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Stem Cell Reports. 2014 May 22;2(6):751-60. doi: 10.1016/j.stemcr.2014.04.016. eCollection 2014 Jun 3.

Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions.

Author information

1
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.
2
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.
3
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.
4
Intensive Care Unit, The University of Tokyo, Tokyo 113-0033, Japan.
5
Center for Disease Biology and Integrative Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
6
Department of Reconstructive Sciences, School of Dental Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA.
7
Department of Sensory and Motor System Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
8
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.

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.

PMID:
24936463
PMCID:
PMC4050355
DOI:
10.1016/j.stemcr.2014.04.016
[Indexed for MEDLINE]
Free PMC Article

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