Establishment of human immortalized mesenchymal stem cells lines for the monitoring and analysis of osteogenic differentiation in living cells

Takashi Narai; Ryohei Watase; Yuji Nakayama; Isamu Kodani; Toshiaki Inoue; Kenji Kokura

doi:10.1016/j.heliyon.2020.e05398

Establishment of human immortalized mesenchymal stem cells lines for the monitoring and analysis of osteogenic differentiation in living cells

Heliyon. 2020 Oct 31;6(10):e05398. doi: 10.1016/j.heliyon.2020.e05398. eCollection 2020 Oct.

Authors

Takashi Narai¹, Ryohei Watase², Yuji Nakayama³, Isamu Kodani¹, Toshiaki Inoue², Kenji Kokura^{2

4}

Affiliations

¹ Division of Oral and Maxillofacial Surgery, Department of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago, 683-8504, Japan.
² Division of Human Genome Science, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago, 683-8503, Japan.
³ Division of Radioisotope Science, Research Initiative Center, Organization for Research Initiative and Promotion, Tottori University, Yonago, 683-8503, Japan.
⁴ Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan.

Abstract

Mesenchymal stem cells (MSCs) are expected to be useful in bone regeneration treatment for various diseases and conditions, including cleft lip and palate, fracture, and bone absorption. However, to date, MSCs have failed to produce satisfactory results in clinical settings. This is primarily due to the low rate of induced osteogenic differentiation. To realize MSC potential, it is necessary to establish methods for the isolation of MSC-derived living osteoblasts. However, no osteoblast markers have been reported to date. In an attempt to develop a method for the assessment of osteoblast differentiation, we established reporter human immortalized MSC (hiMSC) lines for in vitro monitoring of bone gamma-carboxyglutamate protein (BGLAP, osteocalcin) expression. To this end, we successfully knocked-in an enhanced green fluorescent protein (EGFP) gene cassette immediately downstream of the first ATG of BGLAP via CRISPR-Cas9, and established hiMSC lines expressing EGFP to monitor osteogenic differentiation. On differentiation day 7, EGFP-positive cells were collected by flow cytometric cell sorting, and the expression of EGFP and endogenous BGLAP was analyzed. During osteogenic differentiation, EGFP upregulation was found to correlate with expression of endogenous BGLAP. Moreover, mineralization was confirmed using Alizarin red-S staining after two weeks of osteogenic differentiation of the modified hiMSC lines. The modified hiMSC lines, as well as the derived differentiated osteoblasts obtained herein, are valuable tools for the monitoring osteoblast gene and protein expression, and can be used to develop novel methods for isolating living osteoblasts.

Keywords: Bone gamma-carboxyglutamate protein; CRISPR-Cas9; Cell biology; Cell culture; Cell differentiation; Mesenchymal stem cells; Osteogenic differentiation; Promoter; Regenerative medicine; Reporter cells; Stem cell research.