Periodic Reporting for period 3 - CellViewer (CellViewer: super-resolution systems microscopy to assess pluripotency and differentiation of stem cells at single cell level)
Okres sprawozdawczy: 2018-08-01 do 2020-09-30
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.
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.
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.