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.

Reference:
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

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  1. Two papers published in the June issue of Cell describe the identification and characterization of a new transcription factor specifically expressed in embryonic stem (ES) cells.

    ES cells are derived in vitro from the inner cell mass (ICM) of the early embryo at the blastocyst stage. ES cells have been first obtained from the mouse embryo in the early 80’s (Evans and Kaufman, 1981; Martin, 1981), and have been more recently isolated from human embryos (Thomson et al., 1998). These cells can be propagated indefinitely in vitro, that is, they self-renew. Most importantly, they are pluripotent; they retain the capacity to differentiate into virtually any cell type under appropriate conditions. These exceptional properties make ES cells very powerful tools for basic research. They are studied to gain precious information about various biological processes such as embryonic development, cell differentiation, and more specifically, molecular events involved in the maintenance of the stem cell properties. ES cells also offer tremendous potential for the development of cell therapy strategies that could allow us to treat many human diseases.

    Major molecular characteristics of mouse ES cells are 1) they express Oct 3/4, a POU domain transcription factor whose expression is downregulated upon ES cell differentiation, and 2) they require cytokines of the LIF (Leukemia Inhibitory Factor) family for the maintenance of their properties, through stimulation of the LIF-R/gp130 receptor complex and activation of STAT (signal transducer and activator of transcription)-3 (see Burdon et al., 2002 for a recent review). Although Oct 3/4 is of critical importance for the maintenance of the mouse ES cell phenotype, it is not sufficient: ES cells constitutively expressing Oct 3/4 from an exogenous promoter still differentiate and require LIF for their propagation in vitro (Niwa et al., 2000). Thus, LIF was the only known factor able to sustain the in vitro maintenance of mouse ES cells, although evidence has suggested that the LIF-R/gp130 pathway is not fundamental for self-renewal and pluripotency in vivo in knockout mice (Li et al., 1995; Stewart et al., 1992; Ware et al. 1995; Yoshida et al. 1996).

    Now, Mitsui et al. (2003) and Chambers et al. (2003) describe the identification and characterization of a new transcription factor, named Nanog after the Celtic legend “Tir na nÓg” or “land of the ever young,” which is able to overcome the ES cell requirement for LIF (see also Cavaleri and Scholer, 2003). These two groups utilized very different means to identify Nanog. Mitsui et al. (2003) analyzed the UniGen database, which allows the performing of a differential display digitally, i.e., a way of finding genes specifically expressed in ES cells by comparing their genomic signatures with those of other somatic tissues or cells. Although this type of method has proven to be successful in identifying new genes in cancer (Scheurle et al., 2000; De Young et al., 2002), it is important to set the analysis with the right entries to avoid as many false positives as possible. Unfortunately, there is no information on the way Mitsui et al. performed their analysis (unlike another paper from the same group using the same approach Tokuzawa et al., 2003); that makes it hard to reproduce. They report a list of 20 genes differentially expressed in ES cells, including Oct 3/4 and others already identified as ES cell-specific markers. After confirming the ES cell-specific expression of nine previously uncharacterized genes, the authors experimentally overexpressed them individually in ES cells, and screened for undifferentiated colonies that could develop in the absence of LIF. Among the nine genes, only overexpression of Nanog was able to prevent the differentiation of ES cell colonies grown without LIF.

    In contrast, the analysis by Chambers et al. (2003) was truly experimental. They first generated a clone of ES cells devoid of the LIF-R gene, i.e., unable to maintain the ES cell phenotype even in the presence of LIF. They then transfected these modified ES cells with a cDNA library obtained from self-renewing and undifferentiated ES cells, and screened for colonies that were able to maintain the ES cell phenotype in the absence of LIF. In this way, they found two sets of colonies with morphological characteristics of undifferentiated ES cells, which were both transfected by two versions of the same cDNA, Nanog.

    Nanog is a homeodomain protein that shows little amino-acid identity (up to 50 percent) with other homeodomain proteins. Interestingly, mouse Nanog harbors a very particular motif: Tryptophan residues are repeated every fifth position for a total of 10 times. The function of this motif is unknown.

    Mitsui et al. showed that targeted disruption of Nanog in normal ES cells induces their differentiation into extra-embryonic endoderm, but interestingly, these cells continue to proliferate. The authors were also able to generate Nanog knockout animals, and showed that Nanog is necessary for normal mouse embryonic development, and that no ES cells could be derived from knockout embryos.

    Chambers et al. first analyzed the expression of Nanog mRNA during mouse embryonic development. Nanog is expressed in a temporal wave, being first detected in the compact morula (an early embryonic stage in which the blastomeres compact) and is downregulated at the time of implantation. Furthermore, Nanog expression is restricted to the ICM and later in the epiblast; i.e., the cells from which ES cells can be derived. The authors then performed a thorough in-vitro analysis, trying to build up a hierarchical model of interactions among Nanog, LIF/Stat-3, and Oct 3/4 in ES cells. They report that LIF/Stat-3 pathway does not induce Nanog expression, nor does Nanog influence the level of Stat-3 phosphorylation. However, while Nanog-overexpressing ES cells overcome LIF requirement for their phenotype maintenance, they grow slower than normal ES cells in the presence of LIF. Moreover, addition of LIF to Nanog-overexpressing ES cells leads to the generation of even more undifferentiated ES cell colonies, showing that LIF and Nanog have additional effects. These results suggest that, while there is no direct interaction between Stat-3 and Nanog, LIF/Stat-3 and Nanog pathways are somehow interrelated and might converge at a specific subset of genes. Chambers et al. also point out that Nanog overexpression is not able to overcome the loss of Oct 3/4, showing that Nanog, like LIF, requires the normal expression of Oct 3/4 to exert its effects on ES cells. Finally, the authors utilized a very elegant system allowing removal of extra copies of Nanog in transfected ES cells. They showed that the phenotype of Nanog-overexpressing ES cells can be fully reverted to normal, LIF-dependent, ES cells that do not appear affected by the period of clonal expansion driven by Nanog overexpression. They also showed that such reverted ES cells can be used to generate transgenic animals.

