4D monitoring of stem cell differentiation by dielectric spectroscopic optical coherence tomography

近年来，干细胞的治疗潜力得到了多方的认可。然而，监测它们分化成不同谱系的方法是乏味的，而且具有破坏性，妨碍了三维组织再生的动态研究。在本项目中，我们建议开发一种不使用生物标记剂的成像技术，在单细胞水平上对分化的干细胞进行时间和空间（4D）监测。每一个细胞系都有一个生物电特征，它将细胞对与细胞膜和细胞质的蛋白质含量相关的频率依赖的电磁场的反应转化为生物电特征。这种生物电特征已经在介质光谱和介质电泳中用于细胞分选。分化的干细胞根据其不同的分化状态具有动态特征。EMF激发过程中涉及的生物机制也通过细胞成分的重组产生细胞散射特性的变化。这种光学特性的变化将被利用于光学相干断层扫描（OCT）在单细胞水平上进行3D干细胞分化监测。OCT是一种非侵入性干涉技术，允许在没有标记剂的情况下进行深度组织成像。OCT的最新进展使得在样品光路上具有纳米分辨率的相显微镜成为可能。通过将介电光谱系统与OCT同步，我们将记录频率相关的光学特征（FDOS），并将其与二维和三维的微分事件相关联。商业脂肪来源干细胞（ADSC）将根据2D培养板和支持3D培养的商业海藻酸盐基质中建立的良好协议分化为三种不同的谱系（骨，软骨，脂肪组织）。新成像技术（DSOCT）的多模态将用于实时记录微分事件（光学，介电及其组合（FDOS））。DSOCT的敏感性也将与Hay博士合作，通过将hESC分化为三个不同发育阶段的肝细胞：中胚层、内胚层和肝内胚层来测试。新的成像技术能够在单细胞水平上连续无创地跟踪干细胞分化的各个发育阶段，而无需标记剂，这将导致再生医学的许多突破。

In the recent years the therapeutic potential of stem cells has been recognized multilaterally. Still, the means to monitor their differentiation into different lineages are tedious and destructive precluding dynamic studies of 3D tissue regeneration. In this project, we propose to develop an imaging technology without biological labelling agents to monitor in time and space (4D) differentiating stem cell at a single-cell level. Every cell line has a bioelectrical signature that translates the response of a cell to a frequency dependent electro-magnetic field related to the proteins content of the cell membrane and cytoplasm. This bioelectrical signature is already exploited in dielectric spectroscopy and dielectrophoresis for cell sorting. A differentiating stem cell has a dynamic signature according to its various differentiation states. The biological mechanisms involved during EMF excitation generate also a change of the scattering properties of the cell by the reorganization of the cell components. This variation of the optical properties will be exploited to monitor in 3D at the single-cell level stem cell differentiation by Optical Coherence Tomography (OCT). OCT is a non invasive interferometric technique allowing in depth tissue imaging without labelling agent. Recent advances in OCT enables phase microscopy with nanometer resolution on the sample optical path. By synchronising a dielectric spectroscopic system with an OCT we will record a frequency dependent optical signature (FDOS) and will correlate it to differentiating event in 2D and 3D. Commercially adipose derived stem cells (ADSC) will be differentiated into three different lineages (bone, cartilage, adipose tissue) according to well established protocols in 2D culture plate and in commercial alginate matrixes supporting 3D culture. The multimodality of the new imaging technology (DSOCT) will be used to record differentiating events (optical, dielectric, and their combination (FDOS)) in real time. The sensitivity of the DSOCT will also be tested in collaboration with Dr. Hay by differentiating hESC into liver cells at three different developmental stages: mesendoderm, endoderm and hepatic endoderm. The new imaging technology ability to track various developmental stages of stem cell differentiation continuously and non invasively at the single-cell level without labelling agents will lead to many breakthroughs in regenerative medicine.