The ability of human embryonic stem (hES) cells and induced pluripotent stem (hiPS) cells, collectively termed human pluripotent stem cells (hPSCs), to self-renew indefinitely and to differentiate into essentially all types of human adult cells, renders them potential sources of material for a wide range of clinical applications. However, there remains a real need to develop new cellular markers that will enable both large scale purification of live hPSC cultures, as inputs to clinically relevant differentiation assays, and the stringent removal of residual tumorigenic pluripotent cells from end-point cell populations following differentiation.
Our previously reported FACS-based immunotranscriptional profiling system that identifies a CD9hi/GCTM-2hi/OCT4(POUF1)+ population of enriched hPSCs, combined with membrane polysome translation state array data has enabled the identification of candidate cell surface proteins that had not been previously associated with pluripotent cells, but that we observed were switched off very rapidly upon differentiation1-3.
We have subsequently generated and validated novel monoclonal antibodies (MAbs) that detect 18 of these pluripotency-associated epitopes on live hPSCs. Characterisation studies on both hES and hiPS cell lines by multiparameter immunostaining and flow cytometric analysis demonstrated the correlation of each MAb hPSC profile against our established CD9hi/GCTM-2hi profile. Time course differentiation studies and subsequent FACS profiling in conjunction with OCT4 expression are also in progress for each MAb. In further studies, these novel hPSC-identifying MAbs are being coupled to magnetic beads and to toxins for testing in our hPSC purification and off-target cell purging assays. We anticipate that these new antibodies will be extremely useful tools for the identification, enrichment and removal of hPSCs and that they will enable further exploration of the relationship between the proteins detected by these antibodies and the maintenance and/or loss of pluripotency.
This research is funded jointly by the California Institute for Regenerative Medicine and the State Government of Victoria (CIRM grant TR1-01250).