Publications by authors named "Qi-Long Ying"

51 Publications

The Distinct Role of Tcfs and Lef1 in the Self-Renewal or Differentiation of Mouse Embryonic Stem Cells.

Int J Stem Cells 2020 Jul;13(2):192-201

Department of Life Science, University of Seoul, Seoul, Korea.

Background And Objectives: Tcfs and Lef1 are DNA-binding transcriptional factors in the canonical Wnt signaling pathway. In the absence of -catenin, Tcfs and Lef1 generally act as transcriptional repressors with co-repressor proteins such as Groucho, CtBP, and HIC-5. However, Tcfs and Lef1 turn into transcriptional activators during the interaction with -catenin. Therefore, the activity of Tcfs and Lef1 is regulated by -catenin. However, the intrinsic role of Tcfs and Lef1 has yet to be examined. The purpose of this study was to determine whether Tcfs and Lef1 play differential roles in the regulation of self-renewal and differentiation of mouse ES cells.

Methods And Results: Interestingly, the expression of Tcfs and Lef1 was dynamically altered under various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcf and Lef1 may vary in ES cells. Ectopic expression of Tcf1 or the dominant negative form of Lef1 (Lef1-DN) contributes to ES cells to self-renew in the absence of leukemia inhibitory factor (LIF), whereas ectopic expression of Tcf3, Lef1 or Tcf1-DN did not support ES cells to self-renew. Ectopic expression of either Lef1 or Lef1-DN blocked neuronal differentiation, suggesting that the transient induction of Lef1 was necessary for the initiation and progress of differentiation. ChIP analysis shows that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog.

Conclusions: The overall data suggest that Tcf1 plays a critical role in the maintenance of stemness whereas Lef1 is involved in the initiation of differentiation.
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http://dx.doi.org/10.15283/ijsc20044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378906PMC
July 2020

Long-term self-renewal of naïve neural stem cells in a defined condition.

Biochim Biophys Acta Mol Cell Res 2019 06 9;1866(6):971-977. Epub 2019 Mar 9.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology & Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, United States of America. Electronic address:

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naïve NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.
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http://dx.doi.org/10.1016/j.bbamcr.2019.03.001DOI Listing
June 2019

A Chemical-Genetic Approach Reveals the Distinct Roles of GSK3α and GSK3β in Regulating Embryonic Stem Cell Fate.

Dev Cell 2017 12;43(5):563-576.e4

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. Electronic address:

Glycogen synthase kinase 3 (GSK3) plays a central role in diverse cellular processes. GSK3 has two mammalian isozymes, GSK3α and GSK3β, whose functions remain ill-defined because of a lack of inhibitors that can distinguish between the two highly homologous isozymes. Here, we show that GSK3α and GSK3β can be selectively inhibited in mouse embryonic stem cells (ESCs) using a chemical-genetic approach. Selective inhibition of GSK3β is sufficient to maintain mouse ESC self-renewal, whereas GSK3α inhibition promotes mouse ESC differentiation toward neural lineages. Genome-wide transcriptional analysis reveals that GSK3α and GSK3β have distinct sets of downstream targets. Furthermore, selective inhibition of individual GSK3 isozymes yields distinct phenotypes from gene deletion, highlighting the power of the chemical-genetic approach in dissecting kinase catalytic functions from the protein's scaffolding functions. Our study opens new avenues for defining GSK3 isozyme-specific functions in various cellular processes.
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http://dx.doi.org/10.1016/j.devcel.2017.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851779PMC
December 2017

Myeloid Notch1 deficiency activates the RhoA/ROCK pathway and aggravates hepatocellular damage in mouse ischemic livers.

Hepatology 2018 03 24;67(3):1041-1055. Epub 2018 Jan 24.

The Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA.

Notch signaling plays an emerging role in the regulation of immune cell development and function during inflammatory response. Activation of the ras homolog gene family member A/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates ras homolog gene family member A/ROCK-mediated immune responses in liver ischemia and reperfusion (IR) injury. This study investigated intracellular signaling pathways regulated by Notch receptors in the IR-stressed liver and in vitro. In a mouse model of IR-induced liver inflammatory injury, we found that mice with myeloid-specific Notch1 knockout showed aggravated hepatocellular damage, with increased serum alanine aminotransferase levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and proinflammatory mediators compared to Notch1-proficient controls. Unlike in the controls, myeloid Notch1 ablation diminished hairy and enhancer of split-1 (Hes1) and augmented c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), JNK, ROCK1, and phosphatase and tensin homolog (PTEN) activation in ischemic livers. Disruption of JSAP1 in myeloid-specific Notch1 knockout livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and toll-like receptor 4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed toll-like receptor 4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain promoted Hes1 and inhibited JSAP1 in lipopolysaccharide-stimulated bone marrow-derived macrophages. Hes1 deletion enhanced JSAP1/JNK activation, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated JSAP1 knockout diminished ROCK1/PTEN and toll-like receptor 4 signaling.

