Self-renewing Embryonic Stem Cells Research Articles

Nanog transforms NIH3T3 cells and targets cell-type restricted genes

The transcription factor Nanog is uniquely expressed in embryonic stem (ES) cells and in germ cell tumors and is important for self-renewal. To understand the relation between this and cell transformation, we expressed Nanog in NIH3T3 cells, and these cells showed an increased growth rate and a transformed phenotype as demonstrated by foci formation and colony growth in soft agar. This suggests that Nanog possesses an oncogenic potential that may be related to the role it plays in germ cell tumors and to its function in self renewal of ES cells. We studied the transcription targets of Nanog using microarrays to identify Nanog regulated genes. The list of genes regulated by Nanog was unique for each cell type and more than 10% of the Nanog regulated genes, including transcription factors, are primary Nanog targets since their promoters bind Nanog in ES cells. Some of these target genes can explain the transformation of NIH3T3.

NANOG Changes in Mouse Kidneys with Age

NANOG is essential for mouse and human embryonic stem cell (ESC) pluripotency and selfrenewal. It is also expressed in several adult murine tissues as shown by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. However, human NANOG transcripts have been isolated from adult bone marrow (EST; GenBank accession no. BF893620). Here, we study the NANOG gene expression profile in isolated mouse renal papillary cells by Northern blot and RT-PCR. The whole RNA of mouse renal cells was obtained from fresh renal tissues, renal tissues infused by phosphate-buffered saline (PBS), and isolated renal papillary cells of mouse, respectively, as well as the renal papillary tissue from 18.5 days postcoitum (d.p.c.; fetal), 1-2-week-old (young), 1-8-month-old (adult), and 24-month-old (aging) mice. Our analysis shows that a very low expression level was detected in mouse renal tissues, and the renal papillary cells express more than other tissues as determined with Northern blot and RT-PCR. These data suggest that the kidney has its own cells expressing NANOG, and loss of NANOG expression occurs in an age-dependent manner in the kidney, either due to developmental factors or aging, particularly in renal papillary tissue.

Role of SIRT1 in Proliferation and Self-Renewal of Mouse Embryonic Stem Cells.

A detailed understanding of regulation of self-renewal and ex-vivo expansion of embryonic stem cells is vital to ensure their safe and efficient use for differentiation to hematopoietic cells to be used in various therapeutic methodologies. The maintenance of murine embryonic stem (mES) cell self-renewal is regulated by leukemia inhibitory factor (LIF)-dependent activation of signal transducer and activator of transcription 3 (STAT3) and LIF-independent mechanisms including Nanog, BMP2/4, and Wnt signaling. Here we describe a novel role for the NAD-dependent protein deacetylase, SIRT1, in regulation of mES cell proliferation and self-renewal. SIRT1 mRNA expression in mES cells was induced by LIF through hypoxia-independent activation of hypoxia inducing factor-1α (HIF-1α. We cloned the 2 kb 5′-flanking region of the mouse SIRT1 gene and fused it to the luciferase gene. Overexpression of HIF-1α markedly enhanced SIRT1 promoter activity and a dominant-negative isoform, HIF-1α completely suppressed it. We demonstrated that two putative HIF-1α response element in the SIRT1 gene 5′-flanking region (-216 to -146) is essential for this SIRT1 induction. The two promoter regions, containing HIF-1-binding sequences, were regulated directly by HIF-1 α. These results suggest that HIF-1 enhances transactivation of the SIRT1 gene in mES cells that maintain their undifferentiated state with LIF, implying that stimulation of the SIRT1 promoter by HIF-1 may be responsible for induction of the SIRT1 gene. HIF-1α activation was blocked by the reversible phosphatidylinositol 3-OH kinase (PI3K) inhibitor, LY294002, as well as a more specific inhibititory class I(A) PI3K via regulated expression of dominant negative Deltap85, indicating that PI3K signaling is important for the hypoxia-independent HIF activity. We also demonstrate that SIRT1 deacetylates p53 at Lys 379 and then exports p53 to the cytoplasm, which results in inhibition of p53 transcriptional activity on p21. Inhibition of SIRT1 expression by siRNA translocated p53 to the nucleus and recovered p53 transactivational activity, suggesting that the deacetylated form of p53 at Lys 379 cannot access the p21 promoter and activate p21 transcription. To our knowledge, this is the first description of an influence of SIRT1 on p53 nuclear translocation via its deacetylation activity on p53. Surprisingly, we also found that N-terminal deleted p73 (DeltaNp73), a p53 family member, binds to p53 binding sites on the nanog promoter in normal mES cells, while p53 binds to p53 binding sites on the nanog promoter only in SIRT1-deficient mES cells. We then cloned the 2 kb 5′-flanking region of the mouse nanog gene and fused it to the luciferase gene. Overexpression of DeltaNp73 enhanced nanog promoter activity, but additional overexpression of p53 with DeltaNp73 abolished DeltaNp73-induced enhancement of nanog promoter activity. These data suggest that SIRT1 may regulate binding of two transcription factors, p53 and DeltaNp73, to the p53 binding site of the nanog promoter and turn on/off the nanog gene, which may eventually determine ES cell fate to self-renewal or differentiation. Upon long term inhibition of SIRT1 by SIRT1 siRNA retroviral infection, we observed a reduction in cell proliferation rate, whereas long term overexpression of SIRT by retroviral vector containing SIRT1 gene enhanced cell proliferation. Our results suggest SIRT1 controls self-renewal and proliferation of mES cells by regulation of p53 localization through deacetylation of p53.

