Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing

Human embryonic stem cells (hESCs) can be propagated indefinitely in vitro in an undifferentiated pluripotent state, can differentiate into derivatives of all three germ layers and are of considerable interest for applications in regenerative medicine. Clinical application of hESCs, however, requires reliable protocols for cryopreservation. Current protocols for cryopreservation of hESCs suffer from low recovery rates of hESCs and loss of pluripotency after thawing. We therefore studied the effects of cryopreservation on the viability, proliferation potential, and the pluripotency status of hESCs by combining cellular readouts and transcriptomics. We identified biological processes and pathways affected by cryopreservation in order to understand the limited survival rate of hESCs by comparing transcriptomes of hESCs at different time points after thawing with cells that did not undergo cryopreservation. While the transcriptomes of cells post thawing were very similar to those of control non-frozen hESCs for the early time points, we observed increased expression of genes involved in apoptosis, embryonic morphogenesis, ossification, tissue morphogenesis, regeneration, vasculature development and cell death at later time points. Our data suggest that inhibition of anoikis apoptosis and the stress-induced differentiation pathways are promising targets for improving the survival rate and maintaining pluripotency of hESCs after cryopreservation.