In the present study, we have described a method to enrich phage pools that have specific binding efficiency and selectivity for ES cells. In order to eliminate the possibility of selecting non-relevant phages, we employed two steps of subtraction using dES cells and PMEFs before each selection step. The result showed that the subtraction is rather effective to exclude non-specific binding phages. In addition, the method that we chose must pass through a procedure of detaching cells from culture flasks by dispase or trypsin, which is thought to possibly change the composition and configuration of cell surface proteins 
. To avoid the change of cell surface proteins, we shortened the digestion time as far as possible. We also tried the method using attached cells, however, they were apt to detach during the incubation and the concentration was much lower than that of suspended cells.
After the enrichment step, we picked several phages and sequenced their peptide display gene region. We found an APWHLSSQYSRT peptide sequence was the most accumulated during biopannings and several peptides contain a PWX (L/V) motif. The binding efficiency and specificity of the phage to ES cells was tested by whole-cell ELISA and the result showed that the APW phage served as a better candidate than other phages, which was further verified by the immunofluorescent analysis. We observed a phenomenon that these phages displayed a common motif but showed different binding ability further suggests that the motif is important for the binding while this binding ability is coordinated by flanking sequences. Furthermore, the result that the chemically synthesized APWHLSSQYSRT peptide could inhibit binding of the phage to ES cells indicated that the specific binding ability of APW phages was due to the displayed peptide and independent of the phage itself. A BLAST search of the sequence in the GeneBank showed several existing and/or predicted homologous sequences (Table S1
). In these sequences, we find that several of them are of special interest. For example, human Chorionic Gonadotropin (hCG) that shares the homology with the APWHLSSQYSRT sequence is found to be important for development and leishmanolysin-like protein is reported to have the different expression pattern among different embryonic stem cell lines 
. These findings suggest the APWHLSSQYSRT peptide might simulate protein-protein interaction.
To further characterize the phage and the displayed peptide, we carried out western blotting. The result showed that the phage bound to ES cells by protein-protein interaction, which is consistent with the earlier report that peptides isolated from phage display often bind to the sites by protein-protein interaction 
. This finding also increases the possibility that the APW peptide has the ability to target cell surface proteins, which is possibly a membrane protein with a relatively low molecular weight (about 17 kDa). Further in silico
studies of the peptide structure showed that the peptide contained a hydrophobic site and had a hydrophilic environment on the other side, which suggests that the peptide binds to ES cells via the non-bonding forces. Since generally proteins contain the core hydrophobic residues which are surrounded by a shell of hydrophilic residues, it is possible that the PWX (L/V) motif is important for the maintenance of tertiary structure of the peptide 
. Combined with our previous experiments and the in silico
studies, it is possible that the PWX (L/V) motif serves as an important core motif that binds to the ES cell membrane proteins. Additionally, the in silico
studies of the peptide may open a perspective for improvement of its binding affinity and specificity by redesigning of the peptide. After conjugation with the peptide, the quantum dot showed a much higher binding ability to ES cells compared to the naked quantum dots, suggesting that adding the peptide can increase quantum dot binding affinity and specificity towards ES cells. Moreover, the fact that the peptide-conjugated quantum dots were unable to target mouse ES cell line further suggests their specificity towards a particular cell line.
In general, there are two major concerns in nanoparticles cell-targeting studies: specificity and toxicity. To increase the specificity of nanoparticles to cells, there could be targeting materials designed to link with the nanoparticles. Phage display is a useful tool for searching for the targeting materials. Our group and several other groups have successfully found peptides or antibodies that can specifically recognize a variety of stem cells using phage display 
. However, it is still an open question that how specific these peptides or antibodies are. Although in our study, the selected peptide was unable to bind to the stem cell line of the other species, further proves its specificity, we still observed that several peptides with “homologous core sequence”, namely the PWX (L/V) motif, were able to bind to differentiated ES cells, albeit with low efficiency. Although this might be due to the incomplete differentiation of ES cells or the ES cells slightly differentiated during the selection step, so that some types of differentiated cells can not be distinguished from ES cells, it also possible that the recognition specificity to a particular cell is a relative value. Based on the result of our study, this value is possibly influenced by the flanking sequences of the core motif. By engineering the flanking sequences of the peptide, it is quite possible to increase the specificity of the peptide recognition and the in silico
study might provide a possible direction for the engineering. Another concern of the nanoparticle cell-targeting study is the toxicity. Previous studies have shown that coating is the most important factor for cytotoxicity of the quantum dots 
. Another study has shown that the toxicity and metabolism of the quantum dots are different in different type of cells 
. These studies suggest that the cytotoxicity of quantum dots can be minimized by proper coating technique and selection of correct type of quantum dot material according to the cell type of interest. However, the toxicity of the peptide-quantum dot conjugates remains to be investigated. Although previous studies have shown that targeting of CdSe-ZnS to other type of cells does not change their cellular morphology and physiology 
, the physiological effect of the peptide-quantum dot conjugates on ES cells is unknown.
In summary, we have established a method of screening for ES cell binding peptides by phage display and identified a novel peptide that specifically binds to ES cells. This peptide holds the promise to be a targeting or imaging agent for ES cells and might serve as a novel research tool for studying the ES cell surface as well. However, further studies are needed to understand the biology of the APWHLSSQYSRT peptide. Work is continuing to further assess its specificity using more cell types, to test whether this binding ability is the same in vivo as in vitro, to measure the toxicity of the peptide-quantum dot conjugates on ES cells and determine whether the peptide has a physiological effect on ES cells.