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.
Department of Histology and Embryology, Shinshu University School of Medicine, Nagano, Japan.
Hinako
H
Ichikawa
Susumu
S
Yoshie
Sakiko
S
Shirasawa
Tadayuki
T
Yokoyama
Fengming
F
Yue
Daihachiro
D
Tomotsune
Katsunori
K
Sasaki
D000199:Actins / Q000378:metabolism*; D001483:Base Sequence; D015820:Cadherins / Q000096:biosynthesis*; D002478:Cells, Cultured; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000166:cytology*; D005615:Freezing; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D000199:Actins; D015820:Cadherins; D017931:DNA Primers; D044365:GTP-Binding Protein alpha Subunits, G12-G13