Powell, BE; (2017) Identification of Conditions to Induce and Maintain Naive Human Pluripotency. Doctoral thesis , UCL (University College London).

Abstract

Mouse and human embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts however are distinct in their molecular and biological properties. Historically these discrepancies have been attributed to speciesspecific differences. Recent work capturing human ESC-like cells from postimplantation mouse embryos instead suggests that they represent distinct developmental stages of the pluripotent epiblast. Mouse ESCs resemble the preimplantation epiblast where cells are “naive” and have no differentiation bias. In contrast human ESCs represent a developmental stage postimplantation “primed” for lineage specification. Here a systematic approach was taken to identify conditions to induce and maintain naive human ESCs by screening for small molecules that support self-renewal based on the maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of naive pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors, 5i (MEK, GSK3, BRAF, SRC, ROCK) that induce and maintain OCT4 distal enhancer activity when applied directly to conventional human ESCs. Furthermore, these conditions allowed the capture of naive pluripotent cells from human embryos and somatic cells through reprogramming. The inhibitors described herein capture a population of human pluripotent cells in which transcription factors specific to ground state pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison to published reports of naive human ESCs suggest that previous attempts to isolate naive human pluripotent cells fall short of recapitulating the transcriptional foundations of the naive state. In contrast the conditions presented in this thesis capture a distinct state of human pluripotency that very closely resembles mouse ESCs. Given the stringency of the reporter used and the systematic approach taken, this study should provide a framework for future work in characterizing and consolidating naive human pluripotency.

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