Publications by authors named "Shoudong Ye"

26 Publications

  • Page 1 of 1

Functional genomics study of protein inhibitor of activated STAT1 in mouse hippocampal neuronal cells revealed by RNA sequencing.

Aging (Albany NY) 2021 Mar 24;13(6):9011-9027. Epub 2021 Mar 24.

Foshan Stomatology Hospital, School of Medicine, Foshan University, Foshan 528000, Guangdong, China.

Protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) E3 ligase, was considered to be an inhibitor of STAT1 by inhibiting the DNA-binding activity of STAT1 and blocking STAT1-mediated gene transcription in response to cytokine stimulation. PIAS1 has been determined to be involved in modulating several biological processes such as cell proliferation, DNA damage responses, and inflammatory responses, both and . However, the role played by PIAS1 in regulating neurodegenerative diseases, including Alzheimer's disease (AD), has not been determined. In our study, significantly different expression levels of PIAS1 between normal controls and AD patients were detected in four regions of the human brain. Based on a functional analysis of in undifferentiated mouse hippocampal neuronal HT-22 cells, we observed that the expression levels of several AD marker genes could be inhibited by overexpression. Moreover, the proliferation ability of HT-22 cells could be promoted by the overexpression of . Furthermore, we performed RNA sequencing (RNA-seq) to evaluate and quantify the gene expression profiles in response to overexpression in HT-22 cells. As a result, 285 significantly dysregulated genes, including 79 upregulated genes and 206 downregulated genes, were identified by the comparison of /+ cells with WT cells. Among these genes, five overlapping genes, including early growth response 1 (), early growth response 2 (), early growth response 3 (), FBJ osteosarcoma oncogene () and fos-like antigen 1 (), were identified by comparison of the transcription factor binding site (TFBS) prediction results for STAT1, whose expression was evaluated by qPCR. Three cell cycle inhibitors, p53, p18 and p21, were significantly downregulated with the overexpression of . Analysis of functional enrichment and expression levels showed that basic region leucine zipper domain-containing transcription factors including zinc finger C2H2 (zf-C2H2), homeobox and basic/helix-loop-helix (bHLH) in several signaling pathways were significantly involved in PIAS1 regulation in HT-22 cells. A reconstructed regulatory network under PIAS1 overexpression demonstrated that there were 43 related proteins, notably Nr3c2, that directly interacted with PIAS1.
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http://dx.doi.org/10.18632/aging.202749DOI Listing
March 2021

Regulatory function of praja ring finger ubiquitin ligase 2 mediated by the axis in mouse hippocampal neuronal cells.

Am J Physiol Cell Physiol 2020 06 8;318(6):C1123-C1135. Epub 2020 Apr 8.

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

Praja2 (), a member of the growing family of mammalian RING E3 ubiquitin ligases, is reportedly involved in not only several types of cancer but also neurological diseases and disorders, but the genetic mechanism underlying the regulation of in the nervous system remains unclear. To study the cellular and molecular functions of in mouse hippocampal neuronal cells (MHNCs), we used gain- and loss-of-function manipulations of in HT-22 cells and tested their regulatory effects on three Alzheimer's disease (AD) genes and cell proliferation. The results revealed that the expression of AD markers, including amyloid beta precursor protein (), microtubule-associated protein tau (), and gamma-secretase activating protein (), could be inhibited by overexpression and activated by knockdown. In addition, HT-22 cell proliferation was enhanced by upregulation and suppressed by its downregulation. We also evaluated and quantified the targets that responded to the enforced expression of by RNA-Seq, and the results showed that purinergic receptor P2X, ligand-gated ion channel 3 and 7 ( and ), which show different expression patterns in the critical calcium signaling pathway, mediated the regulatory effect of in HT-22 cells. Functional studies indicated that regulated HT-22 cells development and AD marker genes by inhibiting but promoting , a gene downstream of . In conclusion, our results provide new insights into the regulatory function of the gene in MHNCs and thus underscore the potential relevance of this molecule to the pathophysiology of AD.
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http://dx.doi.org/10.1152/ajpcell.00070.2019DOI Listing
June 2020

A transcriptomic analysis of Nsmce1 overexpression in mouse hippocampal neuronal cell by RNA sequencing.

Funct Integr Genomics 2020 May 2;20(3):459-470. Epub 2019 Dec 2.

Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei, 230601, Anhui, China.

