Heliyon
Volume 6, Issue 10, October 2020, e05398
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Research article
Establishment of human immortalized mesenchymal stem cells lines for the monitoring and analysis of osteogenic differentiation in living cells

https://doi.org/10.1016/j.heliyon.2020.e05398Get rights and content
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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

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

Cited by (0)

1

These authors contributed equally to this work.

2

These authors are co-corresponding authors.