Pertussis toxin enhances colony organization of enzymatic-dissociated single human embryonic stem cells.
Human embryonic stem cells (hESCs) self-renew indefinitely as highly organized pluripotent colonies. Unlike mouse pluripotent stem cell colonies, human colonies form a uniform, flat, epithelium-like monolayer. Interestingly, it has been reported that colony morphology is closely correlated with the maintenance of pluripotency. However, the molecular mechanisms that underlie human pluripotent colony formation and organization are poorly understood. In this study, we used real-time imaging tools to examine the in vitro colony formation of enzymatically dissociated single hESCs under feeder-free conditions. We demonstrate that colony formation consists of 4 stages: attachment, migration, aggregation, and colony formation, which are facilitated in an intracellular, calcium-dependent manner. Moreover, we found that blocking G(i)-coupled G protein-coupled receptor (GPCR) signaling results in enhanced cell-cell interactions and plays an integral role in promoting the survival of hESCs in culture. From the imaging results, we identified the conditions required for colony formation, and we identified the importance of blocking G(i)-coupled GPCR by pertussis toxin in modulating hESC colony formation and organization. These results will likely be useful for engineering hESCs to further study the mechanisms involved in their function.
Stem Cell Research Lab, CHA Stem Cell Institute, CHA University, Seoul, Republic of Korea.
Jung Mo
JM
Kim
Sung-Hwan
SH
Moon
Soon-Jung
SJ
Park
Ha Young
HY
Lee
Ki-Sung
KS
Hong
Joseph
J
Seo
Yoe-Sik
YS
Bae
Hyung-Min
HM
Chung
D000577:Amides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D002450:Cell Communication; D002460:Cell Line; D002465:Cell Movement; D002470:Cell Survival; D002772:Cholera Toxin / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D037342:Pertussis Toxin / Q000494:pharmacology*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
D000577:Amides; D011725:Pyridines; D043562:Receptors, G-Protein-Coupled; C108830:Y 27632; D002772:Cholera Toxin; D037342:Pertussis Toxin; D002118:Calcium