Desai Hetal V, Voruganti Indu S, Jayasuriya Chathuraka, Chen Qian, Darling Eric M
1 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University , Providence, Rhode Island.
Tissue Eng Part A. 2014 Mar;20(5-6):899-907. doi: 10.1089/ten.TEA.2013.0761. Epub 2014 Jan 29.
Adipose-derived stem cells (ASCs) are a widely investigated type of mesenchymal stem cells with great potential for musculoskeletal regeneration. However, the use of ASCs is complicated by their cellular heterogeneity, which exists at both the population and single-cell levels. This study demonstrates a live-cell assay to investigate gene expression in ASCs undergoing osteogenesis using fluorescently tagged DNA hybridization probes called molecular beacons. A molecular beacon was designed to target the mRNA sequence for alkaline phosphatase (ALPL), a gene characteristically expressed during early osteogenesis. The percentage of cells expressing this gene in a population was monitored daily to quantify the uniformity of the differentiation process. Differentiating ASC populations were repeatedly measured in a nondestructive fashion over a 10-day period to obtain temporal gene expression data. Results showed consistent expression patterns for the investigated osteogenic genes in response to induction medium. Peak signal level, indicating when the most cells expressed ALPL at once, was observed on days 3-5. The differentiation response of sample populations was generally uniform when assessed on a well-by-well basis over time. The expression of alkaline phosphatase is consistent with previous studies of osteogenic differentiation, suggesting that molecular beacons are a viable means of monitoring the spatiotemporal gene expression of live, differentiating ASCs.
脂肪来源干细胞（ASCs）是一种被广泛研究的间充质干细胞，在肌肉骨骼再生方面具有巨大潜力。然而，ASCs的使用因细胞异质性而变得复杂，这种异质性在群体水平和单细胞水平上均存在。本研究展示了一种活细胞检测方法，使用称为分子信标的荧光标记DNA杂交探针来研究正在进行成骨分化的ASCs中的基因表达。设计了一种分子信标靶向碱性磷酸酶（ALPL）的mRNA序列，ALPL是一种在早期成骨过程中特征性表达的基因。每天监测群体中表达该基因的细胞百分比，以量化分化过程的均匀性。在10天的时间内，以非破坏性方式对正在分化的ASC群体进行反复测量，以获得时间基因表达数据。结果显示，在所研究的成骨基因对诱导培养基的反应中，表达模式一致。在第3 - 5天观察到峰值信号水平，表明此时最多的细胞同时表达ALPL。随着时间的推移，在逐个孔的基础上评估时，样本群体的分化反应总体上是均匀的。碱性磷酸酶的表达与先前关于成骨分化的研究一致，表明分子信标是监测活的、正在分化的ASCs时空基因表达的一种可行方法。
