Protein kinase A inhibitor, H89, enhances survival and clonogenicity of dissociated human embryonic stem cells through Rho-associated coiled-coil containing protein kinase (ROCK) inhibition.

Study Question: Can cell survival of dissociated human embryonic stem cells (hESCs) be increased during culture?

Summary Answer: A protein kinase A (PKA) inhibitor, H89, can significantly enhance survival and clonogenicity of dissociated hESCs without affecting their pluripotency.

What Is Known Already: hESCs are vulnerable to massive cell death upon cellular detachment and dissociation.

Study Design, Size, Duration: hESCs were dissociated into single cells and then cultured in feeder-dependent and -independent manners. H89 was added to the culture medium at different concentrations for 1 day. The statistical results were obtained from at least three independent experiments (n ≥ 4). The group without treatment was used as the negative control.

Participants/materials, Setting, Methods: 4 µM H89 was added in the culture medium to promote cell survival and colony formation of dissociated hESCs. MTT method and propidium iodide (PI) staining were used to determine cell proliferation, cell death and cell cycle, respectively. To count colony formation, alkaline phosphatase (AP) staining was carried out. Western blot was performed to determine protein expression. Except AP staining, immunofluorescence, RT-PCR and karyotype analysis were used to confirm the pluripotent state of H89 treated hESCs.

Main Results And The Role Of Chance: H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK), myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1), significantly increases cell survival and colony formation, and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro.

Limitations, Reasons For Caution: Appropriate H89 concentration should be used and 1 day of H89 treatment is sufficient for promoting survival and colony formation of dissociated hESCs.

Wider Implications Of The Findings: These results provide an alternative for human pluripotent stem cell (hPSC) culture, broaden the scope of participants in the cell death of single hES cells after dissociation and further enlighten clues to understand the mechanism of dissociation-induced cell death.

Study Funding/competing Interests: This research was supported by the National Natural Science Foundation of China (21176238, 21576266), and Chinese Academy of Sciences. There is no conflict of interest to declare.

Trial Registration Number: Nil.