Time lapse monitoring of hiPSCs cultured on a suspended mesh sheet. (A) Results of immunostaining performed to check for pluripotency of dish cultured hiPSCs before seeding on mesh. Merged image also shows blue pigment from Hoechst staining of cell nuclei. (B) Phase contrast images showing growth and proliferation of hiPSCs on mesh. Last image of the column (Day 15) shows a bubble-like cyst formed as a result of cell differentiation. (C) Phase contrast image showing numerous cysts on a mesh sheet at day 21 of culture. Yellow and red
arrowheads indicate cysts formed on the upper and lower surfaces of a mesh sheet, respectively. (D) Result of RT-PCR analysis performed to compare pluripotency in hiPSCs cultured on mesh and on typical culture dishes for 8 days. Results were normalized by OCT4 expression in hiPSCs cultured on laminin-coated culture dish for 3 days. MAT, Matrigel; LAM, laminin. Scale bar represent 50 μm in (A) 100 μm in (B), and 200 μm in (C). hiPSCs, human pluripotent stem cells; RT-PCR, real-time polymerase chain reaction.