30 May 2003. Embryonic stem (ES) cells have the ability to divide ad infinitum. In today's Cell, two independent research groups report that a key to this "eternal youth" is the protein Nanog, which appears essential not only for cellular self-renewal, but also for maintenance of pluripotency, or the ability of ES cells to generate any specialized cell type.
Because of the source of the material, research on human embryonic stem cells has proven controversial globally, and more difficult in the U.S. since federal restrictions on studying new cell lines were introduced. One way around such obstacles is to generate pluripotent cell lines from nonembryonic stem cells or partially differentiated cells. Knowing what factors are essential for immortality and pluripotency is the first step toward this goal.
Previously, it had been shown that the protein leukemia inhibitory factor (LIF) can bestow immortality on certain mouse ES cells (and LIF also plays a key role in adult neurogenesis-see ARF related news story); however, this protein is not required for self-renewal of human ES cells, indicating that other, heretofore uncharacterized factors are involved. It was in search of these other factors that both groups discovered Nanog.
Shinya Yamanaka and colleagues at the Nara Institute of Science and Technology, Japan, began their search by comparing RNA expression profiles of ES cells and somatic tissue. First author Kaoru Mitsui and colleagues identified nine ES cell-associated transcripts (ecats), which had not yet been characterized. The authors found that, when expressed in ES cells, one of these-ecat4-could maintain division of the cells even in the absence of LIF.
Mitsui and colleagues used genetic recombination to ablate ecat4. This slowed ES cell growth and altered morphology such that the cells took on characteristics of endodermal cells. When the authors made transgenic mice containing only one copy of ecat4, the animals appeared normal, but when they tried to cross these mice they found that homozygous null animals had severe developmental problems and died in utero. The authors, in consultation with the second research group, christened ecat4 Nanog, after the Celtic land of eternal youth, Tir na nÓg.
It was in Celtic Scotland at the University of Edinburgh where Ian Chambers and colleagues screened for self-renewal factors by transfecting LIF-null ES cells with cDNA libraries. Like Mitsui and colleagues, Chambers found that expression of Nanog kept these cells undifferentiated and dividing.
As revealed by both groups, Nanog is a homeodomain protein that is expressed exclusively in pluripotent cells and is absent from differentiated tissues. Nanog has a putative human ortholog which, Chambers and colleagues show, can also drive self-renewal in mouse ES cells, though admittedly not as effectively as the mouse gene.
Chambers and colleagues also explored in depth the relation between Nanog and the LIF pathway. The latter is known to be activated when LIF/LIF receptor binds to another cell surface protein gp130. The formation of this heterodimer leads to activation of the tyrosine kinase Jak, and the Src homology protein Stat3. The authors show that an inhibitor of the Jak/Stat3 pathway, D6665, leads to differentiation of LIF-treated ES cells, while those overexpressing Nanog remained undifferentiated. In addition, while downstream genes like SOCS3 are induced by LIF, Chambers and colleagues found that Nanog levels do not change in response to LIF.
Overall, the results from both groups suggest that Nanog and LIF act independently, but in concert, to keep ES cells perpetually undifferentiated. While Nanog is expressed normally in ES cells, levels are insufficient to drive self-renewal in the absence of LIF. Nevertheless, as suggested by Chambers and colleagues, "The regulation and interactions of Nanog and the nature of its target genes now emerge as central issues in pluripotent stem cell biology."-Tom Fagan.
Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S. The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003 May 30;113:631-642. Abstract
Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003 May 30;113:643-655. Abstract