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

基于干细胞的和更一般的细胞疗法在医学的许多学科中都显示出了有希望的结果。然而，最近的一些临床研究导致了令人失望的结果，因为移植细胞的命运是未知的，其作用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时间的相互作用，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是必不可少的，以获得实时，定量和长期监测移植的细胞和信息的细胞迁移，分布，活力，分化等，以获得在体内单细胞水平的信息，我们需要使用模型生物，适合成像。我们将在这里使用我们完善的鸡胚胎模型，通过直接注射细胞到血管或器官中或通过利用绒毛尿囊膜（CAM）模型。CAM能够支持置于其表面的活组织/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。鸡胚在胚胎第14天之前不受内政部监管，因此有助于替代/减少动物使用。迄今为止的局限性是由于缺乏能够从体外过渡到体内的标记技术，这是由于灵敏度差、光漂白和毒性。StreamBio开发了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，比传统技术亮得多，并且具有用于增强MRI对比度的氧化铁成分，使其具有多模态。它们通过内吞作用被细胞吸收，因此可以标记任何细胞类型。使用先进的成像技术在利物浦临床前成像中心-CPI-和细胞成像中心-CCI-细胞和体内成像的大学;我们将确定如何神经未分化/去分化细胞与免疫细胞，宿主组织和脉管系统相互作用，以及这种环境如何影响他们的身份在分化，生存和迁移能力方面。项目目标：1.优化CPN的长期细胞标记能力及其体外多细胞类型追踪的适用性;使用神经干细胞和脑肿瘤细胞，单独或与免疫细胞共培养。我们将测试CPN的多色标记，并优化其用于细胞迁移跟踪。在2D细胞培养中进行初始表征后，将使用具有多种细胞类型的3D球体模型来测试探针多重化和模拟组织组织。2.用CPN标记并注射到鸡胚中的神经干细胞和神经元癌细胞的短期/中期跟踪。最初，将在注射后以真实的时间进行体内跟踪。将标记的细胞注射到鸡胚外血管中并直接注射到脑中，然后在鸡循环中存活并在鸡器官中定居。3.使用CAM检测和多模式成像进行长期细胞跟踪;通过将神经元细胞类型（干细胞/脑肿瘤细胞）与免疫细胞（每个免疫细胞均用特异性探针标记）植入CAM上。将使用探针的荧光检测和免疫标记监测细胞增殖、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传播到小鸡器官中。稳定的荧光多路复用将能够单独但同时跟踪细胞类型。进一步利用多重化，我们将标记对照细胞和经处理/敲除的细胞的特异性信号传导途径，以获得一种机制性的信号传导途径。

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