title: Application Of Small Molecules Favoring Naïve Pluripotency during Human Embryonic Stem Cell Derivation.
abstract: In mice, inhibition of both the fibroblast growth factor (FGF) mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/Erk) and the Wnt signaling inhibitor glycogen synthase-3β (GSK3β) enables the derivation of mouse embryonic stem cells (mESCs) from nonpermissive strains in the presence of leukemia inhibitory factor (LIF). Whereas mESCs are in an uncommitted naïve state, human embryonic stem cells (hESCs) represent a more advanced state, denoted as primed pluripotency. This burdens hESCs with a series of characteristics, which, in contrast to naïve ESCs, makes them not ideal for key applications such as cell-based clinical therapies and human disease modeling. In this study, different small molecule combinations were applied during human ESC derivation. Hereby, we aimed to sustain the naïve pluripotent state, by interfering with various key signaling pathways. First, we tested several combinations on existing, 2i (PD0325901 and CHIR99021)-derived mESCs. All combinations were shown to be equally adequate to sustain the expression of naïve pluripotency markers. Second, these conditions were tested during hESC derivation. Overall, the best results were observed in the presence of medium supplemented with 2i, LIF, and the noncanonical Wnt signaling agonist Wnt5A, alone and combined with epinephrine. In these conditions, outgrowths repeatedly showed an ESC progenitor-like morphology, starting from day 3. Culturing these "progenitor cells" did not result in stable, naïve hESC lines in the current conditions. Although Wnt5A could not promote naïve hESC derivation, we found that it was sustaining the conversion of established hESCs toward a more naïve state. Future work should aim to distinct the effects of the various culture formulations, including our Wnt5A-supplemented medium, reported to promote stable naïve pluripotency in hESCs.
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .|4 These authors contributed equally to this work.
authors.forename: Margot
authors.initials: M
authors.lastname: Van der Jeught
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .|4 These authors contributed equally to this work.
authors.forename: Jasin
authors.initials: J
authors.lastname: Taelman
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .
authors.forename: Galbha
authors.initials: G
authors.lastname: Duggal
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .
authors.forename: Sabitri
authors.initials: S
authors.lastname: Ghimire
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lierman
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .|2 Department of Anatomy and Embryology, Leiden University Medical Center , 2300 Leiden, The Netherlands .
authors.forename: Susana M
authors.initials: SM
authors.lastname: Chuva de Sousa Lopes
authors.affiliation: 3 Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University , 9000 Ghent, Belgium .
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .
authors.forename: Tom
authors.initials: T
authors.lastname: Deroo
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .
authors.forename: Petra
authors.initials: P
authors.lastname: De Sutter
authors.affiliation: 1 Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital , 9000 Ghent, Belgium .
authors.forename: Björn
authors.initials: B
authors.lastname: Heindryckx
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D004159:Diphenylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004338:Drug Combinations; D004837:Epinephrine / Q000494:pharmacology; D000066449:Human Embryonic Stem Cells / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D051379:Mice; D039904:Pluripotent Stem Cells / Q000502:physiology*; D061251:Primary Cell Culture; D011518:Proto-Oncogene Proteins / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D015398:Signal Transduction; D051153:Wnt Proteins / Q000494:pharmacology; D000071818:Wnt-5a Protein
chemical_list: D001549:Benzamides; C473711:Chir 99021; D004338:Drug Combinations; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D011518:Proto-Oncogene Proteins; D011725:Pyridines; D011743:Pyrimidines; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C506614:mirdametinib; D004159:Diphenylamine; D004837:Epinephrine