title: Passaging techniques and ROCK inhibitor exert reversible effects on morphology and pluripotency marker gene expression of human embryonic stem cell lines.
abstract: Human embryonic stem cells (hESCs) are known for their potential usage in regenerative medicine, but also for handling sensitivity. Much effort has been put into optimizing the culture methods of hESCs. It has been shown that the use of Rho-associated coiled-coil kinase inhibitor (ROCKi) decreases the cellular stress response and the apoptotic cell death in hESC cultures that have been passaged enzymatically. These observations sparked a wide use of ROCKi in hESC cultures. We and others, however, noted that cells passaged enzymatically with the use of ROCKi had a different morphology compared to cells passaged mechanically. Here we show that hESCs that were enzymatically passaged displayed alterations in the nuclear size compared to cultures that were mechanically passaged. Notably, a dramatically decreased expression of the genes encoding common pluripotency markers, such as OCT4/POU5F1 and NANOG were revealed in enzymatically passaged hESCs compared to mechanically passaged, while such differences were not significant when assessing protein levels. The differences in gene expression did not correlate strongly with commonly analyzed histone modifications (H3K4me3, H3K9me3, H3K27me3, and H4K16ac) on the promoters of these genes. Surprisingly, the effects of enzymatic passaging were at least in part reversible as the gene expression profile of enzymatically passaged hESCs that were transferred back to mechanical passaging, showed no significant difference compared to those hESCs that were continuously passaged mechanically. Our results suggest that enzymatic passaging influences parameters associated with hESC characteristics, and emphasizes the importance of using cells handled in the same manner when comparing results both within and between projects.
authors: Department of Neuroscience, Linnaeus Center in Developmental Biology for Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden. fholm@ucsd.edu
authors: Frida
authors: F
authors: Holm
authors: Hero
authors: H
authors: Nikdin
authors: Kristín Rós
authors: KR
authors: Kjartansdóttir
authors: Giulia
authors: G
authors: Gaudenzi
authors: Kaj
authors: K
authors: Fried
authors: Pontus
authors: P
authors: Aspenström
authors: Ola
authors: O
authors: Hermanson
authors: Rosita
authors: R
authors: Bergström-Tengzelius
mesh_terms: D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000096:biosynthesis
chemical_list: D015415:Biomarkers; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D054460:rho-Associated Kinases