CellViewer: super-resolution systems microscopy to assess pluripotency and differentiation of stem cells at single cell level

Projektinformationen

CellViewer

ID Finanzhilfevereinbarung: 686637

DOI

10.3030/686637

Projekt abgeschlossen

EK-Unterschriftsdatum 6 November 2015

Startdatum 1 Februar 2016

Enddatum 30 September 2020

Finanziert unter

• EXCELLENT SCIENCE - Future and Emerging Technologies (FET)

Gesamtkosten

€ 3 988 752,50

EU-Beitrag

€ 3 988 752,50

Koordiniert durch

FUNDACIO CENTRE DE REGULACIO GENOMICA
Spain

Periodic Reporting for period 3 - CellViewer (CellViewer: super-resolution systems microscopy to assess pluripotency and differentiation of stem cells at single cell level)

Berichtszeitraum: 2018-08-01 bis 2020-09-30

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

To deeply understand the biological processes, it is necessary to study the activity and functions of genes, RNAs and proteins within single cells. The microscopy field aims to gain the highest possible resolution “to see” these smallest components inside a cell. Due to the diffraction limit, this can be achieved only with super resolution (SR) microscopy, which is essential for the development of life science research and advanced therapies for different diseases.

CellViewer aimed to develop a fully automated SR microscopy platform capable of visualising thousands of images of DNA, RNA and proteins at a nano-scale resolution in live single cells. To achieve this obejective, it was necessary to: 1) develop targeting strategies to efficiently label specific genes, RNAs, and proteins in order to carry out experiments in multiple single mouse embryonic stem cells cultured in pluripotency or differentiation conditions to obtain SR images; 2) develop integrated software programs to collect and analyse all the SR data and developed predictive models, to detect the pluripotency or differentiation states of the cells; and finally 3) integrate 1 and 2 to build a fully automated SR microscope with high throughput capacity allowing us to determine the state (pluripotent or differentiated) of the cell.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

With CellViewer project we have been able to develop targeting strategies to Halo tag gene loci such as Oct4, Nanog, and β-catenin. In addition, we were able to develop efficient probes to efficiently label DNA, proteins and RNAs for SR imaging. As a result, we were able to visualize the DNA of gene loci and the corresponding mRNAs and protein products in living cells at nanoscale resolution. Further, we characterized the differences in RNA distribution after a treatment with a cell cycle inhibitor. We performed experiments to characterize protein kinetics in ESCs cultured in self-renewal condition and upon their differentiation

WP2 is focused on the visualisation of the dynamics of DNA, mRNA and proteins upon different perturbations. We imaged the spatial organization of DNA compaction in cells perturbed with Actinomycin D and Trichostatin A. Furthermore, we developed particle-tracking approaches and analysis algorithms to follow the dynamic of transcription factors and histone proteins in living mESCs cultured in pluripotency or differentiation conditions.

One of the major successes of CellViewer has been the successful building of a SR microscope prototype, which we expect to commercialise by the end of 2021. During 2020, the prototype will be beta-tested with high-throughput experiments for further improvement. This microscope contains an integrated software with a use-friendly interface to make it easier to use also for the non -expert users.

We have also been developing an integrated SR imaging analysis platform to analyse the high-throughput data obtained with our SR microscope. Indeed, we have developed new methods to extract information on the spatial organisation and architecture of proteins and DNAs within the nucleus of the cells. These imaging analysis methods will be used to extract info from the high-throughput data that will be collected with our prototype.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

We have developed for the first time a fully automatized high-throughput SR microscope. This will soon allow us to carry out complex imaging sessions without human supervision, which is not yet available with any other technology in the market. With this new SR microscope we envision to capitalise on state-of-the-art approaches that will revolutionise the way genes and gene products can be analysed to identify the phenotype of a given cell. For example, if a cell is a stem cell or a cancerous cell.

We expect our findings will transform the way we analyse gene activity, cell phenotype and how these evolve as a response to important stimuli. It will inspect, for example, the mechanisms that regulate lineage decisions in ESCs, which will expedite their use in regenerative medicine, having a huge impact on health and society in general.

The CellViewer research fellows have worked at the edge of biophysics, optic, physics, stem cell biology and engineering to achieve the challenging objectives of CellViewer. Thus, we expect they will become expert technological leaders and pioneers of the next generation of investigators with a very interesting profile for the future in research and technology.

Map of the cellViewer consortium partners