Little is known about the cell-surface molecules that are related to the undifferentiated and pluripotent state of human embryonic stem cells (hESCs). Here, we generated a panel of murine monoclonal antibodies (MAb) against undifferentiated hESCs by a modification of a previously described decoy immunization strategy. H9 hESCs were differentiated in the presence of retinoic acid and used as a decoy immunogen. Twelve Balb/c mice were immunized in the right hind footpads with differentiated H9 cells and in the left hind footpads with undifferentiated H9 cells. After immunization, the left popliteal lymph node cells were collected and were fused with mouse myeloma cells. The fusion resulted in 79 hybridomas secreting MAbs that bound to the undifferentiated H9 cells as shown by flow cytometric analysis. Of these, 70 MAbs bound to the undifferentiated H9 cells, but only weakly or not at all to the differentiated H9 cells. We characterized 37 MAbs (32 IgGs, 5 IgMs) recognizing surface molecules that were down-regulated during embryoid body cell formation. One of the MAbs, L125-C2, was confirmed to immunoprecipitate CD9, previously known as a surface molecule on the undifferentiated hESCs. To investigate the relationship between the MAbs and hESC-specific antibodies, two representative MAbs, viz., L125-C2 and 291-D4, were selected and studied by multi-color flow cytometric analysis. This showed that more than 60% of L125-C2- and 291-D4-positive cells were also positive for the expression of hESC-specific surface molecules such as SSEA3, SSEA4, TRA-1-60, and TRA-1-81, indicating the close relationship between the two MAbs and the hESC-specific surface molecules. Our results suggest that the decoy immunization strategy is an efficient method for isolating a panel of MAbs against undifferentiated hESCs, and that the generated MAbs should be useful for studying the surface molecules on hESCs in the pluripotent and undifferentiated state.

The antifungal activities of the bee venom against Trichophyton mentagrophytes and Trichophyton rubrum were determined by using modified broth dilution assay. The most common dermatophytes, named T. mentagrophytes and T. rubrum, were known to cause a variety of cutaneous infections in humans and animals. The bee venom exhibited prominent antifungal activities against the two dermatophytes tested in this investigation. Moreover, the antifungal activities of the bee venom were much stronger than that of fluconazole, one of the commercial antifungal drugs used in the treatment and prevention of superficial and systemic fungal infections. The result suggests that bee venom could be developed as a natural antifungal drug.

No abstract

No abstract

To study cell surface proteins expressed on human embryonic stem cells (hESCs), we generated a panel of monoclonal antibodies (MAbs) against undifferentiated hESCs by a decoy immunization strategy in a previous study. Two of the MAbs, 63-B6 and 246-D7, bound to human pluripotent stem cells but not to human primary cells such as human peripheral blood mononuclear cells and human lung fibroblasts. They did not bind to either mouse embryonic stem cells or mouse embryonic fibroblasts. The two MAbs had similar binding profiles for many various cancer cells, with few exceptions. Expression of antigens recognized by the two MAbs was rapidly decreased during embryoid body formation of hESCs and gradually increased after initial decrease. The MAbs recognized approximately 130 kDa proteins on the surface of hESCs. Cloning and sequence analysis of antibody genes showed that although the MAbs had exactly the same light chain sequences, they had different heavy chain sequences. Taken together, the results suggest that the two MAbs may recognize two different epitopes of the same or different 130 kDa surface proteins involved in regulating the early differentiation of human pluripotent stem cells and cancer cells.