Mouse Nsmce1 gene is the homolog of non-structural maintenance of chromosomes element 1 (NSE1) that is mainly involved in maintenance of genome integrity, DNA damage response, and DNA repair. Defective DNA repair may cause neurological disorders such as Alzheimer's disease (AD). So far, there is no direct evidence for the correlation between Nsmce1 and AD. In order to explore the function of Nsmce1 in the regulation of nervous system, we have overexpressed or knocked down Nsmce1 in the mouse hippocampal neuronal cells (MHNCs) HT-22 and detected its regulation of AD marker genes as well as cell proliferation. The results showed that the expression of App, Bace2, and Mapt could be inhibited by Nsmce1 overexpression and activated by the knockdown of Nsmce1. Moreover, the HT-22 cell proliferation ability could be promoted by Nsmce1 overexpression and inhibited by knockdown of Nsmce1. Furthermore, we performed a transcriptomics study by RNA sequencing (RNA-seq) to evaluate and quantify the gene expression profiles in response to the overexpression of Nsmce1 in HT-22 cells. As a result, 224 significantly dysregulated genes including 83 upregulated and 141 downregulated genes were identified by the comparison of Nsmce1 /+ to WT cells, which were significantly enriched in several Gene Ontology (GO) terms and pathways. In addition, the complexity of the alternative splicing (AS) landscape was increased by Nsmce1 overexpression in HT-22 cells. Thousands of AS events were identified to be mainly involved in the pathway of ubiquitin-mediated proteolysis (UMP) as well as 3 neurodegenerative diseases including AD. The protein-protein interaction network was reconstructed to show top 10 essential genes regulated by Nsmce1. Our sequencing data is available in the Gene Expression Omnibus (GEO) database with accession number as GSE113436. These results may provide some evidence of molecular and cellular functions of Nsmce1 in MHNCs.
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http://dx.doi.org/10.1007/s10142-019-00728-6DOI Listing
May 2020

Regulatory network reconstruction of five essential microRNAs for survival analysis in breast cancer by integrating miRNA and mRNA expression datasets.

Funct Integr Genomics 2019 Jul 12;19(4):645-658. Epub 2019 Mar 12.

School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong, China.

Although many of the genetic loci associated with breast cancer risk have been reported, there is a lack of systematic analysis of regulatory networks composed of different miRNAs and mRNAs on survival analysis in breast cancer. To reconstruct the microRNAs-genes regulatory network in breast cancer, we employed the expression data from The Cancer Genome Atlas (TCGA) related to five essential miRNAs including miR-21, miR-22, miR-210, miR-221, and miR-222, and their associated functional genomics data from the GEO database. Then, we performed an integration analysis to identify the essential target factors and interactions for the next survival analysis in breast cancer. Based on the results of our integrated analysis, we have identified significant common regulatory signatures including differentially expressed genes, enriched pathways, and transcriptional regulation such as interferon regulatory factors (IRFs) and signal transducer and activator of transcription 1 (STAT1). Finally, a reconstructed regulatory network of five miRNAs and 34 target factors was established and then applied to survival analysis in breast cancer. When we used expression data for individual miRNAs, only miR-21 and miR-22 were significantly associated with a survival change. However, we identified 45 significant miRNA-gene pairs that predict overall survival in breast cancer out of 170 one-on-one interactions in our reconstructed network covering all of five miRNAs, and several essential factors such as PSMB9, HLA-C, RARRES3, UBE2L6, and NMI. In our study, we reconstructed regulatory network of five essential microRNAs for survival analysis in breast cancer by integrating miRNA and mRNA expression datasets. These results may provide new insights into regulatory network-based precision medicine for breast cancer.
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http://dx.doi.org/10.1007/s10142-019-00670-7DOI Listing
July 2019

Telomeric noncoding RNA promotes mouse embryonic stem cell self-renewal through inhibition of TCF3 activity.

Am J Physiol Cell Physiol 2018 06 7;314(6):C712-C720. Epub 2018 Mar 7.

Center for Stem Cell and Translational Medicine, School of Life Sciences, Anhui University , Hefei , People's Republic of China.

Although long noncoding RNAs (lncRNAs) are emerging as new modulators in the fate decision of pluripotent stem cells, the functions of specific lncRNAs remain unclear. Here, we found that telomeric RNA (TERRA or TelRNA), one type of lncRNAs, is highly expressed in mouse embryonic stem cells (mESCs) but declines significantly upon differentiation. TERRA is induced by the Wnt/β-catenin signaling pathway and can reproduce its self-renewal-promoting effect when overexpressed. Further studies revealed that T cell factor 3 ( TCF3) is a potential downstream target of TERRA and mediates the effect of TERRA in mESC maintenance. TERRA inhibits TCF3 transcription, while enforced TCF3 expression abrogates the undifferentiated state of mESCs supported by TERRA. Accordingly, the transcripts of the pluripotency genes Esrrb, Tfcp2l1, and Klf2, repressed by TCF3 in mESCs, are increased in TERRA-overexpressing cells. Our study therefore highlights the important role of TERRA in mESC maintenance and also uncovers a mechanism by which TERRA promotes self-renewal. These data will expand our understanding of the pluripotent regulatory network of ESCs.
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http://dx.doi.org/10.1152/ajpcell.00292.2017DOI Listing
June 2018

Tfcp2l1 safeguards the maintenance of human embryonic stem cell self-renewal.

