Producing mouse totipotent-like cells at high efficiency in vitro

Submission 39

S1-07-Poster Teaser

Presented by: Moyra LAWRENCE

Moyra LAWRENCE ^{1, 2}, Tatsuya Yamakawa ¹, Mio Kabata ¹, Satoko Sakurai ¹, May Nakajima-Koyama ¹, Joonseong Lee ^{1, 2}, Keisuke Okita ¹, Knut Woltjen ¹, Mio Iwasaki ¹, Takuya Yamamoto ^{1, 2, 3}

¹ Centre for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

² Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan

³ Medical-risk Avoidance based on iPS Cells Team, RIKEN Center for Advanced Intelligence Project (AIP), Kyoto, Japan

Objective

Recent advances in stem cell biology have allowed reprogramming to the naïve pluripotent state at high efficiency, as well as efficient primed-to-naïve conversion. Differentiation protocols have also generated pure populations of differentiated cell types from pluripotent ones. However, protocols to generate totipotent cells have lagged behind, generating totipotent cells at lower purity. We aimed to generate totipotent cells at high efficiency from pluripotent cells and investigate the mechanisms behind the transition to totipotency.

Methods

We used a combination of stable transgene overexpression, the activation of repeat elements using Cas9 attached to VP64 and small molecule inhibitors to produce totipotent-like cells, measuring efficiency by intracellular flow cytometry and single cell RNA sequencing. We validated telomere length, gene expression, protein expression and cell cycle in the generated totipotent cells.

Results

We identified a transcription factor overexpression, repeat element activation and small molecule combination which generated totipotent-like cells at 75-94% efficiency, higher than that generated by existing protocols. These cells clustered closer to cells of the early embryo than cells from existing protocols. This highly efficient protocol allowed us to perform the first proteomic studies of the totipotent state, investigating the protein networks underlying the state. Insights provided from this and single-cell RNA sequencing enabled us to characterise the role of translational regulation, protein degradation by the proteasome and post-translational SUMOylation in regulating the transit to the totipotent state.

Conclusion

Using a combination of transgene overexpression, repeat element activation and small molecule inhibitors, we produced totipotent-like cells at 75-94% efficiency. This high efficiency allowed us to investigate the mechanisms underlying the transition, including translational regulation, proteasomal control and post-translational modification. In vitro induced totipotent cells pave the way for new treatments for diseases such as Fascioscapulohumeral dystrophy, where totipotent genes are mis-expressed in adult tissue, and provide a crucial starting cell type for the differentiation of extra-embryonic tissue in vitro.