Tongxiang Lin

p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression

The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53S315A knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53S315A ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.

Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.

(Cell Stem Cell 4, 16–19; January 9, 2009)In our recent article, we unfortunately misquoted the findings in a recent study by Ying et al. (2008)xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al. (2008). When describing previous work using combination of the MEK inhibitor PD0325901 and the GSK3b inhibitor CHIR99021, our statement “Recent studies demonstrated that addition of the FGFR inhibitor PD173074 to the above cocktail is sufficient to maintain mESC pluripotency in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008).” was not accurate. Ying et al., in fact, used PD0325901 to replace PD1730474 and demonstrated maintenance of mESCs in the absence of LIF with these two factors only.The corrected section reads “Indeed, after serial passages, the growth of putative riPSCs treated with only PD0325901 and CHIR99021 declined, and the culture deteriorated due to the expansion of differentiated cells, although recent studies demonstrated that this combination of inhibitors can be used to maintain mESC self-renewal in the absence of LIF (Ying et al., 2008xThe ground state of embryonic stem cell self-renewal. Ying, Q.-L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. Nature. 2008; 453: 519–523Crossref | PubMed | Scopus (1294)See all ReferencesYing et al., 2008). Because the TGFβ/Activin A/Nodal signaling cascade is essential to maintain undifferentiated hESCs and EpiSCs, but dispensable for mESC self-renewal, we tested whether the addition of an inhibitor of the type 1 TGFβ receptor, ALK5 (A-83-01), could help stabilize our riPSC cultures.”In addition, we omitted to mention that while our study was under review, Silva et al. (2008)xPromotion of Reprogramming to Ground State Pluripotency by Signal Inhibition. Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. PLoS Biol. 2008; 6: 2237–2247Crossref | Scopus (456)See all ReferencesSilva et al. (2008) also published the use of these two inhibitors in the generation and propagation of mouse iPSCs.We apologize for any confusion caused.

A chemical platform for improved induction of human iPSCs

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.

Reprogramming of Human Primary Somatic Cells by OCT4 and Chemical Compounds

Induced pluripotent stem cell (iPSC) technology, i.e. reprogramming somatic cells into pluripotent cells that closely resemble embryonic stem cells (ESCs) by introduction of defined transcription factors (TFs), holds great potential in biomedical research and regenerative medicine (Takahashi et al., 2006; Takahashi et al., 2007; Yu et al., 2007). Various strategies have been developed to generate iPSCs with fewer or no exogenous genetic manipulations, which represent a major hurdle for iPSC applications (Yamanaka et al., 2009). With the ultimate goal of generating iPSCs with a defined small molecule cocktail alone, substantial effort and progress have been made in identifying chemical compounds that can functionally replace exogenous reprogramming TFs and/or enhance the efficiency and kinetics of reprogramming (Shi et al., 2008; Huangfu et al., 2008; Lyssiotis et al., 2009; Ichida et al., 2009; Maherali et al., 2009; Lin et al., 2009; Li et al., 2009; Esteban et al., 2010). To date, only neural stem cells (NSCs), which endogenously express SOX2 and cMYC at a high level, have been reprogrammed to iPSCs by exogenous expression of just OCT4 (Kim et al., 2009). However, human fetal NSCs are rare and difficult to obtain. It is therefore important to develop reprogramming conditions for other more accessible somatic cells. Here we report a small molecule cocktail that enables reprogramming of human primary somatic cells to iPSCs with exogenous expression of only OCT4. In addition, mechanistic studies revealed that modulation of cell metabolism from mitochondrial oxidation to glycolysis plays an important role in reprogramming.

Generation of Human Induced Pluripotent Stem Cells in the Absence of Exogenous Sox2

Induced pluripotent stem cell technology has attracted enormous interest for potential application in regenerative medicine. Here, we report that a specific glycogen synthase kinase 3 (GSK‐3) inhibitor, CHIR99021, can induce the reprogramming of mouse embryonic fibroblasts transduced by only two factors, Oct4 and Klf4. When combined with Parnate (also named tranylcypromine), an inhibitor of lysine‐specific demethylase 1, CHIR99021 can cause the reprogramming of human primary keratinocyte transduced with the two factors, Oct4 and Klf4. To our knowledge, this is the first time that human iPS cells have been generated from somatic cells without exogenous Sox2 expression. Our studies suggest that the GSK‐3 inhibitor might have a general application to replace transcription factors in both mouse and human reprogramming. STEM CELLS 2009;27:2992–3000

Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity. Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF-β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021), and TGF-β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs.

