An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti–stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs—the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures
To find a way of purging unwanted ESCs from cultures growing into other cell types, researchers led by Chad Tang and Micha Drukker – working in the lab of Irving Weissman at Stanford University, California – screened a library of antibodies against human ESCs to find those that bound most strongly to the cells. One antibody, which turned out to recognise a previously unknown carbohydrate dubbed SSEA-5, came out “at the top of the heap”, says Weissman.
Next the researchers turned to a method called fluorescence-activated cell sorting (FACS), which can separate cells depending on whether they carry an antibody tagged with a fluorescent label. They stuck such a fluorescent label to their new antibody, before adding it to human ESCs that were developing into blood cells. Then they used FACS to pick out cells the antibody bound to. Finally, they injected the remaining cells into mice to see if they formed teratomas.
By itself, the new antibody did not completely purge the offending cells. But when it was combined with two other antibodies that also target molecules found on ESCs, no teratomas formed.
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