Selection Against Undifferentiated Human Embryonic Stem Cells by a Cytotoxic Antibody Recognizing Podocalyxin-Like Protein-1

Abstract

Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complementindependent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications.

Lonely killers

“…84 mAb was also cytotoxic at 4°C. 51 Interestingly, in the case of 216 mAb cytotoxicity levels increased at this temperature when compared with incubation at 37°C. 44 Additionally, neither the presence of sodium azide nor of EDTA dampened the killing activity of RE2, 70 216 44 and 14F7 66 mAbs, which indicates independence from metabolic energy and calcium influx, respectively.…”

“…Notably, podocalyxin binds to actin through ezrin, 72 and the antigen recognized by 84 mAb is a podocalyxin-like protein. 51 In the case of cells treated with 84 mAb 52 and KID3/RAV12, 48 a disruption of the actin cytoskeleton was observed.…”

“…50 An IgM mAb that recognizes human embryonic stem cells, specifically through the podocalyxin-like protein-1, was found to be directly cytotoxic to these cells. 51 The 84 mAb induced membrane pores in treated cells. 52 Another well characterized antibody exhibiting this effect on plasma membrane is 14F7 mAb, 53 which targets the tumor-associated GM3(Neu5Gc).…”

“…Interestingly, using a pan-caspase inhibitor, it was observed that AD5-10 also induced non-apoptotic cell death, as shown by the absence of the typical DNA fragmentation and the simultaneous demonstration shared by these mAbs is the induction of rapid cell death, often within 5-20 min of incubation. [42][43][44]46,51 Cytoskeleton reorganization in treated cells has been demonstrated for RE2 43,70 and 14F7 66 mAbs. Addition of the actin polymerization inhibitor cytochalasin impaired the cytotoxicity induced by these mAbs.…”

Surface markers of hepatocellular cancer stem cells and their clinical potential

“…Furthermore, in epithelial cell types that lack EpCAM in adults, upregulation of EpCAM coincides with the early stages of neoplastic change [75]. EpCAM was identified in 2008 as a marker for human embryonic stem cells in an antibody-based screening assay [76]. In 2010, Terris et al [75] revealed that the EpCAM (+) AFP (+) HCC subtype had features of hepatic stem/progenitor cells with gene expression and pathway analyses.…”