Open AccessProtein kinase A inhibitor, H89, significantly enhances survival rate of dissociated human embryonic stem cells following cryopreservation
Author(s) -
Zhang Liang,
Xu Yanqing,
Xu Jiandong,
Wei Yuping,
Xu Xia
Publication year - 2016
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/cpr.12278
Subject(s) - cryopreservation , embryonic stem cell , biology , microbiology and biotechnology , kinase , regenerative medicine , stem cell , andrology , immunology , embryo , genetics , medicine , gene
Abstract Objectives Human embryonic stem cells ( hESC s) have huge potential for establishment of disease models and for treating degenerative diseases. However, the extremely low survival level of dissociated hESC s following cryopreservation is been a tremendous problem to allow for their rapid expansion, genetic manipulation and future medical applications. In this study, we have aimed to develop an efficient strategy to improve survival of dissociated hESC s after cryopreservation. Materials and methods Human embryonic stem cells (H9 line), dissociated into single cells, were cryopreserved using the slow‐freezing method. Viable cells and their colony numbers in culture after cryopreservation were evaluated when treated with protein kinase A inhibitor H89. Western blotting was carried out to investigate mechanisms of low survival levels of dissociated hESC s following cryopreservation. Immunofluorescence, reverse transcription‐polymerase chain reaction ( RT ‐ PCR ), in vitro and in vivo differentiation were performed to testify to pluripotency and differentiation ability of hte cryopreserved cells treated with H89. Results H89 significantly improved survival level of dissociated hESC s after cryopreservation through ROCK inhibition. H89‐treated cells still maintained their pluripotency and differentiation capacity. Conclusions This new approach for cryopreservation of single hESC s, using H89, can promote potential use of hESC s in regenerative medicine in the future.