KLF17 acts as a dual reporter for tracking naive pluripotency and 8-cell-like states

Abstract

Human pluripotent stem cells (hPSCs) can be stabilized in states corresponding to successive stages of preimplantation development. Conventional primed hPSCs resemble the post-implantation epiblast, naive hPSCs the pre-implantation epiblast, and rare 8-cell-like cells (8CLCs) arising within naive cultures recapitulate features of zygotic genome activation (ZGA) at the 8-cell stage of early human embryonic development. To enable monitoring of these successive states, we exploited the expression of KLF17, which is absent from primed hPSCs and present in the human 8-cell embryo at levels exceeding those of the epiblast. We hypothesized that its expression level, rather than its presence alone, would report position along the continuum of early human development. We therefore generated an EGFP knock-in at the endogenous KLF17 locus and asked whether expression level could resolve two consecutive transitions in the same line. During conversion of primed cells to the naive state, KLF17-positive cells showed elevated expression of naive-associated genes including SUSD2, DNMT3L, DPPA3, DPPA5, TFCP2L1 and CD75 relative to the negative fraction, and formed blastoids at higher efficiency. Higher KLF17 levels in turn marked the naive-to-8C-like transition: following TSA and DZNep treatment, cells sorted into negative, low, mid and high fractions showed a progressive increase in ZGA-associated transcripts such as ZSCAN4, LEUTX, TPRX1, MBD3L2, DUXA and RFPL2. KLF17 expression level thus provides a single continuous readout of position along early human development, from the primed state through naive identity to the 8C-like state.