Selective Removal of Undifferentiated Human Embryonic Stem Cells Using Magnetic Activated Cell Sorting Followed by a Cytotoxic Antibody

Abstract

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.

Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to Eliminate Teratoma Formation

“…An example of a cytotoxic antibody that detects and removes hESCs is the monoclonal antibody mAB-84 [17], which binds to PODXL (Podocalyxin-like protein 1) on hESCs and initiates a sequence of events that leads to hESC-membrane damage by formation of leaking pores [18]. It has been proposed that using the monoclonal antibody mAB-84 in a two-step cell-cell separation approach can eliminate teratoma-forming hESC from differentiated cell types [19]. In this strategy, an initial depletion of hESCs was achieved via MACS using a panel of commonly used hESC cell-surface markers, which was followed by selective elimination of residual undifferentiated stem cells post-MACS using the cytotoxic antibody mAB-84, an approach that appears to increase the safety of cell transplantation [19].…”

“…It has been proposed that using the monoclonal antibody mAB-84 in a two-step cell-cell separation approach can eliminate teratoma-forming hESC from differentiated cell types [19]. In this strategy, an initial depletion of hESCs was achieved via MACS using a panel of commonly used hESC cell-surface markers, which was followed by selective elimination of residual undifferentiated stem cells post-MACS using the cytotoxic antibody mAB-84, an approach that appears to increase the safety of cell transplantation [19].…”

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

“…Therefore, if the aim is to use antibodies to detect human pluripotent cells and purge them from a sample of hPSC-derived cells, selected antibodies should not also react with antigens on the specific differentiated cell types intended for transplantation. Unfortunately, there are limited numbers of antibodies that detect cell surface markers on live hPSCs surface marker is not sufficient to eliminate all hPSC, suggesting that any attempt to eliminate all hPSC pluripotent subpopulations should rely on methods that use two or more antibodies detecting different epitopes expressed by hPSCs [9][10] . As mentioned above, only hPSC-derived cells that could be determined as pluripotent stem cell-free cell populations are appropriate for human transplantation.…”

Label-free density difference amplification-based cell sorting

“…6,7 Similarly, magnetic-activated cell sorter (MACS) uses magnetic. 8,9 Alternatively, label-free cell separation methods have exploited inherent a) J. Song and M. Song contributed equally to this work.…”