Conclusion: Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).
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http://dx.doi.org/10.1002/hep.29593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826840PMC
March 2018

represses multiple lineage commitment of mouse embryonic stem cells through MTA1 and LEF1.

J Cell Sci 2017 Nov 5;130(22):3809-3817. Epub 2017 Oct 5.

Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei 230601, PR China

TFCP2L1 is a transcription factor that is crucial for self-renewal of mouse embryonic stem cells (mESCs). How TFCP2L1 maintains the pluripotent state of mESCs, however, remains unknown. Here, we show that knockdown of in mESCs induces the expression of endoderm, mesoderm and trophectoderm markers. Functional analysis of mutant forms of TFCP2L1 revealed that TFCP2L1 depends on its N-terminus and CP2-like domain to maintain the undifferentiated state of mESCs. The N-terminus of TFCP2L1 is mainly associated with the suppression of mesoderm and trophectoderm differentiation, while the CP2-like domain is closely related to the suppression of endoderm commitment. Further studies showed that MTA1 directly interacts with TFCP2L1 and is indispensable for the TFCP2L1-mediated self-renewal-promoting effect and endoderm-inhibiting action. TFCP2L1-mediated suppression of mesoderm and trophectoderm differentiation, however, seems to be due to downregulation of expression. Our study thus provides an expanded understanding of the function of TFCP2L1 and the pluripotency regulation network of ESCs.
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http://dx.doi.org/10.1242/jcs.206532DOI Listing
November 2017

Sp5 induces the expression of Nanog to maintain mouse embryonic stem cell self-renewal.

PLoS One 2017 29;12(9):e0185714. Epub 2017 Sep 29.

Center for Stem Cell and Translational Medicine, School of Life Science, Anhui University, Hefei, PR China.

Activation of signal transducer and activator of transcription 3 (STAT3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal. Our previous study showed that trans-acting transcription factor 5 (Sp5), an LIF/STAT3 downstream target, supports mESC self-renewal. However, the mechanism by which Sp5 exerts these effects remains elusive. Here, we found that Nanog is a direct target of Sp5 and mediates the self-renewal-promoting effect of Sp5 in mESCs. Overexpression of Sp5 induced Nanog expression, while knockdown or knockout of Sp5 decreased the Nanog level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of Nanog eliminated the mESC self-renewal-promoting ability of Sp5. Finally, we demonstrated that the self-renewal-promoting function of Sp5 was largely dependent on its zinc finger domains. Taken together, our study provides unrecognized functions of Sp5 in mESCs and will expand our current understanding of the regulation of mESC pluripotency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185714PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621696PMC
November 2017

β-catenin coordinates with Jup and the TCF1/GATA6 axis to regulate human embryonic stem cell fate.

Dev Biol 2017 11 21;431(2):272-281. Epub 2017 Sep 21.

Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei 230601, PR China. Electronic address:

β-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of human embryonic stem cells (hESCs). However, the results are controversial and the mechanism by which β-catenin regulates the hESC fate remains unclear. Here, we report that β-catenin and γ-catenin are functionally redundant in mediating hESC adhesion and are required for embryoid body formation, but both genes are dispensable for hESC maintenance, as the undifferentiated state of β-catenin and γ-catenin double deficient hESCs can be maintained. Overexpression of β-catenin induces rapid hESC differentiation. Functional assays revealed that TCF1 plays a crucial role in hESC differentiation mediated by β-catenin. Forced expression of TCF1, but not other LEF1/TCF family members, resulted in hESC differentiation towards the definitive endoderm. Conversely, knockdown of TCF1 or inhibition of the interaction between TCF1 and β-catenin delayed hESC exit from pluripotency. Furthermore, we demonstrated that GATA6 plays a predominant role in TCF1-mediated hESC differentiation. Knockdown of GATA6 completely eliminated the effect of TCF1, while forced expression of GATA6 induced hESC differentiation. Our data thus reveal more detailed mechanisms for β-catenin in regulating hESC fate decisions and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.
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http://dx.doi.org/10.1016/j.ydbio.2017.09.004DOI Listing
November 2017

The transcription factor Gbx2 induces expression of Kruppel-like factor 4 to maintain and induce naïve pluripotency of embryonic stem cells.

