title: Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media.
abstract: Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.
authors.affiliation: Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Fukuta
authors.affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nakai
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Kirino
authors.affiliation: Department of Reprogramming Science, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Nakagawa
authors.affiliation: Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Sekiguchi
authors.affiliation: Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
authors.forename: Sanae
authors.initials: S
authors.lastname: Nagata
authors.affiliation: Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Matsumoto
authors.affiliation: Department of Reprogramming Science, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Katsutsugu
authors.initials: K
authors.lastname: Umeda
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Heike
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Okumura
authors.affiliation: Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Koizumi
authors.affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
authors.forename: Tatsutoshi
authors.initials: T
authors.lastname: Nakahata
authors.affiliation: Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
authors.forename: Megumu
authors.initials: M
authors.lastname: Saito
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Otsuka
authors.affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Morio
authors.initials: M
authors.lastname: Ueno
authors.affiliation: Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ikeya
authors.affiliation: Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Toguchida
mesh_terms: D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured / Q000166:cytology; D019902:Chondrocytes / Q000166:cytology; D003315:Cornea / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology; D042783:Endothelial Cells / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D007328:Insulin / Q000378:metabolism; D008544:Melanocytes / Q000166:cytology; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D009432:Neural Crest / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D010012:Osteogenesis; D039904:Pluripotent Stem Cells / Q000166:cytology*; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D019485:Bone Morphogenetic Proteins; D003470:Culture Media; D018076:DNA, Complementary; D007328:Insulin; D016212:Transforming Growth Factor beta