Prospective Isolation of Mesenchymal Stem Cells from Human Bone Marrow Using Novel Antibodies Directed Against Sushi Domain Containing 2

Abstract

Several strategies have been developed to facilitate the prospective isolation of bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) based on the selective expression or absence of surface markers. Recently, we described the monoclonal antibodies W3D5 and W5C5, which selectively react with BM-MSCs, but not with hematopoietic cells. Both antibodies showed an identical reactivity pattern, indicating that they may recognize the same molecule. To identify the cognate antigen, cultured MSCs were sorted for cells expressing either very high levels of W5C5/W3D5 antigen or for cells which were negative for this antigen. Further processing of these cells for microarray analysis revealed a 20-fold enrichment of the type 1 integral membrane protein Sushi domain containing 2 (SUSD2) in the in W5C5(+) subset. To confirm the identity of the W5C5/W3D5 antigen to SUSD2, HEK293 cells were transfected with the full-length coding sequence of human SUSD2 followed by reactivity analysis of W5C5 and W3D5 antibodies with the transfected line. Flow cytometric analysis showed that both antibodies selectively recognized HEK293/huSUSD2 cells, but not the parental cell line. In line with this, SUSD2 siRNA treatment of SUSD2(+) WERI-RB-1 retinoblastoma cells reduced the expression levels of W3D5 and W5C5 antigens to ~39% and 37%, respectively. Finally, FACSorting and colony assays revealed that only SUSD2(+), but not SUSD2(-) BM cells give rise to colony-forming units-fibroblasts and are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In conclusion, we identified SUSD2 as a novel and specific marker for the prospective isolation of BM-MSCs.

Isolation and Localization of Mesenchymal Stem Cells in Human Palatine Tonsil by W5C5 (SUSD2)

“…W5C5 could be used as a novel marker for the prospective isolation of endometrial MSCs from biopsy, curettage, or hysterectomy tissue. In addition, W5C5 expressed with high selectivity on human bone marrow-derived MSCs [3]. In the present study, W5C5 was identified as a novel single marker for purifying T-MSCs, which self-renew and differentiate into adipogenic, osteogenic, and chondrogenic cell lineages.…”

“…W5C5, a known epitope of the sushi domain containing two (SUSD2) molecules, was recently identified as the transmembrane protein of 820 amino acids consisting of a large extracellular region containing a Somatomedin B, an adhesion-associated domain in MUC4 and other proteins, a von Willebrand factor, and a Sushi domain [3]. Sushi domains are components of a variety of complement and adhesion proteins.…”

“…Mesenchymal stem cells (MSCs) are multipotent cells that are able to self-renew and differentiate into several cell types of the same mesenchymal lineage, including osteocytes, adipocytes, and chondrocytes [1][2][3]. MSCs are ideal candidates for cell-based therapy and regenerative medicine because of their self-renewal abilities and their multipotent and paracrine effects [4,5].…”

“…W5C5 has been introduced as a novel marker for the prospective isolation of highly clonogenic endometrial MSCs that were able to reconstitute endometrial stromal tissues in vivo [3,9,17]. However, there have been no reports on the expression and functional differences of W5C5 in MSCs derived from tonsil tissues.…”

Low Osteogenic Differentiation Potential of Placenta-Derived Mesenchymal Stromal Cells Correlates with Low Expression of the Transcription Factors Runx2 and Twist2

“…In addition to these antigens, expression of the stage-specific embryonic antigen 4 (SSEA-4) or fibroblast activation protein-a (FAPA) were suggested as marker antigens for MSC [7,8]. However, other antigens such as CD146 (alias MCAM, MUC18), the sushi-containing domain 2 protein (susd2, alias W5C5 antigen) [9], or the molecule detectable by monoclonal antibody clone W12D1 (protein unknown) generated distinct histogram patterns, indicating that MSC prepared by conventional techniques present a complex mixture of cells [10,11].…”