J Biol Chem 2017 10 28;292(41):17121-17128. Epub 2017 Aug 28.

From the Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei 230601, China and

The transcription factor Gbx2 (gastrulation brain homeobox 2) is a direct target of the LIF/STAT3 signaling pathway, maintains mouse embryonic stem cell (mESC) self-renewal, and facilitates mouse epiblast stem cell (mEpiSC) reprogramming to naïve pluripotency. However, the mechanism by which Gbx2 mediates its effects on pluripotency remains unknown. Here, using an RNA-Seq approach, we identified Klf4 (Kruppel-like factor 4) as a direct target of Gbx2. Functional studies indicated that Klf4 mediates the self-renewal-promoting effects of Gbx2, because knockdown of expression abrogated the ability of Gbx2 to maintain the undifferentiated state of mESCs. We also found that Gbx2 largely depends on to reprogram mEpiSCs to a mESC-like state. In summary, our study has uncovered a mechanism by which Gbx2 maintains and induces naïve pluripotency. These findings expand our understanding of the pluripotency control network and may inform the development of culture conditions for improved ESC maintenance and differentiation.
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http://dx.doi.org/10.1074/jbc.M117.803254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641878PMC
October 2017

Cytoplasmic and Nuclear TAZ Exert Distinct Functions in Regulating Primed Pluripotency.

Stem Cell Reports 2017 09 24;9(3):732-741. Epub 2017 Aug 24.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. Electronic address:

Mouse epiblast stem cells (mEpiSCs) and human embryonic stem cells (hESCs) are primed pluripotent stem cells whose self-renewal can be maintained through cytoplasmic stabilization and retention of β-catenin. The underlying mechanism, however, remains largely unknown. Here, we show that cytoplasmic β-catenin interacts with and retains TAZ, a Hippo pathway effector, in the cytoplasm. Cytoplasmic retention of TAZ promotes mEpiSC self-renewal in the absence of nuclear β-catenin, whereas nuclear translocation of TAZ induces mEpiSC differentiation. TAZ is dispensable for naive mouse embryonic stem cell (mESC) self-renewal but required for the proper conversion of mESCs to mEpiSCs. The self-renewal of hESCs, like that of mEpiSCs, can also be maintained through the cytoplasmic retention of β-catenin and TAZ. Our study indicates that how TAZ regulates cell fate depends on not only the cell type but also its subcellular localization.
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http://dx.doi.org/10.1016/j.stemcr.2017.07.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599246PMC
September 2017

The Art of Capturing Pluripotency: Creating the Right Culture.

Stem Cell Reports 2017 06;8(6):1457-1464

Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address:

Embryonic stem cells (ESCs) are a unique tool for genetic perturbation of mammalian cellular and organismal processes additionally in humans offer unprecedented opportunities for disease modeling and cell therapy. Furthermore, ESCs are a powerful system for exploring the fundamental biology of pluripotency. Indeed understanding the control of self-renewal and differentiation is key to realizing the potential of ESCs. Building on previous observations, we found that mouse ESCs can be derived and maintained with high efficiency through insulation from differentiation cues combined with consolidation of an innate cell proliferation program. This finding of a pluripotent ground state has led to conceptual and practical advances, including the establishment of germline-competent ESCs from recalcitrant mouse strains and for the first time from the rat. Here, we summarize historical and recent progress in defining the signaling environment that supports self-renewal. We compare the contrasting requirements of two types of pluripotent stem cell, naive ESCs and primed post-implantation epiblast stem cells (EpiSCs), and consider the outstanding challenge of generating naive pluripotent stem cells from different mammals.
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http://dx.doi.org/10.1016/j.stemcr.2017.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470336PMC
June 2017

Depletion of and maintains mouse embryonic stem cell self-renewal.