J Cell Physiol 2018 09 11;233(9):6944-6951. Epub 2018 Apr 11.

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

Tfcp2l1 is a transcription factor critical for mouse embryonic stem cell (mESC) maintenance. However, its role in human ESCs (hESCs) remains unclear. Here, we investigated the role of Tfcp2l1 in controlling hESC activity and showed that Tfcp2l1 is functionally important in the maintenance of hESC identity. Tfcp2l1 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Forced expression of Tfcp2l1 promoted hESC self-renewal. Functional analysis of the mutant forms of Tfcp2l1 revealed that both the CP2- and SAM-like domains are indispensable for Tfcp2l1 to maintain the undifferentiated state of hESCs. Notably, the CP2-like domain is closely related to the suppression of definitive endoderm and mesoderm commitment. Accordingly, knockdown of Tfcp2l1 significantly induced differentiation preferentially into definitive endoderm and mesoderm. Further studies found that inhibition of Wnt/β-catenin signaling pathway by IWR1 is able to eliminate the differentiation caused by Tfcp2l1 downregulation. Taken together, these findings reveal the unique and crucial role of Tfcp2l1 in the determination of hESC fate and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.
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http://dx.doi.org/10.1002/jcp.26483DOI Listing
September 2018

A transcriptomic study of myogenic differentiation under the overexpression of PPARγ by RNA-Seq.

Sci Rep 2017 11 10;7(1):15308. Epub 2017 Nov 10.

School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong, China.

To study the cellular and molecular function of peroxisome proliferator-activated receptor γ (PPARγ) in skeletal muscle differentiation, we have generated inducible gain-of-function to overexpress PPARγ in C2C12 myoblasts. In order to identify PPARγ targets, RNA sequencing (RNA-seq) was used to evaluate and quantify the transcriptomes and expression patterns during myogenic differentiation under the overexpression of PPARγ. The formation of myotubes and the expression of muscle-specific myogenic genes such as MyoD and MyoG may be inhibited by PPARγ overexpression. Multiple genes and pathways were significantly involved in this process, including 11 genes such as Fndc9 and Slc14a1 with fundamental change of regulation modes, 9 genes of which were validated by the data of qRT-PCR. Our studies demonstrate that PPARγ would play critical roles on myoblasts differentiation, mediating crosstalk among several pathways and transcription factors. Our data is available in the Gene Expression Omnibus (GEO) database with the accession number as GSE99399.
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http://dx.doi.org/10.1038/s41598-017-14275-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681552PMC
November 2017

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

Inhibition of Wnt/β-catenin signaling by IWR1 induces expression of Foxd3 to promote mouse epiblast stem cell self-renewal.

Biochem Biophys Res Commun 2017 08 16;490(3):616-622. Epub 2017 Jun 16.

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

Inhibition of Wnt/β-catenin signaling facilitates the derivation of mouse epiblast stem cells (EpiSCs), as well as dramatically promotes EpiSC self-renewal. The specific mechanism, however, is still unclear. Here, we showed that IWR1, a Wnt/β-catenin signaling inhibitor, allowed long-term self-renewal of EpiSCs in serum medium in combination with ROCK inhibitor Y27632. Through transcriptome data analysis, we arrived at a set of candidate transcription factors induced by IWR1. Among these, Forkhead box D3 (Foxd3) was most abundant. Forced expression of Foxd3 could recapitulate the self-renewal-promoting effect of IWR1 in EpiSCs. Conversely, knockdown of Foxd3 profoundly compromised responsiveness to IWR1, causing extinction of pluripotency markers and emergence of differentiation phenotype. Foxd3 thus is necessary and sufficient to mediate self-renewal downstream of Wnt/β-catenin signaling inhibitor. These findings highlight an important role for Foxd3 in regulating EpiSCs and will expand current understanding of the primed pluripotency.
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http://dx.doi.org/10.1016/j.bbrc.2017.06.086DOI Listing
August 2017

Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

Life Sci 2017 Apr 22;175:37-46. Epub 2017 Mar 22.

