Smus Justyna P, Moura Catarina Costa, McMorrow Emma, Tare Rahul S, Oreffo Richard O C, Mahajan Sumeet
Department of Chemistry and Institute for Life Sciences , Highfield Campus , University of Southampton , SO17 1BJ , UK . Email:
Centre for Human Development , Stem Cells and Regeneration , Institute of Developmental Sciences , University of Southampton , SO16 6YD , UK . Email:
Chem Sci. 2015 Dec 1;6(12):7089-7096. doi: 10.1039/c5sc02168e. Epub 2015 Sep 9.
Coherent anti-Stokes Raman scattering (CARS) is a chemically selective label-free imaging technique which is rapidly emerging as a powerful alternative to conventional microscopy in biomedicine. The strength of this imaging approach is the provision of rapid insight into chemical distribution especially of small biomolecules such as lipids. The label-free, non-destructive and non-invasive nature of CARS lends itself for use with stem cells, as labelling or staining will render them otherwise unsuitable for therapy. Isolation, enrichment and characterisation of skeletal stem cells (SSCs) and their progeny is of tremendous significance in regenerative medicine. However, SSCs differentiation into bone, cartilage or fat cell types, is currently assessed using several invasive and, typically, destructive methodologies. Thus, CARS presents an exciting alternative to interrogate the differentiation of SSCs in their natural state. In the current study, we have examined the adipogenic differentiation of SSCs over time using CARS imaging and verified the observed differentiation using molecular analysis of gene expression as well as compared the results to conventional Oil Red O lipid staining. We find that the CARS analysis provides an enhanced resolution and definition of lipid droplets, detectable as early as 24 hours and 72 hours after adipogenic induction. Quantification of the CARS image data sets also showed a change in lipid droplet size distribution during the course of adipogenesis over 14 days. Furthermore, CARS provided a superior and facile approach to monitor changes in SSCs as a result of chemical modulation of adipogenic differentiation. The current studies pave the way for the use of CARS as a powerful chemical imaging tool in therapeutics, regenerative medicine and skeletal stem cell biology.
相干反斯托克斯拉曼散射（CARS）是一种具有化学选择性的无标记成像技术，在生物医学领域正迅速崛起，成为传统显微镜的有力替代方法。这种成像方法的优势在于能够快速洞察化学物质分布，尤其是脂质等小分子生物分子的分布。CARS的无标记、非破坏性和非侵入性特点使其适用于干细胞研究，因为标记或染色会使干细胞不适用于治疗。在再生医学中，分离、富集和鉴定骨骼干细胞（SSCs）及其子代具有极其重要的意义。然而，目前评估SSCs向骨、软骨或脂肪细胞类型的分化，使用的是几种侵入性且通常具有破坏性的方法。因此，CARS为研究自然状态下SSCs的分化提供了一种令人兴奋的替代方法。在本研究中，我们使用CARS成像随时间检查了SSCs的脂肪生成分化，并通过基因表达的分子分析验证了观察到的分化，同时将结果与传统的油红O脂质染色进行了比较。我们发现，CARS分析能够提高脂质小滴的分辨率和清晰度，在脂肪生成诱导后24小时和72小时即可检测到。对CARS图像数据集的定量分析还显示，在14天的脂肪生成过程中，脂质小滴的大小分布发生了变化。此外，CARS提供了一种优越且简便的方法来监测由于脂肪生成分化的化学调节导致的SSCs变化。目前的研究为将CARS用作治疗学、再生医学和骨骼干细胞生物学中的强大化学成像工具铺平了道路。
