Eom Jennifer, Feisst Vaughan, Ranjard Louis, Loomes Kerry, Damani Tanvi, Jackson-Patel Victoria, Locke Michelle, Sheppard Hilary, Narayan Pritika, Dunbar P Rod
School of Biological Sciences, The University of Auckland.
Research School of Biology, The Australian National University.
J Vis Exp. 2018 Mar 31(133):57153. doi: 10.3791/57153.
Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.
目前有几种染料可用于检测间充质细胞向脂肪细胞的分化。诸如油红O等染料价格便宜、使用方便，被分析间充质细胞成脂潜能的实验室广泛使用。然而，它们并非特异性针对基因转录的变化。我们开发了一种基因特异性分化检测方法，以分析间充质细胞何时将其命运转变为脂肪生成谱系。针对脂肪酸结合蛋白4（FABP4）（一种脂肪生成分化的谱系特异性标志物）进行免疫标记，能够实现对分化细胞的可视化和定量分析。以96孔微孔板形式定量间充质细胞成脂分化潜能的能力，对许多应用具有广阔的前景。数百项临床试验涉及成人间充质基质细胞的使用，目前很难在这些临床试验内部尤其是之间关联治疗结果。这种简单的高通量FABP4检测方法为评估患者来源细胞的分化潜能提供了一种定量检测方法，并且是比较不同分离和扩增方法的有力工具。鉴于人们越来越认识到间充质细胞产品中用于治疗患者的细胞的异质性，这一点尤为重要。该检测方法在高通量药物筛选中也具有潜在用途，特别是在肥胖症和糖尿病前期研究中。
