conjugated antibody. In this study, this system has been adapted for viral entry through cell-surface markers expressed on the hES and iPS cells. The antibody-directed transduction system utilizes a modified Sindbis virus envelope, termed m 168, pseudotyped onto lentiviral particles. The modifications include the replacement of the Targeted Gene Delivery to Human ES and iPS Cells cell staining. Here we describe a robust technique for delivering reporter genes into human iPS cells through the Abdirected targeted transduction system during reprogramming of somatic fibroblast cells to the pluripotent state. The successfully reprogrammed iPS cells can be specifically infected by the targeting Ab, marked by enhanced green fluorescent protein, and enriched under puromycin selection. This provides a relatively easy tool for monitoring and identifying potential iPS cells, as well as hES cells within a mixed heterogeneous population. Results Optimization of gene transduction using VSV-G pseudotyped lentiviral vectors on the H9 human ES cell line Poor viral transgene expression in hES cells is a well-known phenomenon. Conditions were optimized to increase viral infection and expression in the undifferentiated and differentiated hES cells. Maximal viral 
transduction was obtained when hES cells were dispersed into single cells with Accutase ” followed by the addition of the ROCK inhibitor Y-27632 to protect cells from apoptosis and increases colony formation. Variation in the lentiviral vector backbone can also contribute to efficiency of gene transfer and cell expression profiles. Two vectors were compared for expression of eGFP: pHR’CMVGFPW expressed GFP from ” an internal cytomegalovirus immediate-early promoter and pSin-EF2-GFP-Puro expressed GFP from the buy c-Met inhibitor 2 elongation factor-1 a promoter. Virus bearing both vectors delivered and expressed high levels of GFP into 293T cells. In our experimental system, a vector was desired which 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. 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 recogniz
