Freeze-thawing single human embryonic stem cells induce e-cadherin and actin filament network disruption via g13 signaling

Poor adhesion of single human embryonic stem (hES) cells after freeze-thawing causes death. To investigate mechanisms responsible for this, Rho-dependent protein kinase (ROCK) inhibitor Y-27632-treated and untreated single hES cells were analyzed for E-cadherin and F-actin distribution by immunostaining and phalloidin staining respectively and for G13 signaling pathway components by DNA microarray and quantitative polymerase chain reaction (PCR). Y-27632-treated cells clustered rapidly and maintained E-cadherin and F-actin distribution without losing Oct-3/4. Immediately after thawing, E-cadherin in untreated hES cells dotted along the membrane and then displayed eccentric cytoplasmic localization. Bleb formation and early Oct-3/4 loss occurred after F-actin network condensation in the cytoplasm. Microarray analyses and quantitative PCR indicated upregulation of two actin reorganization-associated components of the G13 signaling pathway, Arhgdib and Cdc42, in untreated cells. Considering these findings and that cell death was partly interrupted by Y-27632, E-cadherin and actin cytoskeleton network disruption through the G13 signaling pathway may cause hES cell death after freeze-thawing.