Reprogramming of Primed Human Pluripotent Stem Cells into a Naïve State

Abstract—Human pluripotent stem cells (PSCs) can be maintained in a naïve or primed state of pluripotency in vitro. Being in one state or another, PSCs have different potentials of differentiation into extra-embryonic and embryonic cells. In terms of the expression profile and epigenetic pattern of the genome, naïve PSCs are comparable to the cells of the inner cell mass of the blastocyst, while primed PSCs are similar in their characteristics to the cells of the postimplantation epiblast. Reprogramming of primed PSCs into the naïve state and maintenance of naïve PSCs in culture is a crucial issue in studying the epigenetic processes of preimplantation development of the human embryo and methods for efficient differentiation of PSCs into derivatives of embryonic and extra-embryonic cells. The aim of this work is to reprogram primed induced pluripotent stem cells (iPSCs) into a naïve pluripotent state to obtain a homogeneous population of iPSCs according to the state of pluripotency in culture. The task of this work is to develop a protocol and conditions for reprogramming primed iPSCs into a naïve state of pluripotency. In this work, naïve iPSCs were obtained under conditions of application of growth factors FGF2 and TGFβ1 and inhibition of GSK3β and the MEK/ERK signaling pathway (2iF medium). Pretreatment of primed iPSCs with histone deacetylase inhibitors (HDACi) changes the cell morphology and gene expression profile of PSCs towards an earlier state of pluripotency. Using pretreatment of HDACi primed iPSCs followed by maintenance in 2iF medium, the authors obtained naïve iPSCs comparable in colony morphology and expression profile of naïve state marker genes to control naïve iPSCs obtained in RSeT medium. In order to confirm the naïve state of pluripotency of iPSCs obtained 2iF conditions, it is necessary to carry out single cell RNA sequencing.