De Melo Nigel, McGinlay Sarah, Markus Robert, Macri-Pellizzeri Laura, Symonds Michael E, Ahmed Ifty, Sottile Virginie
Wolfson STEM Centre, School of Medicine, The University of Nottingham, Nottingham NG7 2RD, UK.
School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.
Biomimetics (Basel). 2019 Jul 10;4(3):48. doi: 10.3390/biomimetics4030048.
Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media, modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells.
间充质干细胞（MSCs）是形成骨的成骨细胞和储存脂质的脂肪细胞的祖细胞，这两种主要细胞谱系共存于骨髓中。当在体外分离时，如果用特殊培养基处理，这些干细胞可重现成骨细胞或脂肪细胞的形成过程，模拟这些细胞谱系在体内的相互作用方式。成骨分化的特征是细胞外基质中积累矿物质沉积，通常使用组织学技术进行评估。脂肪生成伴随着细胞内脂质的积累，这可以通过油红O染色常规观察到。在这两种情况下，染色都需要固定细胞，因此仅限于终点评估。在此，开发了一种活体染色方法，用于同时检测分化培养物中的矿物质沉积和脂滴。诱导分化的干细胞产生了包含脂肪细胞和类骨结节的混合培养物，两周后，将活细胞培养物分别与盐酸四环素和硼二吡咯孵育，以标记含矿物质和脂质的结构。荧光显微镜显示在活细胞培养物中同时观察到矿化区域和充满脂质的脂肪细胞。结合核染料Hoechst 33258，这种方法进一步实现了对散布在矿化基质中的脂肪生成细胞进行活细胞共聚焦成像。在随后的时间点重复这种多重标记，证明了这种新方法在对活干细胞进行实时高精度成像方面的潜力。
