Production of a Recombinant Antibody Specific for i Blood Group Antigen, a Mesenchymal Stem Cell Marker

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.

Umbilical Cord Blood-Derived Mononuclear Cells Exhibit Pericyte-Like Phenotype and Support Network Formation of Endothelial Progenitor Cells In Vitro

“…Inhibitory factors secreted by SS-MNCs to prevent further EPC network formation is consistent with the physiological function of pericytes to stabilize nascent microvessel structures. 1,2 The Matrigel™ matrix is also limited in its ability to offer thorough analysis of SS-MNC pericyte function due to the lack of EC network stability we observed after 24 h. The immature SS-MNCs may require additional time to differentiate towards a pro-angiogenic mural cell type, which we observed by 2 weeks of coculture with EPCs based on the support of EPC network formation (Fig. 6).…”

“…Recently, groups have reported methods to improve the purity of MSCs by using surface antigens. 4,20 While this approach can yield pure populations of MSCs, the time and cell doubling rate required to reach therapeutically-relevant cell numbers can lead to senescent cells that no longer possess angiogenic characteristics of MSCs observed at earlier passages. 13,24,44 In this work, we found a highly proliferative population of MNCs from cord blood morphologically resembling pericytes was possible.…”

“…Another possible explanation for the delayed onset of EPC network formation found in SS-MNC cocultures in comparison to SMC cocultures could be due to differences in Angiopoietin-1 and Tie-2 signaling, critical for the regulation of pericyte and EC interactions. 2 There may also exist differences in the extracellular matrix composition between SMC and SS-MNC cocultures that could affect subsequent EPC network formation. For example, collagen I can induce EC capillary morphogenesis whereas laminin-1 can suppress EC activation and subsequent microvessel formation.…”

Glycan Markers of Human Stem Cells Assigned with Beam Search Arrays*[S]

“…In the light of the reported expression of i antigen on human mesenchymal stem cells (55), it was opportune to investigate the effect of sulfation on its expression. Earlier studies from our group (36) showed that i antigen expression on the unbranched T2-T2 sequence is masked on KS chains that are fully 6-sulfated on Gal and GlcNAc residues.…”

“…Here we use an array of sequence-defined glycans to make for the first time a close comparison of the glycan-binding specificities of the five antibodies that recognize human stem cells: mAbs TRA-1–60, TRA-1–81 and FC10.2 that broadly recognize ES, iPS and EC cells, an anti-i P1A ELL directed at linear poly- N -acetyllactosamine sequences (29), which according to ref (30) is predicted to recognize mesenchymal stem cells, and R-10G that recognizes ES and iPS cells. To assign the determinant of mAb R-10G, which is clearly different from those of the four other antibodies, we use the Beam Search microarray approach (28) using an antigen-positive KS polysaccharide as the macromolecular start-point.…”