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 CH₃ (2935 cm⁻¹) 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.

中文翻译：

---

人脂肪来源的间充质干细胞向多个谱系分化的原位无标记监测

干细胞分化成特定谱系的精确表征，特别是以非破坏性和非侵入性的方式，对于生成患者特异性细胞而无分化细胞的质量损失极为重要。在这里，我们报告了一种能够原位检测的新方法干细胞分化为多个谱系的无标记定量分析，即使在单个细胞水平也是如此。首先将人脂肪来源的间充质干细胞（hADMSC）分化为两种不同类型的细胞（成骨细胞和脂肪细胞），然后通过显微拉曼光谱法对这些分化的细胞进行深入分析。有趣的是，发现分配给脂质滴和羟基磷灰石的拉曼峰对脂肪细胞（脂肪细胞）和成骨细胞（骨细胞）具有高度特异性，因此可用于与CH结合生成无标记的单细胞拉曼图像₃（2935厘米^-1）峰，以可视化细胞形状。值得注意的是，基于这些拉曼图像，我们发现分化9天后即可确定并量化hADMSC的成骨作用，这比典型的茜素红染色方法早了一周。在脂肪形成的情况下，在整个脂肪形成期间，可以清楚地观察到并检测到单个细胞水平上细胞质中脂质滴的增加，而这无法使用任何其他当前可用的方法（例如油红O和免疫染色）来实现。因此，这项研究中报道的新方法作为一种精确用于原位监测干细胞分化的分析工具非常有前途，并且可以促进基于干细胞的材料用于再生治疗。