Nanog turns stem cells into a heterogenous bunch

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(Nat Cell Biol, Nov 2012) Study explores how normal fluctuations in the expression of a key cell pluripotency factor affect stem cells.

stem cells are maintained by a small but highly interconnected network of commonly known as the core pluripotency network (CPN). Researchers have known for a while that some CPN factors show oscillatory expression at the single cell level, but it remains unclear what effect such oscillations have on the biology of stem cells.

Recently, Ben MacArthur and a group of collaborators led by Ihor Lemischka at the University of Southampton, in the United Kingdom set out to understand how fluctuations in the expression of one of these transcription factors, Nanog, affects the biology of stem cells (1). It turned out that Nanog fluctuations make stem cells slightly different from each other, which may have important implications for their differentiation potential.

Lemischka’s team used a clever trick to control Nanog expression and mimic its fluctuations in stem cells: they permanently expressed a to inhibit the endogenous Nanog gene, while simultaneously introducing an inducible Nanog transgene, which allowed them to control the re-expression of Nanog in these cells. were then used to analyze the response of the stem cells to Nanog removal and re-expression.

MacArthur and colleagues first noticed that most CPN factors seem able to withstand the natural fluctuations in Nanog expression. While the endogenous levels of Nanog had been reported to drop for only less than a day, the researchers found that the expression of most CPN factors remained the same even a couple of days after the full removal of Nanog. Of note, Oct4, Sox2 and Klf4, the three famous partners of Nanog in the reprogramming of induced pluripotent stem cells (or ), seemed particularly unaffected by the transient Nanog depletion. On the other hand, Nanog downregulation led to a significant increase in the expression of lineage commitment markers. Because these markers were rapidly turned off following Nanog re-expression, the authors speculate that naturally occuring dips in Nanog levels may induce a “reversible primed state” in stem cells that prepares them to differentiate should the lack of Nanog persist and the CPN eventually break down.

Up to this point, the researchers had worked with tens of thousands of cells in their samples, which they recognize can mask important information lost to sample averaging. Therefore, they next turned their attention to the behavior of single cells, focusing on the expression of a specific set of genes in response to Nanog removal. Surprisingly, their measurements did not show any significant similarities across samples, suggesting that Nanog depletion causes a random drift of gene expression among stem cells rather than an invariant cascade of transcriptional changes.

The authors hit their home run when seeking the source of such heterogeneity. Given its high connectivity, the CPN is full of , most of which include Nanog. According to the CPN model, removing Nanog should wreak havoc on almost 70% of the feedback loops. But Lemiscka’s group went a little further… To replace the endogenous Nanog protein that is lost to the constant inhibition by shRNAi, they used an inducible Nanog transgene that can make Nanog protein but does not include any of the from the endogenous Nanog gene, which makes this transgene ‘invisible’ to the other factors in the CPN. In other words, MacArthur and colleagues generated cells that remain pluripotent because they have Nanog protein, but with a much reduced number of CPN feedback loops (ie. those that would normally include regulation of the Nanog gene). To their surprise, these cells turned out to be less heterogeneous than their normal counterparts, suggesting that the CPN feedback loops that include Nanog would contribute to gene expression heterogeneity in normal stem cells.

Whether any of this holds up in vivo remains to be seen, of course. But this is certainly an unexpected turn of events that may open the door to a series of exciting biology questions and, hopefully, a better understanding of stem cells in the context of regenerative medicine.

(1) MacArthur BD et al. Nanog-dependent feedback loops regulate murine embryonic stem cell heterogeneity. Nat Cell Biol, 2012 Nov;14(11):1139-47. doi: 10.1038/ncb2603.

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In developmental biology, ‘pluripotency’ refers to the potential of a precursor cell to differentiate into multiple different cell types. Some stem cells can only give rise to a very limited number of cell types, but pluripotent stem cells can give rise to most cell types in an organism.
Transcription factors (TFs) are proteins that recognize and bind specific sequences in DNA to recruit and/or modulate the activity of other proteins that regulate gene expression. Thus, TFs can turn the expression of a gene on, off, or modulate its expression levels.
shRNA stands for short-hairpin RNA. When scientists want to inhibit the expression of a given protein, they can genetically engineer a cell to produce RNA molecules that fold back and bind to themselves, giving rise to short stretches of double stranded RNA (dsRNA) held by a loop (a structure that vaguely resembles a rather primitive hairpin). Making a long story short, these shRNAs will destroy endogenous mRNA molecules that contain a sequence (or “seed”) that is identical to some of the sequence in their dsRNA stretch. Thus, when researchers want to inhibit the expression of a given gene, they design DNA constructs that, when transcribed in a cell, will form an RNA hairpin with a sequence that is present in the mRNA that they want to destroy (and preferably absent from all other mRNAs in the cell).
Researchers can engineer transgenes to be ‘immune’ to inhibition by a given dsRNA. They do so by carefully introducing changes to its sequence, such that the protein encoded by the modified transgene is left unaltered, while its mRNA does not contain the “seed sequence” (see shRNA), making it “invisible” to the given dsRNA.
“Microarrays” refers to a technique that researchers use to simultaneously measure the abundance of thousands of expressed RNA molecules present in a sample.
Until somewhat recently, human pluripotent stem cells were obtained from the consensual donation of human embryos created through in vitro fertilization. In 2006, however, researchers used genetic engineering to induce the conversion of otherwise fully differentiated cells (such as adult skin cells) into pluripotent stem cells – and hence their name.
Feedback loops refer to cases in which a given factor A affects the activity of another factor B, which (directly or indirectly) affects the activity of factor A in return. In the context of transcriptional networks, feedback loops typically refer to a transcription factor A affecting the expression of a transcription factor B, and vice versa.
cis-regulatory information refers to sequences in DNA bound by transcription factors that regulate the expression of a gene. cis means ‘on the same side’, and it refers to the fact that DNA sequences are ‘on the same side of the gene’ (as in the same molecule), as opposed to the transcription factors that bind to them, which are referred to as ‘trans-regulators’.

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