Department of Oncology and Diagnostic Sciences, School of Dentistry, University of Maryland, Baltimore, MD, USA.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
J Tissue Eng Regen Med. 2018 Feb;12(2):437-446. doi: 10.1002/term.2470. Epub 2017 Aug 11.
Metformin, a first-line antidiabetic drug used by millions of patients, has been shown to have potential osteogenic properties. The present study was performed to test the hypothesis that clinically relevant doses of metformin promote the osteogenic differentiation and mineralization of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). iPSC-MSCs were treated with metformin (10 μm) to assess cell viability, osteogenic differentiation, mineralization and activation of the LKB1/AMP-activated protein kinase (AMPK) signalling pathway, a surrogate marker of metformin action. To determine its potential application in MSC-based bone and periodontal tissue engineering, iPSC-MSCs were also treated with metformin when seeded on to calcium phosphate cement (CPC) scaffolds. Immunoblotting and cellular uptake assays showed that iPSC-MSCs express functional organic cation transporter-1 (OCT-1), a transmembrane protein that mediates the intracellular uptake of metformin. Although metformin treatment did not impair iPSC-MSC viability, it significantly stimulated alkaline phosphatase activity, enhanced mineralized nodule formation and increased expression of osteogenic markers, including Runt-related transcription factor 2 (RUNX2) and osterix. Inhibition of LKB1 activity, a common upstream AMPK kinase, markedly reversed metformin-induced AMPK activation, RUNX2 expression and nuclear localization. Moreover, metformin substantially increased mineralized nodule formation of iPSC-MSC seeded on CPC scaffolds. Collectively, functional OCT-expressing iPSC-MSCs responded to metformin by inducing an osteogenic effect in part mediated by the LKB1/AMPK pathway. Considering the widespread use of metformin in diabetics, this work may lead to novel tissue-engineering platforms where autogenous OCT-expressing iPSC-MSCs might be used to enhance bone and periodontal regeneration in diabetic patients prescribed with daily doses of metformin.
二甲双胍是一种被数百万患者使用的一线抗糖尿病药物，已被证明具有潜在的成骨特性。本研究旨在检验以下假设：临床相关剂量的二甲双胍可促进诱导多能干细胞衍生的间充质干细胞（iPSC-MSCs）的成骨分化和矿化。用二甲双胍（10μm）处理 iPSC-MSCs 以评估细胞活力、成骨分化、矿化和 LKB1/AMP 激活蛋白激酶（AMPK）信号通路的激活，该通路是二甲双胍作用的替代标志物。为了确定其在基于 MSC 的骨和牙周组织工程中的潜在应用，当 iPSC-MSCs 接种到磷酸钙水泥（CPC）支架上时，也用二甲双胍处理它们。免疫印迹和细胞摄取试验表明，iPSC-MSCs 表达功能性有机阳离子转运蛋白-1（OCT-1），这是一种跨膜蛋白，可介导二甲双胍的细胞内摄取。尽管二甲双胍处理不会损害 iPSC-MSC 的活力，但它显著刺激碱性磷酸酶活性，增强矿化结节形成并增加成骨标志物的表达，包括 Runt 相关转录因子 2（RUNX2）和骨形成蛋白 2（osterix）。LKB1 活性的抑制，一种常见的 AMPK 上游激酶，显著逆转了二甲双胍诱导的 AMPK 激活、RUNX2 表达和核定位。此外，二甲双胍还大大增加了接种在 CPC 支架上的 iPSC-MSC 的矿化结节形成。总的来说，表达功能性 OCT 的 iPSC-MSCs 通过 LKB1/AMPK 通路部分介导的成骨作用对二甲双胍产生反应。考虑到二甲双胍在糖尿病患者中的广泛使用，这项工作可能会导致新的组织工程平台的出现，其中自体表达 OCT 的 iPSC-MSCs 可能被用于增强糖尿病患者中每天给予二甲双胍治疗的骨和牙周再生。
