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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - ReachingCompleteness (The Molecular Basis of Somatic Nuclear Reprogramming)

Teaser

Summary

The capability to convert skin cells into clinically-relevant cell types such as neurons, heart cells, liver cells and embryonic stem-like cells (induced pluripotent stem cells (iPSCs)) provide an invaluable resource of cells for disease modelling, drug screening, and patient-specific cell-based therapy known as regenerative medicine. However, many of the directly converted cells are not stable, and the vast majority of iPSCs exhibit poor developmental potential as measured by stringent pluripotency tests. This suggests that the prevailing method of reprogramming is not ideal and leads to aberrant/incomplete conversion. To improve the quality of the converted cells, efforts should be focused on the molecular mechanisms that characterize the nuclear reprogramming process. There are two critical hurdles that hinder the progress of deciphering the elements that dictate successful reprogramming: (1) The ability to detect and capture only the rare cells that eventually will be converted and (2) to monitor the transcriptional profile of cells at the single-cell level. Single-cell technology is in its infancy and many of the methods used today are characterized by high noise to signal ratio. In this grant proposal we intend to overcome these limitations by (1) establishing a complex fluorescent knock-in reporter system using the CRISPR/Cas9 method to capture the early rare reprogrammable cells and by (2) employing several cutting-edge single-cell technologies to segregate the real signal from the noise. To identify common and more global elements that facilitate nuclear reprogramming at large, we will trace in parallel, reprogrammable cells from two different somatic cell conversion models, (1. from skin cells into embryonic stem-like cells- induced pluripotent stem cells (iPSCs), and 2. from skin cells into placental stem-like cells- induced trophoblast stem cells (iTSCs)), that reach high degree of nuclear reprogramming, and analyse their transcriptome using sophisticated bioinformatic tools. This study will provide a general overview of the changes that occur during the conversion of various cell types and will uncover the basic features that are essential to reach safe and complete conversion.
Thus, the main objective of this grant proposal is to shed light on the molecular mechanisms that dictate successful reprogramming events in two somatic cell conversion models by employing cutting-edge single cell technologies. The central hypothesis behind this objective is that looking specifically at the transcriptional profile of individual reprogrammable cells in two parallel somatic cell conversion models will illuminate the general criteria that must be fulfilled in a given cell to reach an intact and complete nuclear resetting state.

Work performed

Overall most of our initial aims were completed successfully.
We have completed the characterization of our newly-developed iTSC reprogramming paradigm and publish this new model in the prestigious journal cell stem cell (Benchetrit et al. 2015).
Next, we have proposed to generate a quadruple fluorescence knock-in reporter system to be able to sort out reprogrammable cells for transcriptional analysis and completed this task successfully. Currently we have a system that contains four reporters (Nanog-2A-EGFP- specific for iPSC reprogramming, Elf5-2A-EYFP-NLS- specific for TSC reprogramming, and Utf1-2A-tdTomato and Esrrb-2A-TagBFP- shared between TSC and iPSC reprogramming). This system will refer hereafter as â€œBYKEâ€ system. Note that we had to replace the Esrrb-2A-EBFP reporter with Esrrb-2A-TagBFP reporter since EBFP was too week for clear detection under the microscope. Next, we have generated MEFs from the BYKE system and reprogrammed them either into iPSCs or iTSCs. We noticed that primary infection yielded much better reprogramming efficiency with this system and thus we avoid generating secondary systems as described in the proposal and continued our work using primary infection only.

Next we could determine which combination of reporters has the highest capability to predict which cell will become eventually reprogrammed and form stable iPSC or iTSC.
Knowing the ideal reporter combination allowed us to run the experiment as planed.

We then proposed to sort single cells, based on the predictive markers, undergoing reprogramming into iPSCs or iTSCs and to probe their transcriptome using the C1 machine. Since the grant proposal there has been a great advance in the technology of single cell transcriptomic and currently the Hebrew University purchased the Genomics 10X machine for single cell library preparation. To be able to run several combination of reporters on the same lane of the Genomics 10X the cells need to be marked by additional reporter. For that we are in the middle of introducing luciferase and Renilla RNA for the BYKE system. Once we will have all the marked lines, we will start the reprogramming process and sort out single reprogrammable cells, based on reporter activity, and will run RNA-Seq on the individual cells.

Final results

We have made a lot of progress beyond the state of the art.
1. We developed a new reprogramming model (from skin cells to iTSCs). This is currently the only model that reaches high degree of nuclear reprogramming.
2. We established a very attractive and complicated quadruple fluorescent knock-in reporter system that allows the discrimination between cells undergoing reprogramming to iPSCs and iTSCs.
3. We identified a combination of reporters that can successfully detect reprogrammable cells that will become iPSCs or iTSCs.

Moreover, we understood that probing the transcriptome will not be sufficient to get a clear picture of the reprogramming process and thus we decided to probe also the chromatin structure (ATAC-Seq) and DNA methylation (RRBS) of the reprogrammable cells.
We expect that in the near future we will be able to obtain our single cell transcriptomic data and together with the experiments that we have already performed on the chormatin structure and DNA methylation, we will be able to map accurately the steps that characterize the reprogramming process into iPSCs and into iTSCs.
We believe that the identification of common elements/features between the two models and thier implication in other reprogramming models will aid in achieving intact reprogramming process and to generate high quality converted cells for future clinical use.