h efficiently expressed GFP in both undifferentiated H9 stem cells as well as BMP4 induced trophoblasts. Fig. 1 compares the eGFP expression from pSin-EF2-GFP-Puro and the pHR’CMVGFPW by flow cytometric analysis and fluorescence microscopy after gene transduction by lentiviral particles pseudotyped with the non-selective VSV-G Env during BMP4 driven trophoblast differentiation of hES H9 cells. The 
pSin-EF2GFP-Puro provided maximal GFP expression in both the undifferentiated cells as well as the day 10 differentiated cells with greater than 95% of the cells expressing GFP. In contrast, the pHR’CMVGFPW was silenced in the undifferentiated H9 stem cells but active in the differentiated trophoblast cells. Identical results were observed using fluorescence microscopy as with flow cytometry. Constructs expressing eGFP from the pSinEF2-GFP-Puro based vector were used in subsequent studies. Scopoletin Specific gene delivery to hES cells via antibodyconjugated m 168 pseudotyped lentiviral vectors A key bottleneck in many stem cell applications is the ability to identify, select or counterselect for the stem cells within the mixed population. Specific gene delivery has been achieved using antibody-conjugated systems, in particular lentiviral particles pseudotyped with a modified Sindbis Env, encoding a protein A immunoglobulin G recognition domain . In order to investigate whether the m 168 pseudotyped lentiviral vectors were able to deliver the eGFP gene into the hES cell via a specific monoclonal antibody, we tested a panel of antibodies recognizing hES cell surface marker proteins, including SSEA4, CD24, SSEA3, FZD7, and CD9 . Cell surface expression of all the markers were readily detected on the H9 cells by flow cytometry, SSEA4, CD24, FZD7, CD9, and HLA-1 ). Transduction efficiency was determined by measuring eGFP gene transfer into hES H9 cells. The results indicate that anti-SSEA4, antiCD24, and anti-CD9 antibodies conjugated with lentiviral particles pseudotyped with m 168 were able to transduce hES cells. As a control, eGFP delivery in VSV-G-pseudotyped lentivirus was at a level of 93%. Control infection using an IgG k2 isotype antibody resulted in transduction levels equivalent to the no antibody controls, indicating the absence of background from nonspecific transduction of igG antibodies. Surprisingly, no transduction was observed using the FZD7 IgG antibody, despite being expressed on the 24517231” surface of H9 cells, indicating that not every cell surface protein can serve as an effective receptor for the antibody-mediated transduction. Binding to a receptor is the first step of viral entry leading 8396143 to a complex series of conformational changes required for membrane fusion and viral content release into the cellular cytoplasm, either at the cell surface or during transport through the cellular endosomal pathways. Variations in the endocytosis and recycling of the cell surface receptors therefore can greatly influence the efficiency of the targeted transduction. Transduction using lentiviral particles conjugated with HLA-1 was 47% eGFP. Antibody binding to the ZZ domain is limited predominantly to IgG molecules. Three of the most frequent used antibodies to identify human embryonic stem cells, anti-SSEA3, TRA-1-60 and TRA-1-81, though are IgM molecules and are predicted not to associate with the ZZ domain. Experimentally, the SSEA3 IgM antibody was not effective in targeting entry, yielding eGFP transduction equivalent to the no antibody control. A stra
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