Wang Li-Tzu, Lee Yu-Wei, Bai Chyi-Huey, Chiang Hui-Chun, Wang Hsiu-Huan, Yen B Linju, Yen Men-Luh
Department of Obstetrics and Gynecology, National Taiwan University (NTU) Hospital and College of Medicine, Taipei, Taiwan.
Regenerative Medicine Research Group, Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
Front Cell Dev Biol. 2021 Jan 8;8:607383. doi: 10.3389/fcell.2020.607383. eCollection 2020.
The rapid aging of worldwide populations had led to epidemic increases in the incidence of osteoporosis (OP), but while treatments are available, high cost, adverse effects, and poor compliance continue to be significant problems. Naturally occurring plant-based compounds including phytoestrogens can be good and safe candidates to treat OP, but screening for osteogenic capacity has been difficult to achieve, largely due to the requirement of using primary osteoblasts or mesenchymal stem cells (MSCs), the progenitors of osteoblasts, to conduct time-consuming and osteogenic assay. Taking advantage of MSC osteogenic capacity and utilizing a promoter reporter assay for Runx2, the master osteogenesis transcription factor, we developed a rapid screening platform to screen osteogenic small molecules including natural plant-based compounds. We screened eight plant-derived compounds from different families including flavonoids, polyphenolic compounds, alkaloids, and isothiocyanates for osteogenic capacity using the human RUNX2-promoter luciferase reporter (hRUNX2-luc) transduced into the mouse MSC line, C3H10T1/2, with daidzein-a well-studied osteogenic phytoestrogen-as a positive control. Classical and osteogenesis assays were performed using primary murine and human bone marrow MSCs (BMMSCs) to validate the accuracy of this rapid screening platform. Using the MSC/hRUNX2-luc screening platform, we were able not only to shorten the selection process for osteogenic compounds from 3∼4 weeks to just a few days but also simultaneously perform comparisons between multiple compounds to assess relative osteogenic potency. Predictive analyses revealed nearly absolute correlation of the MSC/hRUNX2-luc reporter platform to the classical functional assay of mineralization using murine BMMSCs. Validation using human BMMSCs with mineralization and osteogenesis assays also demonstrated nearly absolute correlation to the MSC/hRUNX2-luc reporter results. Our findings therefore demonstrate that the MSC/hRUNX2 reporter platform can accurately, rapidly, and robustly screen for candidate osteogenic compounds and thus be relevant for therapeutic application in OP.
全球人口的快速老龄化导致骨质疏松症（OP）的发病率呈流行趋势上升，虽然有相应治疗方法，但高成本、副作用以及低依从性仍是重大问题。包括植物雌激素在内的天然植物性化合物可能是治疗OP的良好且安全的候选物，但由于需要使用原代成骨细胞或间充质干细胞（MSCs，即成骨细胞的祖细胞）来进行耗时的成骨测定，因此难以实现对其成骨能力的筛选。利用MSCs的成骨能力并针对主要的成骨转录因子Runx2采用启动子报告基因检测法，我们开发了一个快速筛选平台，用于筛选包括天然植物性化合物在内的成骨小分子。我们使用转导了人RUNX2启动子荧光素酶报告基因（hRUNX2-luc）的小鼠MSC系C3H10T1/2，以大豆苷元（一种经过充分研究的成骨性植物雌激素）作为阳性对照，筛选了来自不同家族的八种植物衍生化合物，包括黄酮类、多酚类化合物、生物碱和异硫氰酸盐的成骨能力。使用原代小鼠和人骨髓间充质干细胞（BMMSCs）进行经典和成骨测定，以验证这个快速筛选平台的准确性。使用MSC/hRUNX2-luc筛选平台，我们不仅能够将成骨化合物的筛选过程从3至4周缩短至短短几天，还能同时对多种化合物进行比较，以评估相对成骨效力。预测分析显示，MSC/hRUNX2-luc报告基因平台与使用小鼠BMMSCs进行矿化的经典功能测定几乎完全相关。使用人BMMSCs进行矿化和成骨测定的验证也表明与MSC/hRUNX2-luc报告基因结果几乎完全相关。因此，我们的研究结果表明，MSC/hRUNX2报告基因平台可以准确、快速且有力地筛选成骨化合物候选物，因此与OP的治疗应用相关。
