#	Pagina
attuale pagina	/open-h2020/projects/199030/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CellViewer (CellViewer: super-resolution systems microscopy to assess pluripotency and differentiation of stem cells at single cell level)

Teaser

Summary

To deeply decode the biological processes is necessary to study the activity and functions of the small components (genes, mRNAs and proteins) within the cells. The microscopy field aims to gain the highest possible resolution â€œto seeâ€ these smallest components that drive the biological processes inside a cell. For this reason, super resolution (SR) microscopy is essential for the development of life science research.

CellViewer is a FET open project involving 5 partners, which include three research institutes and two companies (see figure 1). The main aims consist in the development of a SR microscopy platform, the CellViewer, capable of visualising DNA, mRNA and proteins at a nano-scale resolution in live single cells. To achieve these objectives, it is necessary first to develop targeting strategies to efficiently label specific genes, mRNAs and proteins. Second, we will carry out experiments in multiple single mouse embryonic stem cells (mESCs) cultured in pluripotency or differentiation conditions to obtain SR images of the stem cell genes, mRNA and proteins of interest. Third, we will develop integrated software programs to collect all the SR solution data and develop predictive models which will inform on the pluripotency or differentiation state of the cells. All this knowledge will be integrated in the CellViewer platform, which will ultimately be capable to collect, analyse and model the SR data with high-throughput to determine the state (pluripotent or differentiated) of the cell.

Work performed

The CellViewer project is organised in six different work packages (WPs) in order to implement the research goals. WP1 focuses on finding the best strategy for targeting gene loci with the use of a universal tag to further obtain SR images of DNA, proteins and mRNA. CRISPR-based approaches to target repetitive sequences have been successfully set up to image repetitive sequences. In addition, some data (still to be optimized for the signal versus noise ratio) were collected to image non-repetitive (i.e. the genes of interest).

WP2 is focused on the visualisation of the dynamics of DNA, mRNA and proteins upon differentiation at single cell level. We established a robust protocol to differentiate mESCs with retinoic acid in multiwall plates. 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. Finally, we developed methods for DNA and RNA visualization in the whole nuclei.

The objective of WP3 is to build a high-throughput SR microscope, the CellViewer, which can visualise DNA, mRNA and proteins in multiple living single cells. The hardware modules of CellViewer SR microscope have been preliminary designed. The designed modules will be essential to build the illumination path and to start the design of the detection path of the image. Moreover, the software modules controlling the CellViewer have also been designed.

We are also developing an integrated SR image analysis platform. It has been developed an initial version of this software platform, which enables to import and export different image data used in SR microscopy as well as it allows to compare results from different sources and to prepare materials ready for presentations. Finally initial modelling approaches to analyse 2D and 3D data have been developed. We also have designed a data management plan in order to efficiently transfer, share and store the large amount of generated data.

The project also deeply takes in consideration the importance of disseminating and outreaching the results to the different stakeholders, which is explained in WP5. For this objective, we have designed a dissemination plan, which details the intended plans for dissemination and outreach, the timing and the indicators we are considering to measure the impact. We have also developed the CellViewer website (www.cellviewer.eu) and twitter account (CellViewer_EU) to enhance the dissemination of the results and events relevant to the project. Finally, we have designed an exploitation plan in order to approach the potential stakeholders.

WP6 takes care of efficiently coordinating and managing of the project. To assure this effectivity and efficiency in the coordination and management we have designed a quality management plan. Moreover, from the very beginning of the project start we have put in place a set of collaborative tools to foster a fluent communication between the consortium researchers.

Final results

After one year, the CellViewer project is indeed on the process to initiate radically new lines of technology. All the obtained results so far indicate that we are moving towards the direction of capitalizing on state-of-the-art approaches to develop new technologies that will revolutionise the way genes and gene products can be analysed to identify the phenotype of a given cell.
The expected impact from the SR microscopy technology side, is to develop a high-throughput, robotic, SR CellViewer to carry out complex imaging sessions without human supervision, starting with the specific application of imaging the mESC system.

The results obtained so far after 1 year from the start of the project support the expected impact of empowering cell biologists with a radically new technology. 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 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. We do not currently foresee any problem in releasing the initial CellViewer prototype, which can be also implemented as a diagnostic tool.

The CellViewer consortium consists of scientists from different research fields and of scientists that have developed a novel expertise. The CellViewer research fellows work 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 they will be the pioneers of the next generation of investigators.