Production of stable pluripotent selectable hES/iPS cell lines for site-specific integration of BAC reporter constructs
Tools and Technologies I
$1 142 640
Human Embryonic Stem (hES) cells are nowadays widely accepted as potential sources of cures to diseases and are commonly used as models to study human development, physiology and pathology. Recently, human induced Pluripotent Stem (hiPS) cells have been obtained by genetically reprogramming adult somatic cells. hiPS cells have similar features to hES cells: they are pluripotent (they differentiate into cell lineages belonging to the three germ layers) and self-renew (they can indefinitely grow in culture without commitment). In addition, they overcome some of the ethical issues raised by the use of hES cells and allow obtaining patient-specific pluripotent cells suitable for cell therapies since no immunological reaction would hinder their use. Overall, the value of pluripotent cells is not to be questioned anymore: they are and will continue to be used for basic and clinical research, as drug discovery platforms or to study development. However researchers face two difficulties while working with those cells: (1) they might spontaneously differentiate in culture, or be composed of an heterogeneous population making it very hard to reproduce results from one laboratory to another, or to judge the quality of a differentiation protocol, (2) there is no routine method to manipulate their genome in a safe and efficient manner. Here we propose a two-step method which allows safe manipulation of the genome of a previously homogenized pluripotent cell population. The method proceeds in two steps: (1) the production of recombinant hES and hiPS cell lines carrying a single random integration of a construct containing a selectable marker controlled by the regulatory sequences of UTF1 (an important transcription factor expressed exclusively in pluripotent cells), allowing the selection of pluripotent cells; (2) the use of an AttP docking site for the PhiC31 integrase included in this construct to site-specifically insert large reporter transgenes (Bacterial Artificial Chromosomes) carrying an AttB site in their backbone. The strengths of this method are multiple: (i) the starting population will be an homogeneous pluripotent cell population; (ii) the integration site after characterization of the right clones will be controllable and unique for any construct targeted in these lines; (iii) any construct could be integrated in this site at will, allowing the study of different differentiation steps in a fully reproducible environment, and close to the physiological regulation, as BAC sequences are long enough to contain most if not all regulatory sequences of a gene. The method we propose to set up here will allow researchers to work with pluripotent cells under standardized conditions, enabling both a better reproducibility of the results and more confidence in the type of the cell population studied. More generally, this proposal presents an efficient, safe and simple method to manipulate the genome of pluripotent cells at will.
Statement of Benefit to California:
Neurological and cardiovascular disorders, autoimmune diseases, diabetes, cancer and osteoporosis strike no less than 10 million Californians each year, causing an incalculable personal toll and an annual economic cost of billions of dollars in medical expenses and lost productivity. One benefit that will be derived from the proposal presented here is the generation of specific tools and methods for reducing medical costs and increasing the quality of life and level of productivity of afflicted Californians. The biggest benefit of the method presented here is indeed to be able to safely manipulate at will the genome of stem cells in order to make advances in the understanding and modeling of genetic processes occurring in physiological conditions or in a variety of pathological conditions such as cardiovascular disorders, autoimmune diseases, diabetes or neurodegenerative diseases, which do not yet have any cures available, and for which stem cell research is the biggest hope available. By offering a method to produce reporter lines in an efficient and controllable way by using engineered Bacterial Artificial Chromosomes as reporter constructs, the proposal opens the window to the systematic generation of reporter pluripotent cell lines allowing fine tuning differentiation protocols in order to be able to apply stem cell technology in therapies for any type of disease. It is also generally accepted that tools allowing work under more standardized conditions would be of great benefit to stem cell research. In this regard the method proposed allows controlling and standardizing the starting cell population of any experiment conducted in the laboratory. The generation of more homogeneous pluripotrent cell lines will significantly improve result reproducibility and collaborations among different groups throughout California and worldwide. Another benefit derived from this research grant proposal is the training of new scientists to serve as educators and researchers for the future, many in the burgeoning area of stem cell biology for which the State of California has emerged as a world leader. Finally, the discoveries derived from innovative and multidisciplinary research on hES/iPS cells described in this proposal, are likely to lead to important new areas of intellectual property that are essential for creating high quality jobs in the biotechnology and pharmaceutical industries in California. The short-term profits are obvious for stem cell research laboratories, but the long-term applications of this method will certainly help the people of California.
This proposal focuses on the development of novel technology for site-specific gene delivery into homogeneous populations of human embryonic cells (hESCs) and induced pluripotent cells (iPSC). The applicant proposes to enable selection of a homogeneous population of hESCs or iPSCs by inserting a fluorescent marker and antibiotic-resistance gene under the control of a pluripotency-specific promoter into the genome of these cells. That construct will also contain a sequence that functions as a docking site for the PhiC31 integrase to allow directed, site-specific integration of a second vector. The second vector contains a sequence that recognizes the docking site and it also contains a reporter gene under the control of regulatory elements provided by a bacterial artificial chromosome (BACs). The PhiC31 integrase then mediates BAC integration at the docking site. The applicant proposes to demonstrate proof-of-principle with a BAC containing regulatory elements specific to pancreatic progenitor cells. The reviewers felt that this proposal could have a broad impact on the stem cell field. This proposal addresses the issues of heterogeneity of pluripotent cell populations and difficulty in genetically manipulating hESC. Reviewers praised the technology and potential utility of being able to insert genes into stem cells in a site-specific fashion. Reviewers expressed concern about the proposal’s feasibility. Although one reviewer stated that the proposal was well thought out, others felt it lacked detail and found it difficult to determine which work the applicant had already completed and which experiments were planned. It was not clear that the team has experience with the proposed recombination technology, nor was it clear that the proposed BAC approach was feasible, and demonstration of its utility with a model system (e.g. mouse ESCs) would have raised enthusiasm. Reviewers were enthusiastic about the quality of the assembled research team. The principal investigator has a strong background in developmental biology and has received numerous national and international honors. Other team members were viewed as outstanding scientists with appropriate backgrounds and abilities to perform the work described in this proposal. Overall, the reviewers were enthusiastic about the potential impact of this proposal and the strength of the research team, but questioned the feasibility of the approach.