Reprogramming of Primed Human Pluripotent Stem Cells into a Naive State

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Abstract

Human pluripotent stem cells (PSCs) can be maintained in a naive or primed state of pluripotency in vitro. Being in one state or another, PSCs have different potentials of differentiation into extra-embryonic and germinal derived cells of the embryo. In terms of the expression profile and epigenetic pattern of the genome, naive 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 naive 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 naive state of pluripotency. In this work, naive iPSCs were obtained under conditions of application of growth factors FGF2, 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 maintaining in 2iF medium, we obtained naive iPSCs comparable in colony morphology and expression profile of naive state marker genes with control naive iPSCs obtained in RSeT medium. In order to confirm the naive state of pluripotency of iPSCs obtained 2iF conditions, it is necessary to carry out single cell RNA sequencing.

About the authors

V. K. Abdyev

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Author for correspondence.
Email: mailtovepa@gmail.com
Russia, 119334, Moscow, ul. Vavilova 26

A. L. Rippa

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: mailtovepa@gmail.com
Russia, 119334, Moscow, ul. Vavilova 26

N. A. Arakelyan

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: mailtovepa@gmail.com
Russia, 119334, Moscow, ul. Vavilova 26

E. A. Vorotelyak

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: mailtovepa@gmail.com
Russia, 119334, Moscow, ul. Vavilova 26

A. V. Vasiliev

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: mailtovepa@gmail.com
Russia, 119334, Moscow, ul. Vavilova 26

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Copyright (c) 2023 В.К. Абдыев, А.Л. Риппа, Н.А. Аракелян, Е.А. Воротеляк, А.В. Васильев