd34 cell derived macrophages also showed a normal capacity to ingest foreign particles in phagocytosis assays using e. coli bioparticles®. in addition to antigen presentation and phagocytosis, macrophages also play a critical role in inflammation and secrete cytokines in response to external stimuli. when exposed to lps, the hes - cd34 cell derived macrophages secreted two important cytokines il - 1 and tnf - α similar to that of fetal liver derived cells. the above data has established that phenotypically and functionally normal macrophages could be derived from hes - cd34 cells. macrophages in addition to playing important physiological roles are also major cell targets for certain viral infections, particularly hiv - 1. here we evaluated the susceptibility of hes - cd34 cell derived macrophages to be productively infected with hiv - 1. similar to that of fetal liver cd34 cell derived cells, the hes - cd34 macrophages also supported hiv - 1 infection although the levels of viral yield differed somewhat. however this should not be a major concern for testing anti-hiv genes in these cells. in all the above experiments, the vector transduced transgenic macrophages also behaved similarly to that of nontransduced cells showing that they were also physiologically normal. the lack of vector toxicity on cellular maturation is encouraging for future work with transduced hes - cd34 cells to derive other important differentiated cells like t cells and dendritic cells relevant for hiv studies. although there are numerous studies on hes cell differentiation into many important end stage mature cells, systematic work on hes cell hematopoietic differentiation and thorough characterization of end stage mature cells that participate in critical immune responses has just begun [ 21, 27 - 29 ]. our current results established that physiologically normal macrophages could be derived from hes cells and that these cells have the potential for use in cellular and gene therapies. to our knowledge this is the first demonstration that hes cell derivatives can be used for infectious disease research. due to the extensive ability for hes cells to self - renew, large numbers of differentiated cells can be derived so that infection studies and evaluation tests can be carried out in a more standar diz ed way. our results showing that both normal and transgenic derivative macrophages support hiv - 1 infection points out to their utility for testing anti-hiv constructs transduced into hes - cd34 cells and pave the way for their application in stem cell based hiv gene therapy. so far a number of studies including our own have tested many gene therapeutic constructs in cd34 cells from conventional sources. these constructs include anti-hiv ribozymes, rna decoys, transdominant proteins, bacterial toxins, anti-sense nucleic acids, and most recently sirnas [ 36 - 50 ]. in addition, a number of cellular molecules that aid in hiv - 1 infection such as cellular receptors and coreceptors cd4, ccr5 and cxcr4 have also been successfully tested in cd34 cell derived macrophages and t cells [ 16, 18, 38 ]. some of these approaches have progressed into clinical evaluations as well [ 14, 51, 52 ]. based on our current results, many of these novel anti-hiv constructs can also be tested in hes - cd34 cells for their potential application. although there are advantages of using hes cell derived cd34 cells for potential cellular therapies, transplantation of these cells constitutes an allogenic source with immune rejection as a major issue. however, a recent study using human leukocyte reconstituted mice suggested that hescs and their derivative cell types were less prone to invoking an allogeneic response [ 53 ]. other recent studies demonstrated successful engraftment of primary and secondary recipients with hes cell derived hematopoietic cells in both immunode ficient mice and in vivo fetal sheep models adding further support that any obstacles could be overcome [ 23, 54, 55 ]. moreover, multiple novel strategies to avoid immune-mediated rejection of hes cell - derived cells have been proposed [ 56, 57 ]. it is not too far in the future that even autologous hes cells may be derived from specific individuals for deriving cd34 cells which can be used for cell replacement therapy. phenotypically 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.