title: 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.
abstract: OBJECTIVE
  Can cell survival of dissociated human embryonic stem cells (hESCs) be increased during culture?
  
  
  CONCLUSIONS
  A protein kinase A (PKA) inhibitor, H89, can significantly enhance survival and clonogenicity of dissociated hESCs without affecting their pluripotency.
  
  
  BACKGROUND
  hESCs are vulnerable to massive cell death upon cellular detachment and dissociation.
  
  
  METHODS
  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.
  
  
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Appropriate H89 concentration should be used and 1 day of H89 treatment is sufficient for promoting survival and colony formation of dissociated hESCs.
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  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.
  
  
  BACKGROUND
  Nil.
authors: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China University of Chinese Academy of Sciences, Beijing 100049, P.R. China.
authors: Liang
authors: L
authors: Zhang
authors: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China.
authors: Yanqing
authors: Y
authors: Xu
authors: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China.
authors: Jiandong
authors: J
authors: Xu
authors: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China.
authors: Yuping
authors: Y
authors: Wei
authors: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China xuxia@ipe.ac.cn.
authors: Xia
authors: X
authors: Xu
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D024722:Cardiac Myosins / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000577:Amides; D007546:Isoquinolines; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D013449:Sulfonamides; C107184:myosin light chain 2; C108830:Y 27632; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D024722:Cardiac Myosins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
keywords: H89; cell survival; dissociation; human embryonic stem cells; pluripotency