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.

A Multilevel Approach to the Causes of Genetic Instability in Stem Cells

“…Even with this supposedly less harmful passage procedure, a relationship between passage number and p53 mutations is noteworthy (Holm et al 2013;Amir et al 2017). Involvement of p53 mutations in ESCs genetic disturbances in culture was also demonstrated when 5 cell lines resulted positive for p53 dominant negative mutations, afterward when 140 lines were analyzed, from that mutated, the allelic fraction in 3 cell lines increased its prevalence to more than 50%, signifying what was interpreted as selective advantage for p53 mutations bearing cells.…”

“…But in spite of the use of Accutase-based passage, deletions were larger and appeared earlier than that in mechanical passages. Duplications and deletion involving chromosomes 20, 12, and 17 were consistently detected with a higher frequency in ESCs and IPSCs that undergo enzymatic passage, and karyotype mosaicism was also detected (Holm et al 2013;Garitaonandia et al 2015;Amir et al 2017). Even when compared with the less harmful Accutase, the mechanical cell detachment is the best choice against the induction of genetic instability.…”

Dynamic Features of Chromosomal Instability during Culture of Induced Pluripotent Stem Cells

“…The temporary application the ROCK inhibitor to cultures after passaging was introduced to promote the attachment of cells during passaging [ 51 ]. Passage methods and the use of the ROCK inhibitor may affect the expression of stem cell genes [ 52 ]. Both WTC-11 and Tuba1-GFP iPSC lines were previously conditioned to enzymatic passaging, as clumps or single cell suspension, respectively.…”

A Video Bioinformatics Method to Quantify Cell Spreading and Its Application to Cells Treated with Rho-Associated Protein Kinase and Blebbistatin

“…inhibitor of myosin II which is downstream of ROCK, can also be used to increase attachment of hESC and thereby improve plating efficiency [19] and may also alter cell morphology [21].…”