Induction of pluripotency in fibroblasts by fusion with enucleated human embryonic stem cell syncytia
Recommended if funds allow
Statement of Benefit to California:
The purpose of this proposal is to explore ways in which cell fusion of somatic cells with human ESCs can be used to produce hESC cell cell lines from adult somatic cells. The strategy to be investigated is the use of VSV glycoprotein-induced cell fusion of hESC cells from existing cell lines to produce large, hESC syncytia. These syncytia will then be fused with adult somatic cells, either before or after the removal of the ESC fused nuclei. It is hypothesized that the abundant cytoplasm in the syncytia will provide an adequate source of factors for reprogramming the adult nucleus. The project has two specific aims: the first aim will be to create and characterize hESC syncytia of various sizes; the second will be to investigate methods of creating cell fusion products between the syncytia and adult fibroblast cells that contain only the fibroblast nuclei and to characterize the pluripotency of the fusion product. The investigator will use both NIH-approved and non-approved cell lines in this work. SIGNIFICANCE AND INNOVATION: The PI proposes to create hESC syncytia to use as a vehicle for somatic cell reprogramming. If successful, this will represent a novel and accessible method for creating patient-specific pluripotent cells. This is a highly innovative project of high significance. STRENGTHS: The approach is novel and signficant as stated above. The experiments appear to be well conceived and preliminary data is presented. The PI and team are proficient in the techniques, and Dr. Renee Reijo Pera will serve as a collaborator and consultant for the hESC work. UCSF is an excellent institution for this project. PI has carefully considered experiments and potential pitfalls. Solutions and alternative approaches are discussed and seem feasible. Methodology to create hESC synctia of various sizes using VSV-G and nucleofector has a good chance of success. WEAKNESSES: Lack of detail in describing analysis of extent of reprogramming and developmental potential - Aim 2b. A discussion of the use of Hoechst 33342 as a measure of ploidy and the proposed methodology is necessary. This dye is a substrate for the multi-drug resistance transporter and may be pumped out of the cells if the syncytia have active transporters, which it is expected they will. Will the cells be kept on ice after loading? Will an inhibitor of the transporter be used? More details are necessary. DISCUSSION: The goal of this work is to create patient-specific fibroblasts by reprogramming. It is a novel and significant approach, and the preliminary data are compelling. There were a number of questions regarding this proposal. Even if these experiments worked, what would we do with the cells? There are other ways to make patient cells. Is the use of VSVG realistic, or clinically acceptable? Reviewer 2 felt that these experiments have a good chance of success, but the grant was weakened by a lack of detail in describing the reprogramming analysis. How is this better than making a cytoplasmic extract and doing dose response-type experiments for reprogramming. The PI has some expertise in cell biology, specifically with respect to cell fusion, and is a recently appointed assistant professor.