Understanding induced pluripotent stem cells.
-Puri MC, Nagy A.2012. Concise review: Embryonic stem cells versus induced pluripotent stem cells: the game is on. Stem Cells. 30(1):10-4.
-Ji J, Ng SH, Sharma V, Neculai D, Hussein S, Sam M, Trinh Q, Church GM, McPherson JD, Nagy A, Batada NN. 2012. Elevated coding mutation rate during the reprogramming of human somatic cells into induced pluripotent stem cells. Stem Cells. 30(3):435-40.
-Woltjen K, Hämäläinen R, Kibschull M, Mileikovsky M, Nagy A. 2012. Transgene-free production of pluripotent stem cells using piggyBac transposons. Methods Mol Biol. 767:87-103.
Question 1: About Dr. Andras Nagy
Dr. Nagy can you please tell us a bit about yourself, your career path over the years and how you began studying cellular reprogramming with IPSCs?
Question 2: PiggyBac transposon/transposase method
Why exactly is the piggyBac transposon/transposase system so much better than any other method used before, such as Cre-mediated excisions etc.? And if this is such a great method, then how come this has never been used for iPSCs before?
Question 3: Permissive Cell Types
In the article “Transgene-Free Production of Pluripotent Stem Cells Using piggyBac Transposons” you state that the permissiveness of a particular cell type to DNA transfection initially governs its ability to convert to iPSCs. Are there specific cell types that are more permissive than others? If yes are researchers today using this cell type?
Question 4: iPSCs vs ESCs in disease modeling
Even though ESCs are considered the gold standard of pluripotentcy, are there any times when iPSCs can be of more use than ESCs, specifically in disease modeling?
Question 5: iPSCs vs hESCs in differentiation, efficiency, kinetics, safety
You mention that hESCs are the golden standard for pluripotency because they have the potential to differentiate into any types of cell in the body, and accordingly efficiency and fidelity of iPSCs are always compared to that of ESCs. However, is it possible for iPSCs to be better than hESCs? (in terms of differentiation potential, efficiency, kinetics, safety, etc.) For example, ESCs also have differing methylation patterns and epigenetic profile that may influence their differentiation potential. If we chemically wipe out the methylation patterns, would it make differentiation better or worse? If we understand which aspects of ESCs make them the way they are, maybe we can engineer the iPSCs to become better in that aspect.
Question 6: Molecular differences between pluripotentent and totipotent cells
Why is it that pluripotent stem cells are used instead of totipotent cells since “development proceeds from a state of totipotency”? Would some of the issues that hinder disease modeling using iPSCs, such as epigenetic memory, be resolved with the use of totipotent cells? Also do you think it is possible to create totipotent stem cells from pluripotent stem cells? We have learned from the generation of iPSCs that different developmental stages of cells are not permanently irreversible. What are the molecular differences between the two and what prevents pluripotent cells (like hESCs) from differentiating into cells of the placenta, etc.?
Question 7: Reprogramming factors in fibroblasts/iPSCs
How crucial is the exact timing of washing out the reprogramming factors from the fibroblasts/iPSCs? Because they factors are oncogenic, could leaving them in for just an extra few minutes play a role in inducing mutations?
Question 8: Controlling cellular reprogramming
You conclude that reprogramming of somatic cells creates many mutations within a cell. Are there ways to control the rate at which reprogramming occurs? Perhaps reducing the stress that cells undergo during reprogramming could lead to higher fidelity iPSCs.
Question 9: Reducing iPSC mutations
After your discovery that a majority of iPSC mutations are due to experimental conditions, what specific processes in the lab are you going to try to improve upon to reduce these mutations? Do you believe that this number can be significantly reduced in the near future? Enough so that these cells can be safely used in a clinical setting?
Question 10: Hela cells
Do you work with tumorogenic strains of IPSCs and have experience with these types of cells could you please touch on both why hela cells are not accurate models for assessing human diseases, testing novel drug treatments, and generally not representative of processes in normal human cells and also the benefits they have provided the medical community in the last few decades.
Question 11: Stem cell changes over the past decade
Could you comment on how stem cells are currently changing? What changes have you personally seen in the last decade, especially for the use of modeling human diseases?
Question 12: Pressing questions in the cellular reprogramming field
Currently in your opinion what are the most pressing unanswered questions in your field of cellular reprogramming and use of IPSCs?