Suppressing P16Ink4a and P14ARF pathways overcomes apoptosis in individualized human embryonic stem cells.

Dissociation-induced apoptosis is a striking phenomenon in human embryonic stem cells (hESCs), but not in naive mouse ESCs. Rho-associated kinase-dependent actin-myosin hyperactivation is an underlying mechanism that triggers apoptosis in dissociated hESCs; however, in this study, we show that the Ink4A-ARF-mediated senescence pathway is another mechanism to cause apoptosis in individualized hESCs. We show that P16INK4A and P14ARF are immediately induced in hESCs upon dissociation, but not in mouse ESCs. Overexpression of BMI1, a suppressor for Ink4A-ARF, greatly promotes survival and cloning efficiency of individualized hESCs mechanistically via direct binding the H3K27me3-marked Ink4A-ARF locus. Forced expression of BMI1 in hESCs does not reduce the actin-myosin activation that is triggered by dissociation, which indicates it is an independent pathway for hESC survival. Furthermore, dual inhibition of both Ink4A-ARF and actin-myosin hyperactivation enables successful passaging of hESCs via gelatin, a nonbioactive matrix. In sum, we provide an additional mechanism that underlies cell death in individualized hESCs that might help to fully understand the differential cell characteristics between naive and primed ESCs.-Wang, W., Zhu, Y., Huang, K., Shan, Y., Du, J., Dong, X., Ma, P., Wu, P., Zhang, J., Huang, W., Zhang, T., Liao, B., Yao, D., Pan, G., Liu, J. Suppressing P16Ink4a and P14ARF pathways overcomes apoptosis in individualized human embryonic stem cells. © FASEB.