Is the Analysis of Gene Expression Based on an Erroneous Assumption?

The MIT-based researcher Rick Young is one of the world’s top molecular biologists. His laboratory at the Whitehead Institute for Biomedical Research has helped define many of the key principles of how gene expression is regulated, especially in stem cells and cancer cells. At a symposium organized by the International Society for Stem Cell Research (ISSCR), Rick presented some very provocative data today, which is bound to result in controversial discussions about how researchers should assess gene expression.

Ptolemey’s world map from Harmonica Macrocosmica

It has become very common for molecular biology laboratories to use global gene expression analyses to understand the molecular signature of a cell. These global analyses can measure the gene expression of thousands of genes in a single experiment. By comparing the gene expression profiles of different groups of cells, such as cancer cells and their healthy counterparts, many important new genes or new roles for known genes have been uncovered. The Gene Expression Omnibus is a public repository for the huge amount of molecular information that is generated. So far, more than 800,000 samples have been analyzed, covering the gene expression in a vast array of organisms and disease states.

Rick himself has extensively used such expression analyses to characterize cancer cells and stem cells, but at the ISSCR symposium, he showed that most of these analyses are based on the erroneous assumption that the total RNA content in cells remains constant. When the gene expression in cancer cells is compared to that of healthy non-cancer cells, the analysis is routinely performed by normalizing or standardizing the RNA content. The same amount of RNA from cancer cells and non-cancer cells is obtained and the global analyses are able to detect relative differences in gene expression. However, a problem arises when one cell type is generating far more RNA than the cell type it is being compared to.

In a paper that was published today in the journal Cell entitled “Revisiting Global Gene Expression Analysis”, Rick Young and his colleagues discuss their recent discovery that the cancer-linked gene regulator c-Myc increases total gene expression by two to three-fold. Cells expressing the c-Myc gene therefore contain far more total RNA than cells that don’t express it. This means that most genes will be expressed at substantially higher levels in the c-Myc cells. However, if one were to perform a traditional gene expression analysis comparing c-Myc cells versus cells without c-Myc, one would “control” for these differences in RNA amount by using the same amount of RNA for both cell types. This traditional standardization makes a lot of sense; after all, how would one be able to compare the gene expression profile in the two samples, if we loaded different amounts of RNA? The problem with this common-sense standardization is that it misses out on global shifts of gene expression, such as those initiated by potent regulators such as c-Myc. According to Rick Young, one answer to the problem is to include an additional control by “spiking” the samples with defined amounts of known RNA. This additional control would allow us to then analyze if there is also an absolute change in gene expression, in addition to the relative changes that current gene analyses can detect.

In some ways, this seems like a minor technical point, but I think that it actually points to a very central problem in how we perform gene expression analysis, as well as many other assays in cell biology and molecular biology. One is easily tempted to use exciting large scale analyses to study the genome, epigenome, proteome or phenome of cells. These high-tech analyses generate mountains of data and we spend an inordinate amount of time trying to make sense of the data. However, we sometimes forget to question the very basic assumptions that we have made. My mentor Till Roenneberg taught me how important it was to use the right controls in every experiment. The key word here is “right” controls, because merely including controls without thinking about their appropriateness is not sufficient. I think that Rick Young’s work is an important reminder for all of us to continuously re-evaluate the assumptions we make, because such a re-evaluation is a pre-requisite for good research practice.


The Importance of Being Embryonic

Human ESC colony – Wikimedia

There are three broad categories of human stem cells: 1) adult stem cells, 2) embryonic stem cells (ESCs) and 3) induced pluripotent stem cells (iPSCs). Adult stem cells can be found in selected adult tissues, such as the hematopoietic stem cells in the bone marrow which give rise to a variety of blood cells on a daily basis in an adult. Such adult stem cells are quite rare and, when compared to ESCs, somewhat limited in the type of cells they can generate. Hematopoietic stem cells, for example, routinely produce leukocytes (white blood cells) and erythrocytes (red blood cells), but most researchers agree that they cannot give rise heart muscle cells. On the other hand, human ESCs are pluripotent, which refers to the fact that they can differentiate into nearly all cell types, from neurons to insulin-producing pancreatic cells or even heart muscle cells.

Human ESCs are usually derived from human eggs that were created in an in vitro fertilization clinic but never implanted in a woman. Such clinics often generate far more fertilized human eggs than they actually implant, because it is difficult to predict how many implantation attempts are necessary before a successful pregnancy can be achieved. The “back-up” eggs remain in a freezer at the in vitro fertilization clinic and the donors can then decide whether they want these eggs to be used for the generation of human ESCs, which can be used for either research or ESC-based therapies. The informed consent of the donors is critical and needs to be documented before the ethics committees at the research institutions permit their usage. In spite of these regulations, some religious groups in the US have voiced concerns about using the ESCs, because they feel that even though the donated fertilized egg was never implanted in a woman, it could have been implanted and that its fertilized state already indicates a degree of personhood that requires protection. When the fertilized egg is cultured in a lab and ESCs are derived from it, the fertilized egg is invariably destroyed and from a certain religious perspective, this constitutes a destruction of a human life. Due to concerns about the ethics of using human ESCs, multiple US-based Christian groups have championed the use of adult stem cells to help repair injured tissues and organs. However, since adult stem cells are very rare and limited in their differentiation potential, most stem cell biologists do not see adult stem cells as a suitable alternative to ESCs.