Biol Open 2017 Apr 15;6(4):511-517. Epub 2017 Apr 15.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Mouse and rat embryonic stem cell (ESC) self-renewal can be maintained by dual inhibition of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK). Inhibition of GSK3 promotes ESC self-renewal by abrogating T-cell factor 3 (TCF3)-mediated repression of the pluripotency network. How inhibition of MEK mediates ESC self-renewal, however, remains largely unknown. Here, we show that inhibition of MEK can significantly suppress lymphoid enhancer factor 1 (LEF1) expression in mouse ESCs. Knockdown or knockout of partially mimics the self-renewal-promoting effect of MEK inhibitors. Moreover, depletion of both and enables maintenance of undifferentiated mouse ESCs without exogenous factors, cytokines or inhibitors. Transcriptome resequencing analysis reveals that LEF1 is closely associated with endoderm specification in ESCs. Thus, our study adds support to the notion that the key to maintaining the ESC ground state is to shield ESCs from differentiative cues.
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http://dx.doi.org/10.1242/bio.022426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399551PMC
April 2017

Blockade of Notch signaling promotes acetaminophen-induced liver injury.

Immunol Res 2017 06;65(3):739-749

The Dumont-UCLA Transplant Center, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.

Liver injury after experimental acetaminophen treatment is mediated both by direct hepatocyte injury through a P450-generated toxic metabolite and indirectly by activated liver Kupffer cells and neutrophils. This study was designed to investigate the role of Notch signaling in the regulation of innate immune responses in acetaminophen (APAP)-induced liver injury. Using a mouse model of APAP-induced liver injury, wild-type (WT) and toll-like receptor 4 knockout (TLR4 KO) mice were injected intraperitoneally with APAP or PBS. Some animals were injected with γ-secretase inhibitor DAPT or DMSO vehicle. For the in vitro study, bone marrow-derived macrophages (BMMs) were transfected with Notch1 siRNA, TLR4 siRNA, and non-specific (NS) siRNA and stimulated with LPS. Indeed, paracetamol/acetaminophen-induced liver damage was worse after Notch blockade with DAPT in wild-type mice, which was accompanied by significantly increased ALT levels, diminished hairy and enhancer of split-1 (Hes1), and phosphorylated Stat3 and Akt but enhanced high mobility group box 1 (HMGB1), TLR4, NF-κB, and NLRP3 activation after APAP challenge. Mice receiving DAPT increased macrophage and neutrophil accumulation and hepatocellular apoptosis. However, TLR4 KO mice that received DAPT reduced APAP-induced liver damage and NF-κB, NLRP3, and cleaved caspase-1 activation. BMMs transfected with Notch1 siRNA reduced Hes1 and phosphorylated Stat3 and Akt but augmented HMGB1, TLR4, NF-κB, and NLRP3. Furthermore, TLR4 siRNA knockdown resulted in decreased NF-κB and NLRP3 and cleaved caspase-1 and IL-1β levels following LPS stimulation. These results demonstrate that Notch signaling regulates innate NLRP3 inflammasome activation through regulation of HMGB1/TLR4/NF-κB activation in APAP-induced liver injury. Our novel findings underscore the critical role of the Notch1-Hes1 signaling cascade in the regulation of innate immunity in APAP-triggered liver inflammation. This might imply a novel therapeutic potential for the drug-induced damage-associated lethal hepatitis.
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http://dx.doi.org/10.1007/s12026-017-8913-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464368PMC
June 2017

The myeloid heat shock transcription factor 1/β-catenin axis regulates NLR family, pyrin domain-containing 3 inflammasome activation in mouse liver ischemia/reperfusion injury.

Hepatology 2016 11 29;64(5):1683-1698. Epub 2016 Aug 29.

The Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA.

Heat shock transcription factor 1 (HSF1) has been implicated in the differential regulation of cell stress and disease states. β-catenin activation is essential for immune homeostasis. However, little is known about the role of macrophage HSF1-β-catenin signaling in the regulation of NLRP3 inflammasome activation during ischemia/reperfusion (I/R) injury (IRI) in the liver. This study investigated the functions and molecular mechanisms by which HSF1-β-catenin signaling influenced NLRP3-mediated innate immune response in vivo and in vitro. Using a mouse model of IR-induced liver inflammatory injury, we found that mice with a myeloid-specific HSF1 knockout (HSF1 ) displayed exacerbated liver damage based on their increased serum alanine aminotransferase levels, intrahepatic macrophage/neutrophil trafficking, and proinflammatory interleukin (IL)-1β levels compared to the HSF1-proficient (HSF1 ) controls. Disruption of myeloid HSF1 markedly increased transcription factor X-box-binding protein (XBP1), NLR family, pyrin domain-containing 3 (NLRP3), and cleaved caspase-1 expression, which was accompanied by reduced β-catenin activity. Knockdown of XBP1 in HSF1-deficient livers using a XBP1 small interfering RNA ameliorated hepatocellular functions and reduced NLRP3/cleaved caspase-1 and IL-1β protein levels. In parallel in vitro studies, HSF1 overexpression increased β-catenin (Ser552) phosphorylation and decreased reactive oxygen species (ROS) production in bone-marrow-derived macrophages. However, myeloid HSF1 ablation inhibited β-catenin, but promoted XBP1. Furthermore, myeloid β-catenin deletion increased XBP1 messenger RNA splicing, whereas a CRISPR/CRISPR-associated protein 9-mediated XBP1 knockout diminished NLRP3/caspase-1.

