title: Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.
abstract: Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Galbha
authors.initials: G
authors.lastname: Duggal
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Sharat
authors.initials: S
authors.lastname: Warrier
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Sabitri
authors.initials: S
authors.lastname: Ghimire
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.|Laboratory of Cellular Metabolism and Metabolic Regulation Vesalius Research Center (VIB3), Herestraat 49, 300, Leuven, Belgium.
authors.forename: Dorien
authors.initials: D
authors.lastname: Broekaert
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Margot
authors.initials: M
authors.lastname: Van der Jeught
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lierman
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Deroo
authors.affiliation: Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Luc
authors.initials: L
authors.lastname: Peelman
authors.affiliation: Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
authors.affiliation: Department of Basic Medical Science, Ghent University, Ghent, Belgium.
authors.forename: Ria
authors.initials: R
authors.lastname: Cornelissen
authors.affiliation: Center for Medical Genetics, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
authors.forename: Björn
authors.initials: B
authors.lastname: Menten
authors.affiliation: Center for Medical Genetics, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Mestdagh
authors.affiliation: Center for Medical Genetics, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.affiliation: Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Matthias
authors.initials: M
authors.lastname: Roost
authors.affiliation: Department of Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Roderick C
authors.initials: RC
authors.lastname: Slieker
authors.affiliation: Department of Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Bastiaan T
authors.initials: BT
authors.lastname: Heijmans
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Petra
authors.initials: P
authors.lastname: De Sutter
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.|Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Susana Chuva
authors.initials: SC
authors.lastname: De Sousa Lopes
authors.affiliation: Ghent Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Björn
authors.initials: B
authors.lastname: Heindryckx
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D000066449:Human Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000502:physiology*
chemical_list: D036341:Intercellular Signaling Peptides and Proteins
keywords: Human embryonic stem cells; Naïve; Pluripotency; Signaling; Small molecules