Selective depletion of SSEA-3- and TRA-1-60-positive undifferentiated human embryonic stem cells by magnetic activated cell sorter (MACS)

Abstract

The capacity of indefinite self-renewal and pluripotency make human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) an attractive source for potential regenerative medicine. However, teratoma formation is one of the major obstacles for implementing hESC-based therapeutics in a clinical setting. While this tumorigenic capacity is known to be lost upon lineage differentiation in vitro, there is a potential risk that any residual undifferentiated hESCs or progenitor cells may form tumors upon transplantation. To ensure the safety of hESCs in clinical application, we thus explored the magnetic activated cell sorter (MACS) as a tool for separating undifferentiated hESCs from a mixed population of hESCs and human blood mononuclear cells. Labeled with single or combinations of two fluorsecein-labeled monoclonal antibodies (SSEA-3 and TRA-1-60) and subsequently stained with anti-FITC and/or anti-PE magnetic microbeads, cells were subjected to MACS separation. While a reduction of hESCs by depletion with a single marker was observed, there was still a significant fraction of residual hESCs in the flow-through fraction. However, two consecutive MACS separations upon simultaneous staining of two different hESC markers, SSEA-3 and TRA-1-60, completely depleted hESCs, as validated by flow cytometer, real-time PCR and immunofluoresence microscopic analyses. The maximum efficacy of hESC removal using this protocol was higher than 99.9%. No teratoma formation was observed in hESC-depleted cell injection to NOD/SCID mice. These results indicate that the current MACS protocol with two antibodies can efficiently eliminate undifferentiated cells from differentiated cells and greatly alleviate concerns about tumor formation by hESC-derived cellular therapeutics.