Conclusion: The myeloid HSF1-β-catenin axis controlled NLRP3 activation by modulating the XBP1 signaling pathway. HSF1 activation promoted β-catenin, which, in turn, inhibited XBP1, leading to NLRP3 inactivation and reduced I/R-induced liver injury. These findings demonstrated that HSF1/β-catenin signaling is a novel regulator of innate immunity in liver inflammatory injury and implied the therapeutic potential for management of sterile liver inflammation in transplant recipients. (Hepatology 2016;64:1683-1698).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074868PMC
http://dx.doi.org/10.1002/hep.28739DOI Listing
November 2016

Efficient Assessment of Developmental, Surgical and Pathological Lymphangiogenesis Using a Lymphatic Reporter Mouse and Its Embryonic Stem Cells.

PLoS One 2016 9;11(6):e0157126. Epub 2016 Jun 9.

Division of Plastic and Reconstructive Surgery, Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.

Several lymphatic reporter mouse lines have recently been developed to significantly improve imaging of lymphatic vessels. Nonetheless, the usage of direct visualization of lymphatic vessels has not been fully explored and documented. Here, we characterized a new Prox1-tdTomato transgenic lymphatic reporter mouse line, and demonstrated how this animal tool enables the researchers to efficiently assess developmental, surgical and pathological lymphangiogenesis by direct visualization of lymphatic vessels. Moreover, we have derived embryonic stem cells from this reporter line, and successfully differentiated them into lymphatic vessels in vivo. In conclusion, these experimental tools and techniques will help advance lymphatic research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157126PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900649PMC
July 2017

Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9.

Sci Rep 2016 Feb 22;6:21918. Epub 2016 Feb 22.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology &Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

Chromosomal translocation is the most common form of chromosomal abnormality and is often associated with congenital genetic disorders, infertility, and cancers. The lack of cellular and animal models for chromosomal translocations, however, has hampered our ability to understand the underlying disease mechanisms and to develop new therapies. Here, we show that site-specific chromosomal translocations can be generated in mouse embryonic stem cells (mESCs) via CRISPR/Cas9. Mouse ESCs carrying translocated chromosomes can be isolated and expanded to establish stable cell lines. Furthermore, chimeric mice can be generated by injecting these mESCs into host blastocysts. The establishment of ESC-based cellular and animal models of chromosomal translocation by CRISPR/Cas9 provides a powerful platform for understanding the effect of chromosomal translocation and for the development of new therapeutic strategies.
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http://dx.doi.org/10.1038/srep21918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761995PMC
February 2016

Wnt/β-catenin and LIF-Stat3 signaling pathways converge on Sp5 to promote mouse embryonic stem cell self-renewal.

J Cell Sci 2016 Jan 23;129(2):269-76. Epub 2015 Nov 23.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Activation of leukemia inhibitor factor (LIF)-Stat3 or Wnt/β-catenin signaling promotes mouse embryonic stem cell (mESC) self-renewal. A myriad of downstream targets have been identified in the individual signal pathways, but their common targets remain largely elusive. In this study, we found that the LIF-Stat3 and Wnt/β-catenin signaling pathways converge on Sp5 to promote mESC self-renewal. Forced Sp5 expression can reproduce partial effects of Wnt/β-catenin signaling but mimics most features of LIF-Stat3 signaling to maintain undifferentiated mESCs. Moreover, Sp5 is able to convert mouse epiblast stem cells into a naïve pluripotent state. Thus, Sp5 is an important component of the regulatory network governing mESC naïve pluripotency.
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http://dx.doi.org/10.1242/jcs.177675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732286PMC
January 2016

Stem cell maintenance by manipulating signaling pathways: past, current and future.

