Narai Takashi, Watase Ryohei, Nakayama Yuji, Kodani Isamu, Inoue Toshiaki, Kokura Kenji
Division of Oral and Maxillofacial Surgery, Department of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago, 683-8504, Japan.
Division of Human Genome Science, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago, 683-8503, Japan.
Heliyon. 2020 Oct 31;6(10):e05398. doi: 10.1016/j.heliyon.2020.e05398. eCollection 2020 Oct.
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 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.
间充质干细胞（MSCs）有望用于治疗各种疾病和病症的骨再生，包括唇腭裂、骨折和骨吸收。然而，迄今为止，MSCs在临床环境中未能产生令人满意的结果。这主要是由于诱导成骨分化的比率较低。为了实现MSCs的潜力，有必要建立分离源自MSCs的活成骨细胞的方法。然而，迄今为止尚未报道有成骨细胞标志物。为了尝试开发一种评估成骨细胞分化的方法，我们建立了用于监测骨γ-羧基谷氨酸蛋白（BGLAP，骨钙素）表达的报告人永生化MSC（hiMSC）系。为此，我们通过CRISPR-Cas9成功地在BGLAP的第一个ATG的紧邻下游敲入了一个增强型绿色荧光蛋白（EGFP）基因盒，并建立了表达EGFP以监测成骨分化的hiMSC系。在分化第7天，通过流式细胞术细胞分选收集EGFP阳性细胞，并分析EGFP和内源性BGLAP的表达。在成骨分化过程中，发现EGFP上调与内源性BGLAP的表达相关。此外，在修饰的hiMSC系进行两周成骨分化后，使用茜素红-S染色确认了矿化。本文获得的修饰的hiMSC系以及衍生的分化成骨细胞是监测成骨细胞基因和蛋白表达的有价值工具，可用于开发分离活成骨细胞的新方法。
