Single cell live imaging in vivo, to understand cell activity in the context of regenerative medicine and cancer biology.

以干细胞为基础的更普通的细胞疗法已经在医学的许多学科中显示出令人振奋的结果。然而，最近的几项临床研究导致了令人失望的结果，这是因为移植细胞的命运尚不清楚，支持其效果的基本机制也不清楚。该项目将监测和跟踪体内移植的多种细胞类型随着时间的推移的相互作用，以及它们如何与宿主组织相互作用。非侵入性细胞成像技术对于实时、定量和长期监测移植细胞以及细胞迁移、分布、存活、分化等信息是必不可少的。为了获得体内单细胞水平的信息，我们需要使用适合成像的模型生物。在这里，我们将使用我们建立的成熟的鸡胚模型，要么直接将细胞注入血管或器官中，要么利用绒毛尿囊膜(CAM)模型。CaM能够支持放置在其表面的活组织/细胞的生长和维持，使其成为理想的生物反应器和评估再生医学和癌症研究中生物过程的核心资源。雏鸡胚胎在胚胎14天之前不受内政部的监管，因此有助于取代/减少动物的使用。到目前为止，由于灵敏度、光漂白和毒性较差，缺乏能够从体外过渡到体内的标记技术。StreamBio已经开发出共轭聚合物纳米颗粒(CPN)，这是一种高度稳定的荧光标记探针，比传统技术亮度高得多，并且含有用于增强MRI对比度的氧化铁成分，使其具有多模式。它们通过内吞作用被细胞摄取，因此可以标记任何细胞类型。使用利物浦大学临床前成像中心(CPI)和细胞成像中心(CCI)的先进成像技术进行细胞和体内成像；我们将确定神经未分化/去分化细胞如何与免疫细胞、宿主组织和血管系统相互作用，以及这种环境如何在分化、存活和迁移能力方面影响它们的身份。项目目标：1.优化CPN的长期细胞标记能力及其在体外对多种细胞类型示踪的适用性；单独使用神经干细胞和脑瘤细胞，或与免疫细胞共同培养。我们将测试CPN的多种颜色标记，并优化它们在细胞迁移跟踪中的使用。在2D细胞培养的初始特征之后，具有多种细胞类型的3D椭圆形模型将被用于测试探针多路传输和模拟组织结构。2.鸡胚胎注射CPN标记的神经干细胞和神经癌细胞的中短期示踪。最初，体内跟踪将在注射后实时进行。标记的细胞将被注射到雏鸡的胚胎外血管中，并直接注射到大脑中，随后活着进入雏鸡的循环，并在雏鸡的器官中稳定下来。3.使用CAM分析和多模式成像的长期细胞跟踪；通过将带有免疫细胞的神经细胞类型(干细胞/脑肿瘤细胞)植入CAM，每个免疫细胞都用特定的探针标记。使用探针的荧光检测和免疫标记将监测细胞的增殖、存活/死亡和分化。重要的是，我们还将能够通过使用纳米探针的荧光和磁性来跟踪细胞向鸡器官的扩散。稳定的荧光多路传输将使单独但同时跟踪细胞类型成为可能。进一步利用多路传输，我们将为特定的信号通路标记控制细胞和经药物/击倒处理的细胞，以获得机械性的Under

Stem cell-based, and more general cell therapies have shown promising results across many disciplines in medicine. However, several recent clinical studies have led to disappointing results, due to the fact that the fate of the transplanted cells is unknown and the fundamental mechanisms underpinning their effects not understood. This project will monitor and track the interaction over time of multiple cell types transplanted in vivo and how they interact with the host tissues. Non-invasive cell imaging techniques are essential to obtain real-time, quantitative, and long-term monitoring of transplanted cells and information on cell migration, distribution, viability, differentiation etc. To obtain information at the single cell level in vivo, we need to use model organisms, amenable to imaging. We will here use our well-established chick embryo model either by direct injection of cells in the vasculature or organs or by exploiting the chorioallantoic membrane (CAM) model. CAM has the ability to support the growth and maintenance of live tissue/cells placed on its surface, making it an ideal bioreactor and a core resource to evaluate biological processes in regenerative medicine and cancer research. The chick embryo is not under home office regulation until Embryonic day14, thereby contributing to Replace / Reduce animal use. The limitation to date has been down to the lack of a labelling technology capable of transitioning from in vitro to in vivo due to poor sensitivity, photobleaching and toxicity. StreamBio has developed Conjugated Polymer Nanoparticles (CPNs), which are highly stable, fluorescent labelling probes immensely brighter than conventional technologies, and have an iron oxide component for MRI contrast enhancing, making them multi-modal. They are taken up by cells through endocytosis and can therefore label any cell type. Using the advanced imaging technologies at the University of Liverpool Centre for Preclinical Imaging -CPI- and Centre for Cell Imaging -CCI- for cellular and in vivo imaging; we will determine how neural undifferentiated/dedifferentiated cells interact with immune cells, the host tissue and vasculature, and how this environment impact on their identity in terms of differentiation, survival and migratory capabilities. Project objective: 1. Optimisation of long-term cell labelling capabilities of CPNs and their suitability for multiple cell type tracking in vitro; using neural stem cells and brain tumour cells, on their own or in co-culture with immune cells. We will be testing multiple-colour labelling with the CPNs and optimise their use for cell migration tracking. Upon initial characterisation in 2D cell culture, 3D spheroid models with multiple cell types will be used to test probe multiplexing and mimic tissue organisation. 2. Short/medium-term tracking of neural stem cells and neuronal cancer cells labelled with the CPNs and injected in the chick embryo. Initially, tracking in vivo will be performed in real time upon injection. Labelled cells will be injected in the chick extra-embryonic blood vessels and directly in the brain, and followed live in the chick circulation and upon settling in the chick organs. 3. Long-term cell tracking using the CAM assay and multimodal imaging; by implanting neuronal cell types (stem cells / brain tumour cells), with immune cells, each labelled with specific probes, onto the CAM. Cell proliferation, survival/death and differentiation using fluorescence detection of the probes and immunolabelling will be monitored. Importantly, we will also be able to track cell dissemination into the chick organs by using both the fluorescence and magnetic properties of the nanoprobes. The stable fluorescence multiplexing will enable individual yet simultaneously tracking of cell types. Exploiting further the multiplexing we will label both control cells and cells treated pharmacologically / knocked-down for specific signalling pathways to obtain a mechanistic unde