The Creation and Use of Induced Pluripotent Stem Cells
Marius Wernig Alexander Meissner, Ruth Foreman, Tobias Brambrink, Manching Ku, Konrad Hochedlinger, Bradley E. Bernstein and Rudolf Jaenisch. 2007. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318-325.
Briefly tell us a bit about yourself and your career path over the years leading to your investigations of somatic cell reprogramming and embryonic stem cells. Additionally can you please comment generally on how only 4 genes seem to be required for reprogramming? This is something I never would have imagined, did you?
In 2006 Dr Yamanaka suggested 24 genes that are expressed in mouse ES cells that may be the most important for reprogramming. Why has your lab and Yamanaka’s lab only focused on Oct4, Sox2, c-myc and Klf4? Would you expect that induced expression of all 24 would make a more realistic and effective ES-like cell?
Class wide generated questions about the data:
a) Why was the Retroviral GFP not expressed by the iPS lines (Figure 1)?
b) The microarray data between the ES and iPS cells had almost identical gene expression profiles and were almost complete opposites of the MEF expression profile. Did you initially hypothesize that there would be such a striking similarity between the iPS and ES cells and contrast between the MEF and ES cells as displayed by the microarray data (Figure 2f)?
c) Although these iPS cells show extremely similar gene expression profiles, methylation, and chromatin modifications; there do appear to be some differences such as the chromatin modifications shown in Figure 3a. Can you please explain the nature of these differences? How can you be certain that these cells are in fact similar enough?
Continued class wide questions about data:
d) Overall reprogramming was improved from past studies but still tends to be a sluggish and inefficient process, with ES-like cell colonies developing only after 20 days of culturing. Can you please speculate on the causes for the low efficiency?
e) Although iPS cells produce viable chimeras, long-term differences were not reported. How long do they live, and has their been any health problems with the chimeras over the long term?
One of the potential side effects of using stem cells is the danger of teratomas. In addition, your lab and others used the Myc oncogene as one of the 4 important reprogramming genes, and tumors were reported. How different are the iPS cells potential to form tumors as compared to ES cells? On November 30th, Yamanaka’s lab published new iPS lines that do not rely on the Myc gene and show reduced tumor formation. What do you think of this new data and do you see any other way to reduce the chance of tumor formation derived from iPS or ES cells?
To reprogram a somatic genome into an embryonic pluripotent state, you expressed Oct4, Sox2, c-myc and Klf4 by the use of retroviruses. Could you please explain the barriers the use of retroviral vectors presents to their use in therapeutic application? Can you imagine any other non-retroviral techniques to misexpress these genes in fibroblasts to reprogram them into a pluripotent ES-cell-like state?
In your opinion, does the use of iPS cells completely eliminate the ethical conflicts with the use of natural embryonic stem cells? If not, what other ethical issues continue to surround the use of iPS cells in research and therapy? If so, then do you see the use of iPS cell eventually eliminating the use of ES cells altogether, or minimally having a detrimental impact on the support for ES cell research?
Currently in your opinion what are the most pressing questions in the field of cell reprogramming and embryonic stem cell research? What steps is your lab taking to address those questions?