Abstract
Dr Nagy is currently a Senior Scientist at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Professor in the Department of Molecular Genetics at the University of Toronto and Investigator at the McEwen Centre for Regenerative Medicine. He also holds a Tier I Canada Research Chair in Stem Cells and Regeneration. His research focuses on several areas of interest, which include: functional studies of genes belonging to families with known roles in vessel formation; development of sophisticated genetic manipulation tools in the mouse model; applying genetics to cancer research; derivation, differentiation and genetic modification of both mouse and human embryonic stem cells; and reprogramming of somatic cells to pluripotent stem cells. Dr Nagy‘s research is currently funded by the National Cancer Institute of Canada, Genome Canada, Stem Cell Network, NSERC and the NIH.
What is the main focus of research in your laboratory?
There are two main areas of focus in my laboratory. We are interested in blood vessel development, especially understanding the role of VEGF in blood vessels during development, tumor formation and in normal physiology. For more then 20 years now, we have also been heavily involved in stem-cell biology.
Initially, we studied the developmental potential and limitation of embryonic stem cells (ESCs), and derived several new mouse ESC lines. These ESC lines have been used worldwide for the generation of more than 1000 gene knockouts and are still very popular. Soon after, we developed F1 hybrid ECS lines and the so-called tetraploid complementation assay that allows us to generate completely ESC-derived embryos and animals. We routinely utilize this approach to generate mice directly from ESCs without the traditional problems and the extensive time involved in producing chimeras and wait for germline transmission.
Based on our experience with the mouse, we then moved on to working with human ESCs. Our laboratory was the first in Canada to establish ESCs from human embryos. Our two cells lines, CA1 and CA2, are widely used by researchers in Canada as well as abroad. Around a year ago, we started moving into the induced pluripotent stem cell (iPSC) field.
Your recent paper in Nature demonstrates virus-independent reprogramming of human & mouse cells to iPSCs. Can you tell us a bit more about what led you to this approach?
We had the novel idea of generating these cells without viral transduction using transposons, in particular the piggyBac transposon system. The transfection efficiency of the piggyBac transposon is comparable to that achieved by the viral transduction – this caught my eye and I thought we could possibly use this to transfect cells as efficiently as viruses. Much to our surprise, it worked immediately, so we were able to very quickly come out with two publications showing how to induce these cells without viruses. Most importantly, we also showed how to remove the reprogramming factors from the iPSCs after they have generated reprogrammed cell lines and are no longer needed. For this, we took advantage of the unique properties of the piggyBac transposon that allows a physical removal from the genome, leaving no trace behind.
How efficient was the process? Will it be easily reproducible in other laboratories?
We have not yet made a direct side-by-side comparison with viral transfection, but we have a feeling that our efficiency is very comparable to the viral transduction efficiency. We have tried diluting the transposon DNA to miniscule levels. Typically we transfect 10 µg DNA into ESCs, but we were able to reduce the DNA concentration to 1 ng (10,000× less). Using 1 µg, we generated hundreds of thousands of clones, similar to the numbers achieved with viral transfection. We are convinced that the procedure will be easily reproduced in other laboratories, as I see no reason why other laboratories should have problems getting this to work.
How were the resulting iPSCs tested to establish their stem cell-like properties?
We used all the standard assays and are confident in the stem cell-like properties of the cells. The final proof that human iPSCs generated by the transposon system were fully pluripotent was the formation of teratomas in vivo, a result that was not included in the Nature paper as the results came in after acceptance.
In this study, transposase was used to remove the piggyBac cassette in the mouse iPSCs, but the cassette was not removed from human iPSCs. Why was it not possible to include these data for the human cell lines?
The reason why the human system is lagging behind the mouse is simply because the human cells grow slower. We are currently characterizing our first colonies of human iPSCs after removal of the cassettes. The work is not yet completed, but so far the data look really promising.
Another recently published study, from Rudolf Jaenisch‘s laboratory, used viral vectors to generate iPSCs from skin cells of Parkinson‘s disease patients, and later excised the genes to produce cells without the foreign genes. What are the advantages & disadvantages of this approach in relation to the technique described in your study?
An interesting feature of this study is that they showed the technique applied to patient-derived cells from Parkinson‘s disease patients. It is certainly novel. On the other hand, removing the factors with the Cre–Lox system is not as ‘perfect‘ as with the transposon because it leaves a viral piece behind, a 500 base pair transcriptionally active element, which could interfere with the genome and potentially be mutagenic. That is why I see the next step in iPSC research taking place using the piggyBac system, which leaves no trace. In terms of efficiency, we still need to generate data to support our feeling that the transposon system will be equally efficient as the viral system. However, if this is proven, I can see it very quickly replacing the viral system.
Questions still remain as to whether iPSCs are truly equivalent to ESCs. What research must be carried out before iPSCs can be considered equivalent to ESCs?
Perhaps the more relevant question would be do we care? If iPSCs can be a good source of therapeutically relevant cell types, does it matter whether they are equivalent to ESCs? As far as regenerative medicine is concerned, we might even prefer a cell type that is not equivalent to ESCs, but instead provides cells that are even safer than ESCs. Whether iPSCs are equivalent to ESCs is an interesting scientific question, and certainly should be addressed, but may not be totally relevant to clinical applications of these cells.
How do you see the iPSC field developing over the next 5–10 years? How soon will we see clinical trials with these cells?
It is our expectation that iPSCs will eventually replace ESCs for therapeutic applications, but it is still a long way down the road. At present, we still need ESCs as a standard with which to compare iPSCs. Now that we can seamlessly remove the factors, there is an opportunity to test the clinical possibilities properly. We are now testing the tumorogenicity of iPSCs with and without the factors, and we hope it will show that after removal of the factors, the cells are safer.
In terms of how long it will take before we can use iPSC-derived cells in patients, the field is moving very fast. Now that ESC-derived cells are being used in clinical trials, I can see that if we can generate iPSCs with factors removed, they will follow ESCs into the clinic quite soon.