Reprogramming Pluripotent Stem Cell Towards TotipotencyPublic Deposited
Totipotent cells have the highest developmental potential and can only be created by nuclear transfer into oocytes. Identities of maternal factors that can induce this reprogramming remain a mystery. In this report, we demonstrate induction of totipotency on mouse embryonic stem cells by introducing six factors, Hist1h2aa, H3f3b, H1foo, p-Npm2, Zscan4d, and Ubtfl1. We observed dose-dependent increases in the MuERV-L endogenous retrovirus expression, typically seen in totipotent 2-cell stage blastomere, and adding p150 siRNA and trichostatin A further increased the expression. These cells, which we designated iTLCs (induced totipotent-like cells), had upregulation of totipotent genes but downregulation of pluripotent and differentiation genes, suggesting a distinct shift towards the totipotent state. Furthermore, iTLCs displayed unusually large nuclei, a characteristic of zygotic genome activation (ZGA). Also, iTLCs showed telomere lengthening and were able to be cultured in totipotent condition. Meanwhile, iTLCs did not show malignant transformations as indicated by normal karyotypes, inability to grow in nutrient-deprived condition, and sensitivity to contact inhibition. iTLCs demonstrated expanded cell fate potential by differentiating into all three distinct lineages of the pre-implantation embryo and expressed markers for both embryonic and extraembryonic lineages. RNAseq data showed remarkable similarities between iTLCs and totipotent cells. Early ZGA genes were strongly upregulated in iTLCs, indicating active totipotent state. When reprogrammed with factors only for an extended period, we observed cells resembling various stages of embryogenesis. These data suggest that pluripotent stem cells can be reprogrammed toward totipotent state without the need of oocytes and raise the tantalizing possibility of creating totipotent cells.