A2M induces detachment and cell death in pluripotent hESC. (A) A2M was added to hESC growing on a Matrigel-coated six-well plate at a final concentration of 77 μg/ml. hESC cell detachment and cell death were observed 72 h after adding A2M. In wells without adding A2M, a hESC colony is surrounded by differentiated hESC. (B) In wells with A2M treatment, hESC colonies disappeared and small clumps of dead cells were observed. (C) Differentiated cells still attached to Matrigel have healthy morphology (black arrow), while detached and fragmented hESC clumps appeared (red arrow). (D) Immunostaining after A2M treatment showed that Oct-3/4-positive cells disappeared, and the remaining cells are Oct-3/4 negative. The areas occupied by hESC colonies have no remaining cells. Immunostaining of wells without A2M treatment showed pluripotency marker Oct-3/4 expressed by cells inside the hESC colony, but not by the differentiated cells around it (inset). Red: Oct-3/4; blue: DAPI.