Commonly held ideas are sometimes held because there is a great of evidence to substantiate them. However, other times, an idea is commonly held because simply because it has been repeated over and over and over even though the evidence for it is poor. Thus, when new evidence come to light showing the commonly held believe to be untrue, it becomes incumbent on us to readjust what we think.
When it comes to embryonic stem cells and the genes that keep them pluripotent, the transcription Nanog plays a very critical role in the self-renewal of embryonic stem cells and there is a great deal of evidence for this assertion. However, the expression of the gene that encodes Nanog was thought to follow the same mode of expression as some of the other pluripotency promoting genes. Namely, that only one of the copies of the Nanog gene were thought to be expressed in embryonic stem cells. This turns out to be probably false.
First a little background. In 2007, Ian Chambers and others published a paper in the journal Nature that examined the expression and function of Nanog in embryonic stem cells. Chambers and others found that Nanog expression levels in individual embryonic stem cells from a culture derived from a single cell varied wildly. The figure from the Chambers et al paper is shown below.
The reason for this fluctuation in Nanog levels was uncertain, but Chambers and others showed that Nanog could be deleted from mouse embryonic stem cells without affecting their ability to contribute to various sundry embryonic tissues during mouse development, even though they do not make functional gametes (eggs and sperm). In fact, mouse embryonic stem cells can self-renew under particular conditions without a functional copy of the Nanog gene even though they are prone to differentiation. From this, Chambers and others concluded that Nanog stabilized rather than promoted pluripotency of embryonic stem cells by “resisting or reversing alternative gene expression states.”
Fast forward to 2012 and another Nature paper by Yusuke Miyanari and Maria-Elena Torres-Padilla from the IGBMC in Strasbourg, France, which showed that before mouse embryos implanted into the uterus, only one copy of the Nanog gene was expressed, but after implantation, both copies of the Nanog gene was expressed. Miyanari and Torres-Padilla also made mouse embryonic stem cells that had copies of the Nanog gene labeled with different glowing proteins. This ingenious experiment showed confirmed that Nanog levels were variable, but also showed that only one copy of the Nanog gene was expressed in growing embryonic stem cells in culture.
Now if we fast forward one more year and a paper from the journal Cell Stem Cell and a letter to the same edition of this journal, we have an article by Dina Faddah and others from the laboratory of Rudolf Jaenisch at the Whitehead Institute (MIT, Cambridge, MA), and a supporting letter from Adam Filipczyk and others from Germany and Switzerland. In this article, the authors also double-labeled mouse embryonic stem cells and examined multiple cells and showed that BOTH copies of Nanog were expressed, and that the range of variability of Nanog expression was approximately the same as other pluripotency genes.
Filipczyk and others used a similar approach to examine the expression of Nanog in mouse embryonic stem cells and they came to the same conclusions as those of Faddah and others.
What is the reason for the differences in findings? Faddah and others did an important experiment to answer this question. Some of the cells that with labeled copies of the Nanog gene disrupted the production of a functional Nanog protein. The constructs used in the papers by Faddah and others and by Filipczyk and others did not disrupt Nanog protein production. When Faddah and others tested these other constructs that disrupted Nanog protein production to determine is the amount of glowing protein tracked with the amount of Nanog protein produced, it was clear that the amount of Nanog protein made by the cells did not reflect the amount of glowing protein produced. According to Dina Faddah, “The way the reported was inserted into the DNA seems to disrupt the regulation of the alleles so that when the reported said Nanog isn’t being expressed, it actually is.”
Jaenisch sees this as an instructional tale for all stem cell scientists. He noted: “Clearly, the conclusions for this particular gene need to be reconsidered. And it raises the question for other genes. For some genes, there might be similar issues. For other genes, they might be more resistant to this type of disturbances caused by a reporter.”
Bottom line – read the materials and methods part of the paper carefully because the way these experiments are done can determine if the results are trustworthy.