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Human pluripotent stem cells (hPSCs) exist in multiple states of pluripotency, broadly categorised as naïve and primed states. These provide an important model to investigate the earliest stages of human embryonic development. Naïve cells can be obtained through primed-to-naïve reprogramming; however, there are no reliable methods to prospectively isolate unmodified naïve cells during this process. Moreover, the current isolation strategies are incompatible for enrichment of naïve hPSCs early during reprogramming. Consequently, we know very little about the temporal dynamics of transcriptional changes and remodelling of the epigenetic landscape that occurs during the reprogramming process. To address this knowledge gap, I sought to develop an isolation strategy capable of identifying nascent naïve hPSCs early during reprogramming. Comprehensive profiling of cell-surface markers by flow cytometry in naïve and primed hPSCs revealed pluripotent state-specific antibodies. By compiling the identified state-specific markers into a multiplexed antibody panel, I was able to distinguish naïve and primed hPSCs. Moreover, the antibody panel was able to track the dynamics of primed-to-naïve reprogramming, as the state-specific surface markers collectively reflect the change in pluripotent states. Through using the newly identified surface markers, I found that naïve cells are formed at a much earlier time point than previously realised, and could be subsequently isolated from a heterogeneous cell population early during reprogramming. This allowed me to perform the first molecular characterisation of nascent naïve hPSCs, which revealed distinct transcriptional changes associated with early and late stage naïve cell formation. Analysis of the DNA methylation landscape showed that nascent naïve cells are globally hypomethylated, whilst imprint methylation is largely preserved. Moreover, the loss of DNA methylation precedes X-chromosome reactivation, which occurs primarily during the late-stage of primed-to-naïve reprogramming, and is therefore a hallmark of mature naïve cells. Using the antibody panel at discrete time points throughout reprogramming has allowed an unprecedented insight into the early molecular events leading to naïve cell formation, and permits the direct comparison between different naïve reprogramming methods. Taken together, the identified state-specific surface markers provide a robust and straightforward method to unambiguously define human PSC states, and reveal for the first time the order of transcriptional and epigenetic changes associated with primed to naïve reprogramming.





Rugg-Gunn, Peter


Naïve pluripotency, Human pluripotency, Primed Pluripotency, Epigenetics, X-chromosome, DNA methylation, Reprogramming, Gene regulatory networks, Cell-surface markers, Embryonic Stem Cells


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge