title: Wnt signaling orchestration with a small molecule DYRK inhibitor provides long-term xeno-free human pluripotent cell expansion.
abstract: An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-β (TGFβ) signaling pathways, and none provide for replacement of FGF/TGFβ ligands with chemical compounds. The Wnt/β-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however, the role of Wnt/β-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/β-catenin signaling in mouse embryonic stem cells, we identified a compound, ID-8, that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8, we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFβ activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, California 90033, USA.
authors.forename: Kouichi
authors.initials: K
authors.lastname: Hasegawa
authors.forename: Shin-ya
authors.initials: SY
authors.lastname: Yasuda
authors.forename: Jia-Ling
authors.initials: JL
authors.lastname: Teo
authors.forename: Cu
authors.initials: C
authors.lastname: Nguyen
authors.forename: Michael
authors.initials: M
authors.lastname: McMillan
authors.forename: Chih-Lin
authors.initials: CL
authors.lastname: Hsieh
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Suemori
authors.forename: Norio
authors.initials: N
authors.lastname: Nakatsuji
authors.forename: Masashi
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Miyabayashi
authors.forename: Carolyn
authors.initials: C
authors.lastname: Lutzko
authors.forename: Martin F
authors.initials: MF
authors.lastname: Pera
authors.forename: Michael
authors.initials: M
authors.lastname: Kahn
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000201:enzymology; D055785:Gene Knockdown Techniques; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects* / Q000201:enzymology; D007621:Karyotyping; D051379:Mice; D016513:Mice, SCID; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D034622:RNA Interference; D013997:Time Factors; D060449:Wnt Signaling Pathway / Q000187:drug effects*; D060509:Wnt3A Protein / Q000378:metabolism; D000097603:Dyrk Kinases
chemical_list: D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D060509:Wnt3A Protein; D000097603:Dyrk Kinases