BMB Rep 2015 Dec;48(12):668-76

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retained in various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791322PMC
http://dx.doi.org/10.5483/bmbrep.2015.48.12.215DOI Listing
December 2015

Klf2 and Tfcp2l1, Two Wnt/β-Catenin Targets, Act Synergistically to Induce and Maintain Naive Pluripotency.

Stem Cell Reports 2015 Sep 28;5(3):314-22. Epub 2015 Aug 28.

Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, University of Southern California, Los Angeles, CA 90033, USA. Electronic address:

Activation of Wnt/β-catenin signaling can induce both self-renewal and differentiation in naive pluripotent embryonic stem cells (ESCs). To gain insights into the mechanism by which Wnt/β-catenin regulates ESC fate, we screened and characterized its downstream targets. Here, we show that the self-renewal-promoting effect of Wnt/β-catenin signaling is mainly mediated by two of its downstream targets, Klf2 and Tfcp2l1. Forced expression of Klf2 and Tfcp2l1 can not only induce reprogramming of primed state pluripotency into naive state ESCs, but also is sufficient to maintain the naive pluripotent state of ESCs. Conversely, downregulation of Klf2 and Tfcp2l1 impairs ESC self-renewal mediated by Wnt/β-catenin signaling. Our study therefore establishes the pivotal role of Klf2 and Tfcp2l1 in mediating ESC self-renewal promoted by Wnt/β-catenin signaling.
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http://dx.doi.org/10.1016/j.stemcr.2015.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618593PMC
September 2015

New insights into the conserved mechanism of pluripotency maintenance.

Curr Opin Genet Dev 2015 Oct 13;34:1-9. Epub 2015 Jul 13.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. Electronic address:

Pluripotent stem cells provide a powerful tool for both basic and translational research. The establishment and maintenance of germline-competent pluripotent stem cells in vitro, however, have only succeeded in the mouse and rat. From in vivo studies on pluripotency during embryogenesis and in vitro studies on existing pluripotent stem cells, several mechanisms have been uncovered for maintenance of both the naïve and the primed pluripotent states. Current clues strongly indicate that such mechanisms are likely conserved among different species. A better understanding of how these mechanisms work together to control cell fate choice will guide future research in both stem cell biology and regenerative medicine.
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http://dx.doi.org/10.1016/j.gde.2015.06.002DOI Listing
October 2015

Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells.

Stem Cells Int 2015 5;2015:459301. Epub 2015 Apr 5.

Department of Life Science, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul 130-743, Republic of Korea.

Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor) or negatively (IWR-1-endo, Axin stabilizer) control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.
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http://dx.doi.org/10.1155/2015/459301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402205PMC
May 2015

Molecular basis of embryonic stem cell self-renewal: from signaling pathways to pluripotency network.

Cell Mol Life Sci 2015 May 17;72(9):1741-57. Epub 2015 Jan 17.

Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, Hefei, 230601, PR China.

Embryonic stem cells (ESCs) can be maintained in culture indefinitely while retaining the capacity to generate any type of cell in the body, and therefore not only hold great promise for tissue repair and regeneration, but also provide a powerful tool for modeling human disease and understanding biological development. In order to fulfill the full potential of ESCs, it is critical to understand how ESC fate, whether to self-renew or to differentiate into specialized cells, is regulated. On the molecular level, ESC fate is controlled by the intracellular transcriptional regulatory networks that respond to various extrinsic signaling stimuli. In this review, we discuss and compare important signaling pathways in the self-renewal and differentiation of mouse, rat, and human ESCs with an emphasis on how these pathways integrate into ESC-specific transcription circuitries. This will be beneficial for understanding the common and conserved mechanisms that govern self-renewal, and for developing novel culture conditions that support ESC derivation and maintenance.
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http://dx.doi.org/10.1007/s00018-015-1833-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809369PMC
May 2015

Stat3 signaling regulates embryonic stem cell fate in a dose-dependent manner.

Biol Open 2014 Sep 19;3(10):958-65. Epub 2014 Sep 19.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Present address: Animal Biotechnology Interdisciplinary Group, Center for Veterinary Medicine, United States Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855, USA.

