Silver Bullets to Beating Hearts: CCP-driven Cardiac differentiation of Human Pluripotent Stem cells.
PhD Supervisor(s): Dr James Dixon and Prof Kevin Shakesheff.
This project will build upon the work of the three DTC mini projects that are presently ongoing. Research will translate the protein delivery system for use in the tissue engineering of clinically relevant human heart tissue using CCP-technology.
The tissue engineering group (University of Nottingham) has developed technology to directly program the fate of human embryonic stem cells (HESCs) and induced pluripotent stem cells (HiPSCs) into functional contractile heart tissue (Dixon et al 2011).
Our approach opens many new avenues for cell therapies for a number of heart diseases but as the process involves genetic modification it is not clinically viable. To circumvent this technical disadvantage a protein-based system in which cell-permeable proteins (CPPs) drive this differentiation has been developed. We can now efficiently deliver proteins directly to stem cells and this project will be to further develop this technology and apply it to engineering of cardiac tissue from HESCs and HiPSCs.
During this project differentiation-promoting CPPs will be designed and created using molecular cloning. These will be produced and purified using bacterial, yeast or mammalian expression systems. These recombinant factors will be delivered stem cells to direct their differentiation into cardiac cells. Protein delivery will be monitored by microscopy techniques, as well as flow cytometric and biochemical approaches.
The membrane proteins that mediate CPP transduction are so far unknown and their isolation is important area of research. We have a tractable system which could be exploited to identify these complexes. As retroviruses such as HIV enter the cells through the same mechanism as CPPs the identity of these proteins will not only be useful to create strategies to improve CPP delivery but also as a pharmacological target to prevent HIV-infection. Depending on the student’s interests, proteomic techniques will be used to isolate membrane proteins with interact with CPPs (in collaboration with Dr. Ali Mobasheri/Dr. Susan Liddell, School of Veterinary Science) and these will be identified and functionally confirmed as CPP interacting proteins using cell assays.
