p16High senescence restricts totipotent potential during somatic cell reprogramming

The discovery of four factor (4F)-induced reprogramming of somatic cells into induced pluripotent stem (iPS) cells has revolutionized the fields of cell and regenerative biology. In contrast, the feasibility of a direct conversion of somatic cells into a totipotent state defined as the ability to produce all cell types of an organism, including extraembryonic tissues, is not well established. Using genetic and chemical approaches to manipulate senescent cells, here we found that removal of p16 High cells resulted in 4F-induced reprogramming of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the 2-cell (2C) embryonic state, readily formed implantation-competent blastocyst-like structures, blastoids, and following morula aggregation, contributed to embryonic and extraembryonic lineages in E12.5 embryos. We identified senescence-dependent regulation of nicotinamide N-methyltransferase (NNMT) as a key mechanism controlling the S-adenosyl- l -methionine (SAM) levels during 4F-induced reprogramming that was required for expression of the 2C genes and acquisition of an extraembryonic potential. Our results show that the presence of p16 High senescent cells, high NNMT and low SAM limit cell plasticity during 4F-reprogramming, while their modulation could help to achieve the highest state of stem cell potency, totipotency.