Germ layer induction from embryonic stem cells

Embryonic stem (ES) cells have the potential to develop into all cell types of the adult body. This capability provides the basis for considering the ES cell system as a novel and unlimited source of cells for replacement therapies for the treatment of a wide range of diseases. Before the cell-based therapy potential of ES cells can be realized, a better understanding of the pathways regulating lineage-specific differentiation is required. Current studies suggest that the bone morphogenic protein, transforming growth factor-beta, Wnt, and fibroblast growth factor pathways that are required for gastrulation and germ layer induction in the embryo are also essential for differentiation of ES cells in culture. The current understanding of how these factors influence germ layer induction in both the embryo and in the ES cell differentiation system is addressed in this review.

Live Long and Prosper: Fig. 1.

In this issue of Molecular Pharmacology, Meyn et al. (p. 1320) provide the results of the first comprehensive investigation of the expression pattern of the Src family of nonreceptor tyrosine kinases (SFK) in mouse embryonic stem (ES) cells. They found that self-renewing ES cells express seven of the eight mammalian SFK members and that some undergo distinct expression changes during early differentiation events. One of the most dramatic changes was in Hck transcript levels, which decreased almost 30-fold during the first 3 days of embryoid body formation, a culture system model of early embryogenesis and differentiation. Other SFKs, such as Fyn and Src, remain present and active as ES cells differentiate. Of particular interest was the observation that ES cell self-renewal or differentiation can be manipulated through the selective pharmacological inhibition of SFK members. This information should help in the expanding efforts to exploit ES cells for basic and clinical purposes.

Src Family Kinase Activity Is Required for Murine Embryonic Stem Cell Growth and Differentiation

Self-renewal and differentiation of embryonic stem (ES) cells are regulated by cytokines and growth factors through tyrosine kinase-dependent signaling pathways. In murine ES cells, signals for self-renewal are generated by the leukemia inhibitory factor (LIF). LIF and other growth factors are linked to the activation of the Src family of cytoplasmic protein-tyrosine kinases (SFKs), which consists of eight members having shared structural architecture. In this article, we show that murine ES cells express seven SFKs, three of which (Hck, Src, and Fyn) exhibit constitutive activity in self-renewing ES cells. Differentiation of ES cells to embryoid bodies was associated with rapid transcriptional silencing of Hck and Lck and with the loss of the corresponding kinase proteins. The expression of other family members remained relatively constant, although some loss of Fgr and Lyn proteins was observed during differentiation. Like ES cells, embryoid bodies maintained constitutive Src and Fyn kinase activity. Partial inhibition of endogenous SFK activity with the ATP-competitive inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Src kinase inhibitor-1 induced differentiation of ES cells in the presence of LIF. In contrast, suppression of all SFK activity with higher concentrations of these inhibitors, or with the more potent compound A-419259 (Bioorg Med Chem Lett 12:1683-1686, 2002) blocked differentiation in response to LIF withdrawal. It is surprising that these inhibitor-treated cells remained pluripotent despite the absence of LIF. Our results implicate individual members of the Src kinase family in distinct ES cell renewal and differentiation pathways and show that small-molecule SFK inhibitors can control ES cell fate.

