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Sci Rep. 2019 Jul 4;9(1):9705. doi: 10.1038/s41598-019-46105-y.

Dual usage of a stage-specific fluorescent reporter system based on a helper-dependent adenoviral vector to visualize osteogenic differentiation.

Author information

1
Division of Gene Therapy and Genome Editing, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan.
2
Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.
3
Takara Bio Inc., 7-4-38 Nojihigashi, Kusatsu, Shiga, 525-0058, Japan.
4
Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan.
5
Department of Physiological Science and Molecular Biology, Division of Biomedical Sciences, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan.
6
Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.
7
Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
8
Department of Oral Science, Graduate School of Dentistry, Kanagawa Dental University, 82 Inaoka-cho, Yokosuka, Kanagawa, 238-8580, Japan.
9
Department of Microbial Chemistry, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
10
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
11
Division of Gene Therapy and Genome Editing, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, 350-1241, Japan. mitani@saitama-med.ac.jp.

Abstract

We developed a reporter system that can be used in a dual manner in visualizing mature osteoblast formation. The system is based on a helper-dependent adenoviral vector (HDAdV), in which a fluorescent protein, Venus, is expressed under the control of the 19-kb human osteocalcin (OC) genomic locus. By infecting human and murine primary osteoblast (POB) cultures with this reporter vector, the cells forming bone-like nodules were specifically visualized by the reporter. In addition, the same vector was utilized to efficiently knock-in the reporter into the endogenous OC gene of human induced pluripotent stem cells (iPSCs), by homologous recombination. Neural crest-like cells (NCLCs) derived from the knock-in reporter iPSCs were differentiated into osteoblasts forming bone-like nodules and could be visualized by the expression of the fluorescent reporter. Living mature osteoblasts were then isolated from the murine mixed POB culture by fluorescence-activated cell sorting (FACS), and their mRNA expression profile was analyzed. Our study presents unique utility of reporter HDAdVs in stem cell biology and related applications.

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