An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

Abstract

A significant risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation from residual undifferentiated cells. We have raised a monoclonal antibody (mAb) against hESCs, designated SSEA-5, which binds a novel antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation of SSEA-5 high cells through fluorescence-activated cell sorting (FACS) drastically reduced teratoma formation potential. To ensure complete removal we identified additional markers exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90, and CD200. Immunohistochemistry (IHC) conducted on human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. When applied to incompletely differentiated hESC cultures, immunodepletion with SSEA-5 and 2 additional markers completely removed teratoma formation potential.

Preclinical Studies for Induced Pluripotent Stem Cell-based Therapeutics

“…Extensive epigenetic modifications occurring during reprogramming and differentiation may make iPSCs more prone to causing cancer following transplantation. Development of highly sensitive methods for detection and efficient separation of undifferentiated cells will be needed (86,87). Among new methods potentially limiting the tumorigenicity of iPSCs are increasing the copy number of tumor suppressors (88) and the use of specific drugs such as metformin (89) and pluripotent cell-specific inhibitors (90).…”

Emerging innovation towards safety in the clinical application of ESCs and iPSCs

“…10 Finally, an antibody against stage-specific embryonic antigen-5 (a newly identified PSC-specific surface antigen) can be used to remove undifferentiated cells by fluorescence-activated cell sorting. 11 However, because this method depends on cell sorting, which includes ex vivo manipulation (such as single-cell dissociation and cell-staining techniques), cells might lose viability. New synthesized small molecules (such as JC011), which selectively induce a cytotoxic endoplasmic reticulum stress response in ESCs and iPSCs, have also been reported, but further studies should reveal the precise mechanisms of this pathway.…”

“…Vazquez-Martin et al 9 Chemical inhibitor AMP-activated protein kinase activation Metformin Tang et al 11 Antibody SSEA-5 purging Anti-SSEA-5 monoclonal antibody…”

The Cellular Glycome of Human Induced Pluripotent Stem Cells and Their Specific Probe rBC2LCN

“…TRA-1-60 and TRA-1-81 were first reported to be keratan sulfate (6), but were recently identified by glycan microarray analysis to be type-1 Nacetyl-lactosamine linked to type-2 N-acetyl-lactosamine (Gal β1 3GlcNAcβ1 3Galβ1 4GlcNAc) (7). More recently, SSEA-5 was reported as a novel hiPSC marker, whose structure was determined as an H type-1 glycan (Fucα1 2Galβ1 3GlcNAc) (8). These glyco-markers were identified following the fortuitous development of their specific antibodies, given that most carbohydrate structures are poorly antigenic between mammals.…”