Regulation of Embryonic Stem Cell Self-renewal by Phosphoinositide 3-Kinase-dependent Signaling

The maintenance of murine embryonic stem (ES) cell self-renewal is regulated by leukemia inhibitory factor (LIF)-dependent activation of signal transducer and activator of transcription 3 (STAT3) and LIF-independent mechanisms including Nanog, BMP2/4, and Wnt signaling. Here we demonstrate a previously undescribed role for phosphoinositide 3-kinases (PI3Ks) in regulation of murine ES cell self-renewal. Treatment with the reversible PI3K inhibitor, LY294002, or more specific inhibition of class I(A) PI3K via regulated expression of dominant negative Deltap85, led to a reduction in the ability of LIF to maintain self-renewal, with cells concomitantly adopting a differentiated morphology. Inhibition of PI3Ks reduced basal and LIF-stimulated phosphorylation of PKB/Akt, GSK3alpha/beta, and S6 proteins. Importantly, LY294002 and Deltap85 expression had no effect on LIF-induced phosphorylation of STAT3 at Tyr(705), but did augment LIF-induced phosphorylation of ERKs in both short and long term incubations. Subsequently, we demonstrate that inhibition of MAP-Erk kinases (MEKs) reverses the effects of PI3K inhibition on self-renewal in a time- and dose-dependent manner, suggesting that the elevated ERK activity observed upon PI3K inhibition contributes to the functional response we observe. Surprisingly, upon long term inhibition of PI3Ks we observed a reduction in phosphorylation of beta-catenin, the target of GSK-3 action in the canonical Wnt pathway, although no consistent alterations in cytosolic levels of beta-catenin were observed, indicating this pathway is not playing a major role downstream of PI3Ks. Our studies support a role for PI3Ks in regulation of self-renewal and increase our understanding of the molecular signaling components involved in regulation of stem cell fate.

The Src Family of Tyrosine Kinases Is Important for Embryonic Stem Cell Self-renewal

cYes, a member of the Src family of non-receptor tyrosine kinases, is highly expressed in mouse and human embryonic stem (ES) cells. We demonstrate that cYes kinase activity is regulated by leukemia inhibitory factor (LIF) and serum and is down-regulated when cells differentiate. Moreover, selective chemical inhibition of Src family kinases decreases growth and expression of stem cell genes that mark the undifferentiated state, including Oct3/4, alkaline phosphatase, fibroblast growth factor 4, and Nanog. A synergistic effect on differentiation is observed when ES cells are cultured with an Src family inhibitor and low levels of retinoic acid. Src family kinase inhibition does not interfere with LIF-induced JAK/STAT3 (Janus-associated tyrosine kinases/signal transducer and activator of transcription 3) or p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Together the results suggest that the activation of the Src family is important for maintaining mouse and human ES in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ES cells.

BMP Induction of Id Proteins Suppresses Differentiation and Sustains Embryonic Stem Cell Self-Renewal in Collaboration with STAT3

The cytokine leukemia inhibitory factor (LIF) drives self-renewal of mouse embryonic stem (ES) cells by activating the transcription factor STAT3. In serum-free cultures, however, LIF is insufficient to block neural differentiation and maintain pluripotency. Here, we report that bone morphogenetic proteins (BMPs) act in combination with LIF to sustain self-renewal and preserve multilineage differentiation, chimera colonization, and germline transmission properties. ES cells can be propagated from single cells and derived de novo without serum or feeders using LIF plus BMP. The critical contribution of BMP is to induce expression of Id genes via the Smad pathway. Forced expression of Id liberates ES cells from BMP or serum dependence and allows self-renewal in LIF alone. Upon LIF withdrawal, Id-expressing ES cells differentiate but do not give rise to neural lineages. We conclude that blockade of lineage-specific transcription factors by Id proteins enables the self-renewal response to LIF/STAT3.