Selective removal of undifferentiated human embryonic stem cells using magnetic activated cell sorting followed by a cytotoxic antibody.

One of the most pertinent concerns of using differentiated cells derived from human embryonic stem cells (hESC) is the presence of residual undifferentiated hESC, because they carry a risk of teratoma formation. A new cell-cell separation approach that eliminates teratoma-forming hESC in order to ensure safer cell therapy was developed. By combining antibodies (IgMs or IgGs) for the selective removal of undifferentiated hESC using magnetic activated cell sorting (MACS) followed by selective killing of residual hESC with the unique cytotoxic antibody mAb 84, the required purity of differentiated hESC can be achieved. The applicability and robustness of this separation strategy is shown here in a case study using pools of undifferentiated hESC and human fibroblast cells at different ratios (5%-50% hESC) to reflect the different scenario of contaminating hESC in a differentiated cell population. Notably, 97.2%-99.7% of the hESC were removed after the MACS step and 99.1%-100%, after the mAb 84 treatment step, which was confirmed by double-staining flow cytometry and RT-qPCR analysis. These in vitro findings were further validated in an in vivo severe combined immunodeficiency (SCID) mouse model. Importantly, we observed the absence of teratoma formation in eight out of nine SCID mice 28 weeks postinjection of cells after the MACS step, whereas teratomas were observed in all of the controls. Thus, the combination of MACS with the unique cytotoxic antibody mAb 84 constitutes an indispensible tool for successful and safe cell therapy.