Functional Analysis of the Roles of Posttranslational Modifications at the p53 C Terminus in Regulating p53 Stability and Activity

ABSTRACT Posttranslational modification of the tumor suppressor p53 plays important roles in regulating its stability and activity. Six lysine residues at the p53 C terminus can be posttranslationally modified by various mechanisms, including acetylation, ubiquitination, neddylation, methylation, and sumoylation. Previous cell line transfection studies show that ubiquitination of these lysine residues is required for ubiquitin-dependent degradation of p53. In addition, biochemical and cell line studies suggested that p53 acetylation at the C terminus might stabilize p53 and activate its transcriptional activities. To investigate the physiological functional outcome of these C-terminal modifications in regulating p53 stability and activity, we introduced missense mutations (lysine to arginine) at the six lysine residues (K6R) into the endogenous p53 gene in mouse embryonic stem (ES) cells. The K6R mutation prevents all posttranslational modifications at these sites but conserves the structure of p53. In contrast to conclusions of previous studies, analysis of p53 stability in K6R ES cells, mouse embryonic fibroblasts, and thymocytes showed normal p53 stabilization in K6R cells both before and after DNA damage, indicating that ubiquitination of these lysine residues is not required for efficient p53 degradation. However, p53-dependent gene expression was impaired in K6R ES cells and thymocytes in a promoter-specific manner after DNA damage, indicating that the net outcome of the posttranslational modifications at the C terminus is to activate p53 transcriptional activities after DNA damage.

Acetylation of Mouse p53 at Lysine 317 Negatively Regulates p53 Apoptotic Activities after DNA Damage

ABSTRACT Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53K317R mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53K317R MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53K317R thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53K317R thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Important Roles for E Protein Binding Sites within the Immunoglobulin κ Chain Intronic Enhancer in Activating Vκ Jκ Rearrangement

The immunoglobulin κ light chain intronic enhancer (iEκ) activates κ rearrangement and is required to maintain the earlier or more efficient rearrangement of κ versus lambda (λ). To understand the mechanism of how iEκ regulates κ rearrangement, we employed homologous recombination to mutate individual functional motifs within iEκ in the endogenous κ locus, including the NF-κB binding site (κB), as well as κE1, κE2, and κE3 E boxes. Analysis of the impacts of these mutations revealed that κE2 and to a lesser extent κE1, but not κE3, were important for activating κ rearrangement. Surprisingly, mutation of the κB site had no apparent effect on κ rearrangement. Comparable to the deletion of the entire iEκ, simultaneous mutation of κE1 and κE2 reduces the efficiency of κ rearrangement much more dramatically than either κE1 or κE2 mutation alone. Because E2A family proteins are the only known factors that bind to these E boxes, these findings provide unambiguous evidence that E2A is a key regulator of κ rearrangement.

The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis.

Nanog is a transcription factor required for maintaining the pluripotency of embryonic stem cells, and is not expressed in most normal adult tissues. However, recent studies have indicated that Nanog is overexpressed in many types of human cancers, including breast cancer. To elucidate the physiological roles of Nanog in tumorigenesis, we developed an inducible Nanog transgenic mouse model, in which the expression of Nanog in adult tissues can be induced via LoxP/Cre-mediated deletion. Our findings indicate that overexpression of Nanog in the mammary gland is not sufficient to induce mammary tumor. However, when coexpressed with Wnt-1 in the mouse mammary gland, it promotes mammary tumorigenesis and metastasis. In this context, Nanog promotes the migration and invasion of breast cancer cells. Microarray analysis has shown that the ectopic expression of Nanog deregulates the expression of numerous genes associated with tumorigenesis and metastasis, such as the PDGFRα gene. Our findings demonstrate the involvement of Nanog in breast cancer metastasis, and provide the basis for the reported correlation between Nanog expression and poor prognosis of human breast cancer patients. As Nanog is not expressed in most adult tissues, these findings identify Nanog as a potential therapeutic target in the treatment of Nanog-expressing metastatic breast cancer.