Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density-dependent manner.
BACKGROUND
Rho kinases (ROCKs) mediate cell contraction, local adhesion, and cell motility, which are considered to be important in cell differentiation. We postulated that ROCKs are involved in controlling embryonic stem (ES) cell renewal and differentiation.


RESULTS
CCE, a murine ES cell, was treated with Y-27632 for 48 to 96 hours and colony formation was evaluated. Y-27632 blocked CCE colony formation and induced CCE to grow as individual cells, regardless of the initial seeding cell density either at 10(4)/cm(2) ("high" seeding density) or 2x10(3)/cm(2) ("low" density). However, at high seeding density, Y-27632-treated cells exhibited reduction of alkaline phosphatase (AP) staining and Oct3/4 expression. They expressed SOX-1, nestin, and MAP2c, but not betaIII-tubulin or NG-2. They did not express endoderm or mesoderm lineage markers. After removal of Y-27632, the cells failed to form colonies or regain undifferentiated state. Silencing of ROCK-1 or ROCK-2 with selective small interference RNA induced CCE morphological changes similar to Y-27632. Silencing of ROCK-1 or ROCK-2 individually was sufficient to cause reduction of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs did not augment the effects exerted by individual ROCK siRNA. Y-27632-treated CCE cells seeded at 2x10(3) or 6.6x10(3) cells/cm(2) did not lose renewal factors or express differentiation markers. Furthermore, they were able to form AP-positive colonies after removal of Y-27632 and reseeding. Similar to ROCK inhibition by Y-27632, silencing of ROCK-1 or ROCK-2 in cells seeded at 2x10(3)/cm(2) did not change renewal factors.


CONCLUSIONS
We conclude that ROCKs promote ES cell colony formation, maintain them at undifferentiated state, and prevent them from neural differentiation at high seeding density. ROCK inhibition represents a new strategy for preparing large numbers of neural progenitor cells.
Chang
Tzu-Ching
TC
Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
Chen
Yen-Chung
YC
Yang
Ming-Hua
MH
Chen
Chien-Hung
CH
Hsing
En-Wei
EW
Ko
Bor-Sheng
BS
Liou
Jun-Yang
JY
Wu
Kenneth K
KK
D000469:Alkaline Phosphatase / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000577:Amides; D004791:Enzyme Inhibitors; D007381:Intermediate Filament Proteins; D007546:Isoquinolines; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D011725:Pyridines; D013449:Sulfonamides; C108830:Y 27632; D054460:rho-Associated Kinases; D000469:Alkaline Phosphatase; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide