Non-invasive biophotonics tool for phenotypic identification of pluripotent stem cells and their progeny

用于多能干细胞及其后代表型鉴定的非侵入性生物光子学工具

• 批准号: BB/G010285/1
• 负责人: Ioan Notingher
• 金额: $ 69.09万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG010285%2F1
• 关键词: Non; invasive; biophotonics; tool; phenotypic

The discovery of pluripotent stem cells represented a major scientific breakthrough with immense impact on modern biology and medicine. The ability of these cells to transform into any type of cells found in th body, makes them attractive to many medical applications. Therapies based on cells derived from pluripotent stem cels may provide treatments to many diseases, including Parkinson disease, diabetes and cardiovascular disorders. For example, the function of a heart affected by infarct may be improved by implanting heart cells obtained from stem cells. Pluripotent stem cells may also have an important role in growing replacement tissues in laboratories for repairing diseased or damaged parts of the body. Howhere, the process through which pluripotent stem cells transform into various cell types found in our bodies (differentiation) is not well understood. The factors which affect the decision-making and commitment towards specific cell types are still unclear. For example, why certain stem cells exposed to particular stimuli become heart cells while others, in the same population, do not? Thus, the conditions to derive specific cell types are not standardized, generally producing only low yields of the desired cel types within highly heterogeneous populations that are not suitable for clinical use due to the presence of mainly unwanted cell types. In order to rapidly overcome these obstacles and enable the delivery of validated pluripotent stem cells for clinical use, further technological advances are required, in particular in manufacturing and quality assessment of these therapeutic products. Such technologies need to be robust, automated, to enable integration with existing manufacturing technologies, and to comply with the strict criteria of drug regulatory agencies. Most techniques currently used for assesing cell populations require large number of cells proving average results, which are not suitable for heterogenous cell populations. In addition, most techniques cannot be carried out on living cells. Identification of cell types obtained from pluripotent stem cells is commonly based on specific molecules on the cell surface or genetical modification of cells. These techniques are limited to cell types which have surface specific molecules, while genetic manipulation protocols need to be developed for each cell type and can also interfere with normal behavior of cells. We propose a radically different approach to discriminate single live cells based on the following arguments: In the body, cells are specialized to perform specific functions and therefore they produce specific biochemicals. For example, heart cells contain a large number of myofibrils, bone cells secrete collagen, pancreas cells produce insulin, red blood cells contain haemoglobin, and so on. Two questions arise: is there a technique which could detect these differences between cell types, without killing the cells? If yes, could these biochemical differences be used for identification of various cell types? We will use Raman micro-spectroscopy to discriminate live heart and bone cells obtained from pluripotent stem cells, without use of external chemicals, genetic modification of cells or surface markers. This technique is based on the interaction of laser light with the biomolecules present in the cells to produce 'biochemical fingerprints' of the cells based on their chemical composition. We will determine spectral markers for heart and bone cells obtained from pluripotent stem cells and quantify the time-dependence of these spectral markers during the differentiation of stem cells towards the two cell types. This technique will help the development and refinement of protocols to induce the efficient differentiation of pluripotent stem cells, and has great potential for on-line quality testing as well as separation of end-point differentiated cells of a desired type suitable for clinical applications.

多能干细胞的发现代表了一项重大的科学突破，对现代生物学和医学产生了巨大影响。这些细胞能够转化为体内发现的任何类型的细胞，这使得它们对许多医学应用具有吸引力。基于来自多能干细胞的细胞的治疗可能为许多疾病提供治疗，包括帕金森病、糖尿病和心血管疾病。例如，通过移植从干细胞中获得的心脏细胞，可以改善受梗塞影响的心脏的功能。多能干细胞也可能在实验室培养替代组织以修复身体疾病或受损部位方面发挥重要作用。在哪里，多能干细胞转化为在我们身体中发现的各种细胞类型的过程(分化)还不是很清楚。影响对特定细胞类型的决策和承诺的因素仍然不清楚。例如，为什么暴露在特定刺激下的某些干细胞会变成心脏细胞，而同一人群中的其他干细胞却不会？因此，衍生特定细胞类型的条件没有标准化，通常只在高度异质性的群体中产生所需细胞类型的低产量，由于主要不需要的细胞类型的存在而不适合临床使用。为了迅速克服这些障碍，并能够提供经过验证的多潜能干细胞用于临床，需要进一步的技术进步，特别是在这些治疗产品的制造和质量评估方面。这些技术需要强大、自动化，以实现与现有制造技术的集成，并遵守药品监管机构的严格标准。目前用于评估细胞群体的大多数技术需要大量的细胞来证明平均结果，这不适合于异质细胞群体。此外，大多数技术不能在活细胞上进行。从多能干细胞获得的细胞类型的鉴定通常基于细胞表面的特定分子或细胞的基因修饰。这些技术仅限于具有表面特定分子的细胞类型，而需要为每种细胞类型开发基因操作协议，并且还可能干扰细胞的正常行为。基于以下论点，我们提出了一种截然不同的方法来区分单个活细胞：在人体内，细胞专门执行特定的功能，因此它们产生特定的生物化学物质。例如，心脏细胞含有大量的肌原纤维，骨细胞分泌胶原，胰腺细胞产生胰岛素，红细胞含有血红蛋白等等。出现了两个问题：有没有一种技术可以在不杀死细胞的情况下检测这些细胞类型之间的差异？如果是，这些生化差异能否用于鉴定不同类型的细胞？我们将使用拉曼显微光谱来区分从多能干细胞中获得的活的心脏和骨骼细胞，而不使用外部化学物质、细胞的遗传修饰或表面标记。这项技术是基于激光与细胞中存在的生物分子的相互作用，根据细胞的化学成分产生细胞的“生化指纹”。我们将确定从多能干细胞获得的心脏和骨骼细胞的光谱标记，并量化这些光谱标记在干细胞向这两种细胞类型分化过程中的时间依赖性。这项技术将有助于开发和完善诱导多能干细胞高效分化的方案，并在在线质量检测和分离适合临床应用的所需类型的终点分化细胞方面具有巨大的潜力。

项目成果

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy.

• DOI: 10.1016/j.bbagen.2013.01.030
• 发表时间: 2013-06
• 期刊: Biochimica et biophysica acta
• 作者: Flavius C Pascut;S. Kalra;Vinoj T George;Nathan Welch;C. Denning;I. Notingher

Applications of Raman micro-spectroscopy to stem cell technology: label-free molecular discrimination and monitoring cell differentiation.

• DOI: 10.1140/epjti/s40485-015-0016-8
• 发表时间: 2015
• 期刊: EPJ techniques and instrumentation
• 影响因子: 1
• 作者: Ghita A;Pascut FC;Sottile V;Denning C;Notingher I
• 通讯作者: Notingher I

Tracing amino acid exchange during host-pathogen interaction by combined stable-isotope time-resolved Raman spectral imaging.

• DOI: 10.1038/srep20811
• 发表时间: 2016-02-09
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Naemat A;Elsheikha HM;Boitor RA;Notingher I
• 通讯作者: Notingher I

Visualizing the interaction of Acanthamoeba castellanii with human retinal epithelial cells by spontaneous Raman and CARS imaging

• DOI: 10.1002/jrs.5296
• 发表时间: 2018-03
• 期刊: Journal of Raman Spectroscopy
• 影响因子: 2.5
• 作者: Abida Naemat;F. Sinjab;A. McDonald;A. Downes;A. Elfick;H. Elsheikha;I. Notingher