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

Lead Research Organisation: University of Nottingham
Department Name: Sch of Physics & Astronomy


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.

Technical Summary

While embryonic stem cells are derived from the inner cell mass of blastocyst stage embryos, induced pluripotent stem cells are generated by viral transduction of somatic cells with four key transcription factors. For both pluripotent stem cell types, a large number of cell types can be derived following differentiation, including cardiomyocytes, osteoblasts, neurons, beta-cells, and haematopoietic cells. However, the conditions to derive specific cell types remain suboptimal, reflecting the limited understanding of cell differentiation. Therefore current techniques generally produce only low yields of the desired differentiated lineages within a highly heterogeneous population of mainly unwanted cell types, which are not suitable for clinical applications. In this project, we will develop a non-invasive tool based on Raman micro-spectroscopy for phenotypic discrimination of individual differentiated cells derived from pluripotent stem cells. Since the discrimination will be based on the intrinsic biochemical composition of the cells, the technique has the potential to be used for simultaneously identification of a large number of cell types without affecting their viability. Multivariate methods will be used for the analysis of the spectral markers to establish lineage-specific spectral markers. The quantification of these markers will allow time-course measurements on individual cells to follow the biochemical changes during their differentiation and response to various physiochemical stimuli. The technology will provide on-line information regarding differentiation of pluripotent stem cells and assess their phenotypic characteristics. This will have a huge beneficial impact on refinement and standardisation of differentiation protocols and could help overcoming the current bottlenecks in the manufacturing and quality assessment of cell products, which are key factors for the future advancement and widespread clinical use of regenerative medicine therapies.


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Description We have discovered new Raman spectral markers that can be used in a non-invasive way to monitor the differentiation of human embryonic stem cells towards the cardiac phenotype. We have demonstrated the ability to repeatedly measure the time evolution of these markers and map differentiating cells over a duration of five days, at the point where fully differentiated beating heart cells were obtained. This was the first demonstration of labelling-free monitoring of cells during this biological process.
In addition, we demonstrated the ability of Raman spectroscopy to monitor changes in the mineral components of bone nodules produced by osteoblast generated from mesenchymal cells. This process was monitored over a four-week period.
Exploitation Route 1. Optimising the differentiation methods: the methods can be used to improve the differentiation protocol, whereby Raman spectroscopy readings can be used to provide feedback about the differentiation status of the cells.
2. Development of cell sorting techniques: we have demonstrated that the Raman spectral markers can be used to identify cardiomyocytes with accuracy higher that 95%. Furthermore, the ability to make measurements as short as 1 second for individual cells was demonstrated, paving the way for novel Raman-activated cell sorting technologies.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description Diagnosis of tumours during tissue conserving surgery by multimodal spectral imaging
Amount £1,394,000 (GBP)
Funding ID EP/L025620/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2019
Description Raman spectral imaging for automated Mohs Micrographic surgery of high-risk Basal Cell Carcinoma
Amount £665,143 (GBP)
Funding ID II-LA-0813-20001 
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 07/2014 
End 06/2016