Genome-wide exploration of reprogramming mechanisms using CRISPR/Cas9 and DamID technologies

In our body, skin cells generate only skin, muscle cells make only muscle, bone cells make only bone and so forth. However, our whole body was generated from a single cell, a fertilized egg, in the womb. This cell, the source of life, starts to divide just after fertilization and become 2 cells, 4 cells, 8 cells, 16 cells, continuing to divide and gradually form the body of an embryo. In the beginning of embryo development, until about 1 week after fertilization when the mass of cells still look like a ball, we have cells that can generate all ~300 different cell types which orchestrate the formation of our adult body. However, after this stage, cells in the womb start being specialized to generate specific cell types in the future body. The way cells start making commitments to a specialized cell in a body is often described as a ball rolling down a mountain. On the top of the mountain, the ball can go in to any valley at the bottom. However, once the ball drops from the top of the mountain, the valleys it can go to become restricted to the same side of the ridge. When the ball rolls down to a lower position of the mountain, choices of valleys to be able to travel down become more limited. Similarly, when a baby is born, cells in the body have very limited capacity to generate different cell types. The only way to keep cells with mulit-potential to generate all cell types is taking an embryo from the womb about 1 week after fertilization and put it in a plastic dish with nutrient rich liquid containing optimal conditions to prevent further specialization of the cells. These uncommitted cells that can make all cell types of the body are therefore called 'pluripotent stem cells', and because they arederived from an embryo they are called embryonic stem cells (ESCs).

The general principles of development, whereby stem cells gradually and irreversibly become specialized, were re-written by a very simple experiment in 2006. A group of scientists found that any specialized cells can become uncommitted pluripotent cells like ESCs by artificially manipulating only 4 genes simultaneously. This trick that rewound the cells' biological time clock is called 'reprogramming' and the resulting artificially generated uncommitted cells are called 'induced pluripotent stem cells (iPSCs)'. The findings brought tremendous excitement to the stem cell and medical research community. iPSCs can be generated from any cell in the body (e.g. your skin cells) and in theory can be used for the production of any desired cell types for transplantation, drug screening, toxicology tests and so forth.

At the moment, successful generation of iPSCs is not 100% efficient, the process of reprogramming takes over 1 month and is an expensive procedure. The technology is still far from delivering its merits to the society. In addition, surprisingly, we still do not know why the manipulation of only 4 genes can induce pluripotency, erasing the character of specialized cells. Therefore in this project we aim to illuminate molecular mechanism of reprogramming using state-of-the-art molecular biology techniques. In the first objective, we aim to reveal genes that are inhibitory or essential for efficient reprogramming. In the second objective, we will investigate how the manipulated 4 genes concisely and effectively execute the process of reprogramming. As an outcome of the project we expect to have strategies to generate iPSCs with higher success and in a shorter time period, and also better understanding how we can manipulate specialized cells and/or pluripotent cells to generate different specialized cells required for medical applications.

Ever since the first generation of iPSCs about a decade ago, the most robust reprogramming strategy to establish a pluripotent state is still induction of the 4 Yamanaka factors. Nevertheless, only a handful of genes have been demonstrated to be detrimental or essential for reprogramming. It has also not been shown which genes are directly targeted by the Yamanaka factors at different stages of reprogramming. Thus, this project aims to illuminate molecular mechanisms of reprogramming by 1) performing CRISPR/Cas9-mediated genome-wide knockout screening and 2) dissecting changes of direct targets of the Yamanaka factors during reprogramming with inducible mouse DamID-seq technology. One of the difficulties to investigate the molecular mechanisms of reprogramming is the low efficiency and heterogeneous cell populations during the process. To circumvent these problems, we have identified cell surface markers that allow us to isolate more homogenous reprogramming intermediate subpopulations than simple time course analyses. In addition, we have established a system where robust and homogenous expression of the Yamanaka factors can be induced in mouse embryonic fibroblasts with constitutive expression of Cas9 by administration of doxycycline. We will apply lentiviral gRNA library containing 90,000 gRNAs targeting all protein coding genes in the mouse genome to identify genes detrimental or essential for efficient reprogramming. For the second objective, we have been optimizing DamID-seq technology, which has often been used in Drosophila, in mouse cells. DamID-seq is a complementary technology to ChIP-seq, with an advantage of being able to perform with smaller starting cell numbers due to the absence of the antibody-mediated pulling down step. Through this project we aim to improve the human iPSC derivation technology and identify molecular mechanisms that can be applicable for the swift and efficient generation of various cell types important for medical applications.

The direct beneficiaries of this project are researchers who take advantage of the improved reprogramming systems that will be developed in this project. Researchers in this group are not limited in stem cell biologists. Many researchers who are interested in particular diseases will benefit from the improved reprogramming strategies which decrease cost and time for the generation of iPSCs from patients. Moreover, the genome-wide knockout screenings will provide the research community with extensive lists of genes important for reprogramming, but more than we can validate within this project. We will release enrichment scores of each gene in our screens and the results will be a large resource for the research community. Similarly, results of our DamID-seq could be used to explain phenomena other researches are interested in during reprogramming. Our data will be a platform for many researches to build their hypothesis towards new findings regarding pluripotency induction. DamID-seq technology itself will also have a large impact on any researcher who is interested in transcription factor binding sites in scarce cell populations in mouse. While limited materials and availability of reliable antibodies are often the cause of not being able to perform ChIP-seq, DamiD-seq can overcome both of these problems. We will freely distribute cell lines and plasmids used in this project. The data analysis pipeline will also be integrated into a free online NGS data analysis software, GeneProf, developed by the Tomlinson group (Nature Methods, 2012) and available to any researcher.

In addition to researchers, non-scientists are also beneficiaries of this project. The improvement of reprogramming strategies will decrease costs and time required for the iPSC generation, which will facilitate use of iPSCs in drug screening, toxicology tests, and regenerative medicine. It will also create intellectual properties and patents as well as various associated jobs in the pharmaceutical industry, biotechnology companies, academic and non-academic research institutes across the UK. I will also seek the possibility of licensing technologies and cell lines used and developed in this project with the help of the Edinburgh BioQuarter, which is a commercialization arm of the academic medical centre adjacent to our SCRM building. IP from the proposed research will be handled by the business development of the BioQuarter for rapid commercialization.

While it was not possible to generate pluripotent cells from differentiated cells until about 10 years ago, new biology text books will soon have description about 'reprogramming' by the Yamanaka factors, if they do not have it yet. However, at the moment there is almost nothing to describe the underlying mechanisms of reprogramming, analogous to the mystery of how hereditary transmission occurred before the structure of DNA was discovered. Illuminating the molecular mechanisms of reprogramming through this project will provide a clearer explanation to the public how the rewinding of the biological clock can be achieved by only 4 transcription factors. Through the press release and public outreach, we will be able to deliver the excitement of the state-of-the-art scientific research, which will increase enthusiasm and support for the investment on science in the UK, which is essential to maintain UK as a world-leading country in science.

I will also train postdocs and students through this project. I requested salary of 3 postdocs for 7 years and PhD students will also be involved in the project in the duration of this fellowship. They will have the chance to learn the latest molecular and cellular biology techniques as well as genomics and bioinformatics. Success of the project will give opportunity to the postdocs to start their own labs or senior positions in industries in the near future, where again students and young researchers can be trained to develop their professional skills.