title: Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis.
abstract: Antibody-mediated cell killing has significantly facilitated the elimination of undesired cells in therapeutic applications. Besides the well-known Fc-dependent mechanisms, pathways of antibody-induced apoptosis were also extensively studied. However, with fewer studies reporting the ability of antibodies to evoke an alternative form of programmed cell death, oncosis, the molecular mechanism of antibody-mediated oncosis remains underinvestigated. In this study, a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC) so as to prevent teratoma formation upon transplantation of hESC-derived products. We revealed that A1 induces hESC death via oncosis. Aided with high-resolution scanning electron microscopy (SEM), we uncovered nanoscale morphological changes in A1-induced hESC oncosis, as well as A1 distribution on hESC surface. A1 induces hESC oncosis via binding-initiated signaling cascade, most likely by ligating receptors on surface microvilli. The ability to evoke excess reactive oxygen species (ROS) production via the Nox2 isoform of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical in the cell death pathway. Excess ROS production occurs downstream of microvilli degradation and homotypic adhesion, but upstream of actin reorganization, plasma membrane damage and mitochondrial membrane permeabilization. To our knowledge, this is the first mechanistic model of mAb-induced oncosis on hESC revealing a previously unrecognized role for NAPDH oxidase-derived ROS in mediating oncotic hESC death. These findings in the cell death pathway may potentially be exploited to improve the efficiency of A1 in eliminating undifferentiated hESC and to provide insights into the study of other mAb-induced cell death.
authors.affiliation: Mechanobiology Institute (MBI), National University of Singapore (NUS), T-Lab, No. 10-01, 5A Engineering Drive 1, Singapore 117411, Singapore.
authors.forename: Ji Yun
authors.initials: JY
authors.lastname: Zheng
authors.affiliation: Stem Cell 1 Group, Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, Singapore 138668, Singapore.
authors.forename: Heng Liang
authors.initials: HL
authors.lastname: Tan
authors.affiliation: Mechanobiology Institute (MBI), National University of Singapore (NUS), T-Lab, No. 10-01, 5A Engineering Drive 1, Singapore 117411, Singapore.|Department of Biological Science, Faculty of Science, National University of Singapore (NUS), 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Paul Thomas
authors.initials: PT
authors.lastname: Matsudaira
authors.affiliation: Stem Cell 1 Group, Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, Singapore 138668, Singapore.|Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore (NUS), 9 Engineering Drive 1, Singapore 117575, Singapore.
authors.forename: Andre
authors.initials: A
authors.lastname: Choo
mesh_terms: D000199:Actins / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology*; D017209:Apoptosis; D002240:Carbohydrate Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D000066449:Human Embryonic Stem Cells / Q000276:immunology / Q000648:ultrastructure; D006801:Humans; D008855:Microscopy, Electron, Scanning; D051336:Mitochondrial Membranes / Q000378:metabolism; D000074662:NADPH Oxidase 2 / Q000378:metabolism; D010539:Permeability; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D000939:Epitopes; D017382:Reactive Oxygen Species; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2