The Key Laboratory of Molecular Medicine of Ministry of Education, School of Basic Medical Sciences, Fudan University, Shanghai, China. Electronic address:

Aim: To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system.

Main Methods: Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test.

Key Findings: Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition.

Significance: This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation.
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http://dx.doi.org/10.1016/j.lfs.2017.03.017DOI Listing
April 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

A comprehensive transcriptomic analysis of differentiating embryonic stem cells in response to the overexpression of Mesogenin 1.

Aging (Albany NY) 2016 10;8(10):2324-2336

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

The mutation of somitogenesis protein Mesogenin 1 (Msgn1) has been widely used to study the direct link between somitogenesis and the development of an embryo. Several studies have used gene expression profiling of somitogenesis to identify the key genes in the process, but few have focused on the pathways involved and the coexpression patterns of associated pathways. Here we employed time-course microarray datasets of differentiating embryonic stem cells by overexpressing the transcription factor Msgn1 from the public database library of Gene Expression Omnibus (GEO). Then we applied gene set enrichment analysis (GSEA) to the datasets and performed candidate transcription factors selection. As a result, several significantly regulated pathways and transcription factors (TFs), as well as some of the specific signaling pathways, were identified during somitogenesis under Msgn1 overexpression, most of which had not been reported previously. Finally, significant core genes such as Hes1 and Notch1 as well as some of the TFs such as PPARs and FOXs were identified to construct coexpression networks of related pathways, the expression patterns of which had been validated by our following quantitative real-time PCR (qRT-PCR). The results of our study may help us better understand the molecular mechanisms of somitogenesis in mice at the genome-wide level.
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http://dx.doi.org/10.18632/aging.101049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5115891PMC
October 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

The stromal genome heterogeneity between breast and prostate tumors revealed by a comparative transcriptomic analysis.

Oncotarget 2015 Apr;6(11):8687-97

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

Stromal microenvironment increases tumor cell survival, proliferation and migration, and promotes angiogenesis. In order to provide comprehensive information on the stromal heterogeneity of diverse tumors, here we employed the microarray datasets of human invasive breast and prostate cancer-associated stromals and applied Gene Set Enrichment Analysis (GSEA) to compare the gene expression profiles between them. As a result, 8 up-regulated pathways and 73 down-regulated pathways were identified in the breast tumor stroma, while 32 up-regulated pathways and 18 down-regulated pathways were identified in the prostate tumor stroma. Only 9 pathways such as tryptophan metabolism were commonly up or down regulated, but most of them (including ABC transporters) were specific for these two tumors. Several essential tumors stromal marker genes were also significantly identified. For example, CDH3 was significantly up-regulated in the stromals of both breast and prostate tumors, however EGFR was only significantly down-regulated in the stromal of breast tumor. Our study would be helpful for future therapeutic and predictive applications in breast and prostate cancers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496176PMC
http://dx.doi.org/10.18632/oncotarget.3478DOI Listing
April 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

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

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

Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains.

PLoS One 2012 23;7(4):e35892. Epub 2012 Apr 23.

The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai Medical College, Shanghai, People's Republic of China.

Background: Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains.

Methodology/principal Findings: We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice.

Conclusions/significance: Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035892PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335080PMC
September 2012

All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways.

BMC Cell Biol 2009 Jul 30;10:57. Epub 2009 Jul 30.

Department of Molecular Genetics, Shanghai Medical School, Fudan University, Shanghai, PR China.

Background: All-trans retinoic acid (RA) is one of the most important morphogens with pleiotropic actions. Its embryonic distribution correlates with neural differentiation in the developing central nervous system. To explore the precise effects of RA on neural differentiation of mouse embryonic stem cells (ESCs), we detected expression of RA nuclear receptors and RA-metabolizing enzymes in mouse ESCs and investigated the roles of RA in adherent monolayer culture.

Results: Upon addition of RA, cell differentiation was directed rapidly and exclusively into the neural lineage. Conversely, pharmacological interference with RA signaling suppressed this neural differentiation. Inhibition of fibroblast growth factor (FGF) signaling did not suppress significantly neural differentiation in RA-treated cultures. Pharmacological interference with extracellular signal-regulated kinase (ERK) pathway or activation of Wnt pathway effectively blocked the RA-promoted neural specification. ERK phosphorylation was enhanced in RA-treated cultures at the early stage of differentiation.

Conclusion: RA can promote neural lineage entry by ESCs in adherent monolayer culture systems. This effect depends on RA signaling and its crosstalk with the ERK and Wnt pathways.
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http://dx.doi.org/10.1186/1471-2121-10-57DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728515PMC
July 2009