Thimm Terra N, Squirrell Jayne M, Liu Yuming, Eliceiri Kevin W, Ogle Brenda M
1 Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison , Madison, Wisconsin.
2 Morgridge Institute for Research, University of Wisconsin-Madison , Madison, Wisconsin.
Tissue Eng Part C Methods. 2015 Oct;21(10):995-1004. doi: 10.1089/ten.TEC.2014.0699. Epub 2015 Jun 17.
Components of the extracellular matrix (ECM) have recently been shown to influence stem cell specification. However, it has been challenging to assess the spatial and temporal dynamics of stem cell-ECM interactions because most methodologies utilized to date require sample destruction or fixation. We examined the efficacy of utilizing the endogenous optical signals of two important ECM proteins, elastin (Eln), through autofluorescence, and type I collagen (ColI), through second harmonic generation (SHG), during mouse embryonic stem cell differentiation. After finding favorable overlap between antibody labeling and the endogenous fluorescent signal of Eln, we used this endogenous signal to map temporal changes in Eln and ColI during murine embryoid body differentiation and found that Eln increases until day 9 and then decreases slightly by day 12, while Col1 steadily increases over the 12-day period. Furthermore, we combined endogenous fluorescence imaging and SHG with antibody labeling of cardiomyocytes to examine the spatial relationship between Eln and ColI accumulation and cardiomyocyte differentiation. Eln was ubiquitously present, with enrichment in regions with cardiomyocyte differentiation, while there was an inverse correlation between ColI and cardiomyocyte differentiation. This work provides an important first step for utilizing endogenous optical signals, which can be visualized in living cells, to understand the relationship between the ECM and cardiomyocyte development and sets the stage for future studies of stem cell-ECM interactions and dynamics relevant to stem cells as well as other cell and tissue types.
细胞外基质（ECM）的成分最近被证明会影响干细胞的分化。然而，评估干细胞与ECM相互作用的时空动态一直具有挑战性，因为迄今为止使用的大多数方法都需要对样本进行破坏或固定。我们研究了在小鼠胚胎干细胞分化过程中，利用两种重要的ECM蛋白——弹性蛋白（Eln）通过自发荧光以及I型胶原蛋白（ColI）通过二次谐波产生（SHG）的内源性光学信号的效果。在发现抗体标记与Eln的内源性荧光信号之间存在良好的重叠后，我们利用这种内源性信号绘制了小鼠胚状体分化过程中Eln和ColI的时间变化，发现Eln在第9天之前增加，然后在第12天略有下降，而Col1在12天的时间内稳步增加。此外，我们将内源性荧光成像和SHG与心肌细胞的抗体标记相结合，以研究Eln和ColI积累与心肌细胞分化之间的空间关系。Eln普遍存在，在心肌细胞分化区域富集，而ColI与心肌细胞分化之间呈负相关。这项工作为利用可在活细胞中可视化的内源性光学信号理解ECM与心肌细胞发育之间的关系迈出了重要的第一步，并为未来研究干细胞与ECM的相互作用以及与干细胞以及其他细胞和组织类型相关的动态变化奠定了基础。
