title: Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions.
abstract: Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.
authors.affiliation: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan ; Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Kanke
authors.affiliation: Japan Science Technology Agency, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, The University of Tokyo, Tokyo 108-8639, Japan ; Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Masaki
authors.affiliation: Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan ; Department of Bone and Cartilage Regenerative Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Saito
authors.affiliation: Intensive Care Unit, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Yuske
authors.initials: Y
authors.lastname: Komiyama
authors.affiliation: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Hironori
authors.initials: H
authors.lastname: Hojo
authors.affiliation: Japan Science Technology Agency, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, The University of Tokyo, Tokyo 108-8639, Japan ; Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
authors.affiliation: Department of Reconstructive Sciences, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Lichtler
authors.affiliation: Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Takato
authors.affiliation: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan ; Department of Bioengineering, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Ung-Il
authors.initials: UI
authors.lastname: Chung
authors.affiliation: Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan ; Department of Bioengineering, The University of Tokyo, Tokyo 113-0033, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Ohba
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D003514:Cyclohexylamines / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D014704:Veratrum Alkaloids / Q000494:pharmacology
chemical_list: C473711:Chir 99021; D016895:Culture Media, Serum-Free; D003514:Cyclohexylamines; D011725:Pyridines; D011743:Pyrimidines; C468918:SAG compound; D013876:Thiophenes; D014704:Veratrum Alkaloids; C000541:cyclopamine