    The discovery of Nanog is important for ES cell research. Nanog overexpression is sufficient to maintain ES cell phenotypes, given that they express normal levels of Oct 3/4. Thus, the identification of Nanog target genes is a new avenue for ES cell biology which will certainly bring new fundamental clues about what controls the ES cell phenotype. It will also be of particular interest to find out what factors govern the very specific spatial and temporal expression pattern of Nanog in the developing embryo.

    Could Nanog be used in ES cell-based technology? One application of Nanog’s discovery could be to grow mouse ES cell colonies in laboratories interested in making transgenic and/or knockout animals. Indeed, having ES cell lines overexpressing Nanog with an excision capability system as described in Chambers et al. would prevent the use of purified LIF protein (patented and manufactured only by Chemicon International), or the growth of ES cells as co-cultures with embryonic fibroblasts.

    One question that is not assessed yet is what would be the result of using directly Nanog overexpressing ES cells (not reverted) in transgenic/knockout technology. Would Nanog still be downregulated at the time of implantation? If not, since Nanog overexpression in vitro maintains the ES cell phenotype, it would suggest that such an experiment would probably lead to malformed embryos composed of proliferating and undifferentiated cells resembling teratomas. Indeed, Nanog is associated with pluripotency rather than proliferation, so Nanog overexpression could perturb the normal cell type specification that occurs in the embryo.

    One major drawback of ES cells currently being used is that they first need to be efficiently differentiated into the specific cell type of interest before the are transplanted; otherwise they will form teratomas. Could it be that deletion of Nanog in transplanted ES cells would prevent tumor formation while retaining the cells’ ability to differentiate into the appropriate cell type? While considering this question of a therapeutic use of stem cells in humans, it might be more feasible to use adult stem cells derived from the patients themselves. However, adult stem cells are somewhat differentiated; they can give rise to different cell types of one particular lineage. Could Nanog overexpression (or reexpression) convert these adult stem cells into stem cells with broader differentiative capabilities, so that a minor surgical procedure could be used to isolate one type of adult stem cell that could be reverted to pluripotency and give rise to any cell type for transplantation? The future will teach us.

    References:

    Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol, 2002. 12(9): p. 432-8. Abstract

    Cavaleri F, Scholer HR. Nanog. A new recruit to the embryonic stem cell orchestra. Cell, 2003. 113(5): p. 551-2. 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. 113(5): p. 643-55. Abstract

    De Young MP, Damania H, Scheurle D, Zylberberg C, Narayanan R. Bioinformatics-based discovery of a novel factor with apparent specificity to colon cancer. In Vivo, 2002. 16(4): p. 239-48. Abstract

    Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981. 292(5819): p. 154-6. Abstract

    Li M, Sendtner M, Smith A. Essential function of LIF receptor in motor neurons. Nature, 1995. 378(6558): p. 724-7. Abstract

    Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A, 1981. 78(12): p. 7634-8. Abstract

    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. 113(5): p. 631-42. Abstract

    Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet, 2000. 24(4): p. 372-6. Abstract

    Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol, 2000. 18(4): p. 399-404. Abstract

    Scheurle D, DeYoung MP, Binninger DM, Page H, Jahanzeb M, Narayanan R. Cancer gene discovery using digital differential display. Cancer Res, 2000. 60(15): p. 4037-43. Abstract

    Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, Abbondanzo SJ. Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature, 1992. 359(6390): p. 76-9. Abstract

    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7. Abstract

    Tokuzawa Y, Kaiho E, Maruyama M, Takahashi K, Mitsui K, Maeda M, Niwa H, Yamanaka S. Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development. Mol Cell Biol, 2003. 23(8): p. 2699-708. Abstract

    Ware CB, Horowitz MC, Renshaw BR, Hunt JS, Liggitt D, Koblar SA, Gliniak BC, McKenna HJ, Papayannopoulou T, Thoma B, et al.

    Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development, 1995. 121(5): p. 1283-99. Abstract

    Yoshida K, Taga T, Saito M, Suematsu S, Kumanogoh A, Tanaka T, Fujiwara H, Hirata M, Yamagami T, Nakahata T, Hirabayashi T, Yoneda Y, Tanaka K, Wang WZ, Mori C, Shiota K, Yoshida N, Kishimoto T. Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders. Proc Natl Acad Sci U S A, 1996. 93(1): p. 407-11. Abstract

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References

News Citations

  1. Smells Like Neuronal Proliferation!

Paper Citations

  1. . The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 2003 May 30;113(5):631-42. PubMed.
  2. . Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003 May 30;113(5):643-55. PubMed.

Further Reading

News

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  2. Small Molecules Turn Stem Cells into Neurons, Targeting GSK3β
  3. Adult Neurogenesis in the "Black Substance"
  4. Adult Stem Cells Target Inflammation, Replace Cells in MS Model
  5. Stem Cell Fusion Is for Real
  6. Breaking Away: Human Glial Stem Cells Generate Neurons
  7. Playing Sonic Hedgehog in the Hippocampus
  8. Cholinergic Neurons from Stem Cells—Another Step Toward a Distant Goal

Primary Papers

  1. . The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 2003 May 30;113(5):631-42. PubMed.
  2. . Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003 May 30;113(5):643-55. PubMed.