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

多能干细胞的发现是对现代生物学和医学产生巨大影响的重大科学突破。这些细胞能够转化为人体中发现的任何类型的细胞，这使得它们在许多医学应用中具有吸引力。基于来自多能干细胞的细胞的疗法可以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，受梗死影响的心脏的功能可以通过移植从干细胞中获得的心脏细胞而得到改善。在实验室中，多能干细胞在培养用于修复身体病变或受损部位的替代组织方面也可能发挥重要作用。然而，多能干细胞转化为我们体内各种细胞类型（分化）的过程尚不清楚。影响对特定细胞类型的决策和承诺的因素仍不清楚。例如，为什么某些干细胞暴露在特定的刺激下会变成心脏细胞，而在相同的人群中，其他干细胞却不会？因此，获得特定细胞类型的条件没有标准化，通常只能在高度异质的群体中产生低产量的所需细胞类型，由于主要存在不需要的细胞类型，这些细胞类型不适合临床使用。为了迅速克服这些障碍，使经过验证的多能干细胞能够用于临床，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。这些技术需要是健壮的、自动化的，能够与现有的制造技术集成，并符合药品监管机构的严格标准。目前用于评估细胞群的大多数技术需要大量的细胞来证明平均结果，这不适用于异质细胞群。此外，大多数技术不能在活细胞上进行。从多能干细胞中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遗传修饰。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遗传操作方案，并且还可能干扰细胞的正常行为。基于以下论点，我们提出了一种完全不同的方法来区分单个活细胞：在体内，细胞是专门执行特定功能的，因此它们产生特定的生化物质。例如，心脏细胞含有大量的肌原纤维，骨细胞分泌胶原蛋白，胰腺细胞产生胰岛素，红细胞含有血红蛋白，等等。两个问题出现了：有没有一种技术可以在不杀死细胞的情况下检测出细胞类型之间的差异？如果是，这些生化差异是否可以用于识别各种细胞类型？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中获得的心脏和骨细胞的光谱标记，并量化这些光谱标记在干细胞向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有效分化的方案，并且在在线质量测试以及适合临床应用的所需类型的终点分化细胞的分离方面具有很大的潜力。

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.