Stat3 is essential for mouse embryonic stem cell (mESC) self-renewal mediated by LIF/gp130 receptor signaling. Current understanding of Stat3-mediated ESC self-renewal mechanisms is very limited, and has heretofore been dominated by the view that Stat3 signaling functions in a binary "on/off" manner. Here, in contrast to this binary viewpoint, we demonstrate a contextual, rheostat-like mechanism for Stat3's function in mESCs. Activation and expression levels determine whether Stat3 functions in a self-renewal or a differentiation role in mESCs. We also show that Stat3 induces rapid differentiation of mESCs toward the trophectoderm (TE) lineage when its activation level exceeds certain thresholds. Stat3 induces this differentiation phenotype via induction of Tfap2c and its downstream target Cdx2. Our findings provide a novel concept in the realm of Stat3, self-renewal signaling, and pluripotent stem cell biology. Ultimately, this finding may facilitate the development of conditions for the establishment of authentic non-rodent ESCs.
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http://dx.doi.org/10.1242/bio.20149514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197444PMC
September 2014

Signaling pathways in induced naïve pluripotency.

Curr Opin Genet Dev 2014 Oct 28;28:10-5. Epub 2014 Aug 28.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. Electronic address:

Pluripotent stem cells have become powerful tools for both research and regenerative medicine. To date, however, only mouse and rat embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) have the ability to contribute to the formation of germline-competent chimeras. These stem cells are thus considered as 'naïve' pluripotent stem cells. Several signaling pathways have been identified to play a critical role in the induction and maintenance of this naïve pluripotent state. Understanding how these pathways induce and maintain naïve pluripotency will likely lead to the generation of germline-competent naïve ESCs/iPSCs from humans and animals phylogenetically close to humans.
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http://dx.doi.org/10.1016/j.gde.2014.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262619PMC
October 2014

STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates mouse ESC fates.

Stem Cells 2014 May;32(5):1149-60

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine 727 residue. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF) signaling maintains pluripotency by inducing JAK-mediated phosphorylation of STAT3 Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in regulating mESC identities, using a small molecule-based system to post-translationally modulate the quantity of transgenic STAT3 in STAT3(-/-) mESCs. We demonstrated that pY705 is absolutely required for STAT3-mediated mESC self-renewal, while pS727 is dispensable, serving only to promote proliferation and optimal pluripotency. S727 phosphorylation is regulated directly by fibroblast growth factor/Erk signaling and crucial in the transition of mESCs from pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in significantly reduced neuronal differentiation potential, which could be recovered by a S727 phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem cells to naïve pluripotency, suggesting a dynamic equilibrium of STAT3 pY705 and pS727 in the control of mESC fate.
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http://dx.doi.org/10.1002/stem.1609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181708PMC
May 2014

Induced neural stem cells generated from rat fibroblasts.

Genomics Proteomics Bioinformatics 2013 Oct 27;11(5):312-9. Epub 2013 Sep 27.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Cell and Neurobiology, University of Southern California, Los Angeles, CA 90033, USA; Department of Neurology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China.

The generation of induced tissue-specific stem cells has been hampered by the lack of well-established methods for the maintenance of pure tissue-specific stem cells like the ones we have for embryonic stem (ES) cell cultures. Using a cocktail of cytokines and small molecules, we demonstrate that primitive neural stem (NS) cells derived from mouse ES cells and rat embryos can be maintained. Furthermore, using the same set of cytokines and small molecules, we show that induced NS (iNS) cells can be generated from rat fibroblasts by forced expression of the transcriptional factors Oct4, Sox2 and c-Myc. The generation and long-term maintenance of iNS cells could have wide and momentous implications.
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http://dx.doi.org/10.1016/j.gpb.2013.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908478PMC
October 2013

Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal.

Nat Commun 2013 ;4:2403

1] Department of Cell and Neurobiology, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA [2].

Wnt/β-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal, however, remains poorly understood. Here we show that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely, retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2, a downstream target of β-catenin, or by genetic modifications to β-catenin that prevent its nuclear translocation. We also find that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. Our results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate.
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http://dx.doi.org/10.1038/ncomms3403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183150PMC
April 2014

Embryonic stem cell self-renewal pathways converge on the transcription factor Tfcp2l1.

EMBO J 2013 Oct 13;32(19):2548-60. Epub 2013 Aug 13.

1] Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA [2] Department of Biochemistry and Molecular Biology, Shanghai Medical School and Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, PR China.

