ABSTRACT
Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen-positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology.
ABSTRACT
Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus.