A landmark paper published by Shinya Yamanaka’s group in 2007 provided a new perspective in the gridlock between demands of Christian groups to ban human ESC research and the desire of stem cell biologists to use human ESCs for regenerative medicine.  Yamanaka and his colleagues were able to show that human adult skin fibroblasts could be converted into embryonic-like stem cells (induced pluripotent stem cells or iPSCs). The iPSCs were not adult stem cells with, but actually exhibited the broad differentiation capacity that was previously only seen in human ESCs. From an ethical perspective, iPSCs seemed like a perfect solution since they could be generated without the destruction of any fertilized eggs. Shinya Yamanaka and John Gurdon, whose earlier work had set the stage for Yamanaka’s discovery, received the 2012 Nobel Prize for these exciting findings. Yamanaka’s work was not only lauded by fellow scientists, but also by religious groups, who felt that his work abolished the need for human ESCs. What these religious organizations did not understand was that human ESC research provided the foundation for Yamanaka’s research. All the factors used to reprogram adult skin cells into iPSCs were derived from a careful analysis of ESCs and the culture of human iPSCs was only made possible after the culture of human ESCs had been established in the late 1990s. To this day, the comparison of human ESCs and iPSCs is a topic of active investigation. In many ways, iPSC research is still – pardon the pun – in its embryonic stage. We are still in the process of understanding how an adult cell can be reprogrammed into an iPSC and whether the reprogramming process leaves any kind of marks or blemishes that would affect the generated iPSC.

To understand the biology and nature of iPSCs, researchers routinely use them side-by-side with human ESCs, which still serve as the “gold-standard” for a pluripotent stem cell. At a symposium of the International Society of Stem Cell Research (ISSCR) in San Francisco on August 24, 2012, Yamanaka showed the results of a new study in which he compared the gene expression profiles of 49 different human iPSC lines and 10 different human ESC lines. The comparison revealed that the majority of iPSC lines are indistinguishable from human ESCs, but that there is a minority of iPSC cell lines that behave very differently from human ESCs. Other stem cell researchers have also shown both similarities and differences between ESCs and iPSCs, and definitive conclusions about whether human ESCs and iPSCs are equally suitable for regenerating human tissues and organs cannot yet be drawn.

These new studies remind us that human ESC research is still a very active area of investigation and that in the years to come, research on both ESCs and iPSCs is needed. This was also emphasized in a recent statement by the ISSCR:


Yamanaka’s recent and exciting advances demonstrate that it is possible to reprogram cells in adult human tissues into cells that very closely resemble, but may not be identical to, ES cells. Along with recent progress on redirecting cell fate to enhance tissue repair, these experiments have captured the imagination of the scientific community worldwide. While many scientists are very optimistic about the future of this new research, some people in political circles have incorrectly interpreted this enthusiasm as a verdict that research on human ES cells is no longer necessary. This conclusion is not yet scientifically justified.

At present, and in the foreseeable future, there is a strong scientific and medical consensus that continued research on all types of stem cells is critical to developing research strategies that will ultimately provide new therapies. Supporting all forms of stem cell research is in the best long-term interests of a broad spectrum of patients with debilitating diseases and injuries. In fact, predictions about what might or might not be possible cannot substitute for careful and rigorous research to discover what strategy will provide the most successful therapeutic intervention for a given disease or condition. The basic tools for these discoveries include human ES cells, which remain the benchmark for assessing pluripotency and the ability of cells to develop into all the different cell types of the body.

In the wake of the announcement of the Nobel Prize, the ISSCR (whose current president is Shinya Yamanaka) wanted to pre-empt any attempts to dismiss the importance of human ESC research, which remains a cornerstone of stem cell biology and regenerative medicine. I applaud the ISSCR for this pro-active approach. Taking ethical concerns into account is important, but one also needs to make sure that scientific discoveries are not misused to put forward political or religious agendas. In the next years or decades, we may indeed discover that iPSCs can completely replace human ESCs. On the other hand, we may discover that iPSCs and ESCs will play distinct and complementary roles in the future of regenerative medicine. We will not know the answer to the question until we conduct the research and keep an open mind when we assess the results. The nascent biology of iPSCs and ESCs is a journey into the unknown and this is what makes it such an exciting area of research.