Mouse embryonic stem cell (mESC) self-renewal can be maintained by activation of the leukaemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (Stat3) signalling pathway or dual inhibition (2i) of glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (MEK). Several downstream targets of the pathways involved have been identified that when individually overexpressed can partially support self-renewal. However, none of these targets is shared among the involved pathways. Here, we show that the CP2 family transcription factor Tfcp2l1 is a common target in LIF/Stat3- and 2i-mediated self-renewal, and forced expression of Tfcp2l1 can recapitulate the self-renewal-promoting effect of LIF or either of the 2i components. In addition, Tfcp2l1 can reprogram post-implantation epiblast stem cells to naïve pluripotent ESCs. Tfcp2l1 upregulates Nanog expression and promotes self-renewal in a Nanog-dependent manner. We conclude that Tfcp2l1 is at the intersection of LIF- and 2i-mediated self-renewal pathways and plays a critical role in maintaining ESC identity. Our study provides an expanded understanding of the current model of ground-state pluripotency.
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http://dx.doi.org/10.1038/emboj.2013.175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791365PMC
October 2013

p53 promotes inflammation-associated hepatocarcinogenesis by inducing HMGB1 release.

J Hepatol 2013 Oct 25;59(4):762-8. Epub 2013 May 25.

Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200433, China; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. Electronic address:

Background & Aims: Hepatocellular carcinoma (HCC) develops in response to chronic hepatic injury. Although induced cell death is regarded as the major component of p53 tumor-suppressive activity, we recently found that sustained p53 activation subsequent to DNA damage promotes inflammation-associated hepatocarcinogenesis. Here we aim at exploring the mechanism linking p53 activation and hepatic inflammation during hepatocarcinogenesis.

Methods: p53(-/-) hepatocytes expressing inducible p53 and primary wild type hepatocytes were treated to induce p53 expression. The supernatants were collected and analyzed for the presence of released inflammatory cytokines. Ethyl pyruvate was used in a rat model of carcinogen-induced hepatocarcinogenesis to examine its effect on p53-dependent chronic hepatic injury, inflammation, and tumorigenesis.

Results: Here we show that cytoplasmic translocation and circulating levels of potent inflammatory molecule high-mobility group protein 1 (HMGB1) were greater in wild type rats than in p53(+/-) rats following carcinogen administration. Restoration of p53 expression in p53-null hepatocytes or induction of endogenous p53 in wild type hepatocytes gives rise to the release of HMGB1. Administration of the HMGB1 release inhibitor ethyl pyruvate, which does not affect p53-mediated hepatic apoptosis, substantially prevented carcinogen-induced cirrhosis and tumorigenesis in rat livers.

Conclusions: These results suggest that although p53 is usually regarded as a tumor suppressor, its constant activation can promote pro-tumorigenic inflammation, at least in part, via inducing HMGB1 release. Application of HMGB1 inhibitors when restoring p53 in cancer therapy might protect against pro-tumorigenic effects while leaving p53-mediated clearance of malignant cells intact.
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http://dx.doi.org/10.1016/j.jhep.2013.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805120PMC
October 2013

Gbx2, a LIF/Stat3 target, promotes reprogramming to and retention of the pluripotent ground state.

J Cell Sci 2013 Mar 23;126(Pt 5):1093-8. Epub 2013 Jan 23.

Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

Activation of signal transducer and activator of transcription 3 (Stat3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal and also facilitates reprogramming to ground state pluripotency. Exactly how LIF/Stat3 signaling exerts these effects, however, remains elusive. We identified gastrulation brain homeobox 2 (Gbx2) as a LIF/Stat3 downstream target that, when overexpressed, allows long-term expansion of undifferentiated mESCs in the absence of LIF/Stat3 signaling. Elevated Gbx2 expression also enhanced reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Moreover, overexpression of Gbx2 was sufficient to reprogram epiblast stem cells to ground state ESCs. Our results reveal a novel function of Gbx2 in mESC reprogramming and LIF/Stat3-mediated self-renewal.
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http://dx.doi.org/10.1242/jcs.118273DOI Listing
March 2013

A focused microarray for screening rat embryonic stem cell lines.

Stem Cells Dev 2013 Feb 28;22(3):431-43. Epub 2012 Sep 28.

Department of Anatomy and Physiology, Kansas State University College of Veterinary Medicine, Manhattan, Kansas 66506, USA.

Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future.
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http://dx.doi.org/10.1089/scd.2012.0279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549623PMC
February 2013