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Pluripotency can only be confirmed by tri-lineage differentiation either through in vivo formation of a teratoma containing all three germ layers or by in vitro differentiation to cells of each germ layer.
Our preferred method would be in vitro directed differentiation using:
STEMdiff™ Definitive Endoderm kit for differentiation,
STEMdiff™ NIM for neural differentiation, and
One of many STEMdiff™ APEL™-based protocols.
Expression of markers associated with the undifferentiated state, including expression of Oct3/4, Sox2, Tra-1-60, Tra-1-81, SSEA3 and SSEA4, should be considered a screening tool and not a replacement for a tri-lineage differentiation assay.