School of Integrative Engineering, Chung-Ang University, 84 Heukseuk-ro, Dongjak-gu, Seoul 06974, Republic of Korea.
School of Integrative Engineering, Chung-Ang University, 84 Heukseuk-ro, Dongjak-gu, Seoul 06974, Republic of Korea.
Biomaterials. 2018 Feb;154:223-233. doi: 10.1016/j.biomaterials.2017.11.005. Epub 2017 Nov 5.
Precise characterizations of stem cell differentiation into specific lineages, especially in non-destructive and non-invasive manner, are extremely important for generating patient-specific cells without mass loss of differentiated cells. Here, we report a new method capable of in situ label-free quantification of stem cell differentiation into multiple lineages, even at a single cell level. The human adipose-derived mesenchymal stem cells (hADMSCs) were first differentiated into two different types of cells (osteoblasts and adipocytes) and these differentiated cells were then intensively analyzed by micro-Raman spectroscopy. Interestingly, the Raman peaks assigned to lipid droplets and hydroxyapatite were found to be highly specific to the adipocyte (fat cell) and osteoblast (bone cell) and were thus found to be useful for generating label-free single cell Raman images in combination with CH (2935 cm) peaks for visualizing cell shape. Remarkably, based on these Raman images, we found that the osteogenesis of hADMSCs could be determined and quantified after 9 days of differentiation, which is a week earlier than with the typical alizarin red staining method. In the case of adipogenesis, the increase of lipid droplets in the cytoplasm at the single cell level could be clearly visualized and detected during the entire period of adipogenesis, which is impossible using any other currently available methods such as Oil Red O and immunostaining. Hence, the new method reported in this study is highly promising as an analytical tool for precise in-situ monitoring of stem cell differentiation, and could facilitate the use of stem cell-based materials for the regenerative therapies.
精确描述干细胞向特定谱系的分化，特别是在非破坏性和非侵入性的方式下，对于在不大量损失分化细胞的情况下产生患者特异性细胞是非常重要的。在这里，我们报告了一种新的方法，能够对干细胞向多种谱系的分化进行原位无标记定量，甚至在单细胞水平上也是如此。首先，将人脂肪间充质干细胞（hADMSCs）分化为两种不同类型的细胞（成骨细胞和脂肪细胞），然后通过微拉曼光谱对这些分化细胞进行深入分析。有趣的是，分配给脂滴和羟磷灰石的拉曼峰被发现对脂肪细胞（脂肪细胞）和成骨细胞（骨细胞）具有高度特异性，因此在结合 CH（2935cm）峰生成无标记单细胞拉曼图像以可视化细胞形状方面非常有用。值得注意的是，基于这些拉曼图像，我们发现 hADMSCs 的成骨作用可以在分化后 9 天确定和定量，比典型的茜素红染色方法早一周。在脂肪生成的情况下，细胞质中单细胞水平的脂滴增加可以在整个脂肪生成过程中清晰地可视化和检测到，这是使用任何其他当前可用的方法（如油红 O 和免疫染色）都不可能做到的。因此，本研究中报道的新方法作为一种精确的原位监测干细胞分化的分析工具具有很大的前景，并可以促进基于干细胞的材料在再生疗法中的应用。
