title: Small molecule-directed specification of sclerotome-like chondroprogenitors and induction of a somitic chondrogenesis program from embryonic stem cells.
abstract: Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a and Noggin (Nog). We report that canonical Wnt signaling introduced either by forced expression of activated β-catenin, or the small-molecule inhibitor of Gsk3, CHIR99021, satisfied the need for Wnt3a signaling, and that the small-molecule inhibitor of BMP type I receptors, LDN193189, was able to replace Nog. Mesodermal progeny generated using such small molecules were chondrogenic in vitro, and expressed trunk paraxial mesoderm markers such as Tcf15 and Meox1, and somite markers such as Uncx, but failed to express sclerotome markers such as Pax1. Induction of the osteochondrogenically committed sclerotome from somite requires sonic hedgehog and Nog. Consistently, Pax1 and Bapx1 expression was induced when the isolated paraxial mesodermal progeny were treated with SAG1 (a hedgehog receptor agonist) and LDN193189, then Sox9 expression was induced, leading to cartilaginous nodules and particles in the presence of BMP, indicative of chondrogenesis via sclerotome specification. By contrast, treatment with TGFβ also supported chondrogenesis and stimulated Sox9 expression, but failed to induce the expression of Pax1 and Bapx1. On ectopic transplantation to immunocompromised mice, the cartilage particles developed under either condition became similarly mineralized and formed pieces of bone with marrow. Thus, the use of small molecules led to the effective generation from ESCs of paraxial mesodermal progeny, and to their further differentiation in vitro through sclerotome specification into growth plate-like chondrocytes, a mechanism resembling in vivo somitic chondrogenesis that is not recapitulated with TGFβ.
authors.affiliation: Institute of Molecular Medicine, The University of Texas Health Science Center Medical School at Houston, Houston, TX 77030, USA.
authors.forename: Jiangang
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Australian Stem Cell Centre, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Songhui
authors.initials: S
authors.lastname: Li
authors.affiliation: Institute of Molecular Medicine, The University of Texas Health Science Center Medical School at Houston, Houston, TX 77030, USA.
authors.forename: Suprita
authors.initials: S
authors.lastname: Trilok
authors.affiliation: Australian Stem Cell Centre, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Makoto
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Australian Stem Cell Centre, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Vanta
authors.initials: V
authors.lastname: Jokubaitis-Jameson
authors.affiliation: Australian Stem Cell Centre, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Bei
authors.initials: B
authors.lastname: Wang
authors.affiliation: RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Niwa
authors.affiliation: Institute of Molecular Medicine, The University of Texas Health Science Center Medical School at Houston, Houston, TX 77030, USA Australian Stem Cell Centre, Monash University, Clayton, Victoria 3800, Australia Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia naoki.nakayama@uth.tmc.edu.
authors.forename: Naoki
authors.initials: N
authors.lastname: Nakayama
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002356:Cartilage / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects*; D002469:Cell Separation; D019902:Chondrocytes / Q000166:cytology*; D020219:Chondrogenesis; D003470:Culture Media / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D008648:Mesoderm / Q000166:cytology / Q000502:physiology; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology*; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000378:metabolism; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism
chemical_list: D019485:Bone Morphogenetic Proteins; C473711:Chir 99021; D003470:Culture Media; C554430:LDN 193189; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D016212:Transforming Growth Factor beta; D051176:beta Catenin
keywords: Chondrogenesis; Mouse; Paraxial mesoderm; Pluripotent stem cells; Sclerotome