The primary cultured ESCs we used were confirmed immunocytochemically to be positive for vimentin, a marker for stromal cells, and negative for cytokeratin, a marker for epithelial cells (please see above figure). ESCs at early passages (between 2 and 6 passages) were used for the experiment. In this study, the passaged cells were used without freezing. However, we have confirmed that primary ESCs can be cryopreserved in CELLBANKER, and retain characteristics such as responsiveness to decidual stimuli. These descriptions have been added to the revised manuscript (page 3, lines 96-99). Figure. Immunocytochemistry (ICC) of vimentin and cytokeratin in isolated human ESCs.
Lines 88-98. Cell culture. What characterization was performed to confirm the identity of cells as ESCs? In general, there is very little detail on these primary cultured lines. How long are they held in culture? Are they stored cryogenically? How are they re-seeded into culture?