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Trim28 citrullination maintains mouse embryonic stem cell pluripotency via regulating Nanog and Klf4 transcription

Science China(Life Sciences) 2023年第3期66卷 545-562页

作者： Yaguang Zhang Xiaowen Wan Lei Qiu Lian Zhou Qing Huang Mingtian Wei Xueqin Liu Sicheng Liu Bo Zhang Junhong Han State Key Laboratory of Biotherapy and Cancer Center National Clinical Research Center for Geriatricsand Frontiers Science Center for Disease-related Molecular NetworkWest China HospitalSichuan UniversityChengdu 610041China Department of Gastrointestinal Surgery and Frontiers Science Center for Disease-related Molecular NetworkWest China HospitalSichuan UniversityChengdu 610041China

Protein citrullination,including histone H1 and H3 citrullination,is important for transcriptional regulation,DNA damage response,and pluripotency of embryonic stem cells(ESCs).Tripartite motif containing 28(Trim28),an embryonic development regulator involved in ESC self-renewal,has recently been identified as a novel substrate for citrullination by Padi4.However,the physiological functions of Trim28 citrullination and its role in regulating the chromatin structure and gene transcription of ESCs remain unknown.In this paper,we show that Trim28 is specifically citrullinated in mouse ESCs(m ESCs),and that the loss of Trim28 citrullination induces loss of pluripotency.Mechanistically,Trim28 citrullination enhances the interaction of Trim28with Smarcad1 and prevents chromatin condensation.Additionally,Trim28 citrullination regulates m ESC pluripotency by promoting transcription of Nanog and Klf4 which it does by increasing the enrichment of H3K27ac and H3K4me3 and decreasing the enrichment of H3K9me3 in the transcriptional regulatory region.Thus,our findings suggest that Trim28citrullination is the key for the epigenetic activation of pluripotency genes and pluripotency maintenance of ESCs.Together,these results uncover a role Trim28 citrullination plays in pluripotency regulation and provide novel insight into how citrullination of proteins other than histones regulates chromatin compaction.

关键词： citrullination chromatin embryonic stem cell pluripotency Trim28

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Molecular Cloning and Functional Analysis of ESGP,an embryonic stem cell and Germ cell Specific Protein

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Acta Biochimica et Biophysica Sinica 2005年第12期37卷 7+790-796页

作者： Yan-Mei CHEN Zhong-Wei DU Zhen YAO Laboratory of Molecular and cell Biology.Institute of Biochemistry and cell Biology Shanghai Institute for Biologicol Sciences.Chinese Acadcmy of Sciences.Graduate School of the Chinese Academy of Sciences,Shanghai 200031.China Laboratory of Molecular and cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China

Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have beenidentified using the suppression-subtractive hybridization method.In this study,one of the novel genesencoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA ends.ESGP contains 801 bp encoding an 84 amino acid small protein and has no significant homology to anyknown genes.There is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG)(SeqWeb version 2.0.2,http://***:8080/).The result of immunofluorescence assay suggestedthat ESGP might encode a secretory protein.The expression pattern of ESGP is consistent with the expres-sion of Oct-4 during embryonic development.ESGP protein was detected in fertilized oocyte,from 3.5 daypostcoital (dpc) blastocyst to 17.5 dpc embryo,and was only detected in testis and ovary tissues in adult.Invitro,ESGP was only expressed in pluripotent cell lines,such as embryonic stem cells,embryonic carcinomacells and embryonic germ cells,but not in their differentiated progenies.Despite its specific expression,forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal,and does notaffect the differentiation to three germ layers.

关键词： embryonic stem cell and germ cell specific protein(ESGP)gene embryonic stem cell Oct-4 self-renewal

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Efficient Differentiation of embryonic stem cells into Neurons in Glial cell-conditioned Medium under Attaching Conditions

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Acta Biochimica et Biophysica Sinica 2005年第7期37卷 480-487页

作者： Hai-Bin TIAN~(1,2) Zeng-Liang BAI~1 Hong WANG~2 Jian-Quan CHEN~2 Guo-Xiang CHENG~2 1 Institute of Development Biology,School of Life Science,Shandong University,Jinan 250100,China 2 Shanghai Transgenic Research Center,Shanghai 201203,China Institute of Development Biology School of Life Science Shandong UniversityJinan 250100 China Shanghai Transgenic Research CenterShanghai 201203 China

embryonic stem (ES) cells can differentiate into neurons in vitro,which provides hope for thetreatment of some neurodegenerative diseases through cell transplantation.However,it remains a challengeto efficiently induce ES cells to differentiate into neurons.Here,we show that murine ES cells can efficientlydifferentiate into neurons when cultured in glial cell-conditioned medium (GCM) under attaching conditionswithout the formation of embryoid bodies.In comparison with murine embryonic fibroblast-conditionedmedium,we found that GCM has a positive effect on limiting the generation of non-neuronal cells,such asastrocytes.In addition,compared with suspension conditions,attaching conditions delay the differentiationprocess of ES cells.

关键词： embryonic stem cell glial cell-conditioned medium differentiation neuron attaching conditions

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Study on Transplanting Germ Like cells Differentiated from embryonic stem cell into Mouse Seminiferous Tubules

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Journal of Reproduction and Contraception 2011年第2期22卷 95-105页

作者： Wei-wei ZHENG Guang-hui CUI Xi-gu CHEN Xiang-cai WEI Xing-ming ZHONG Family Planning Research institute of Guangdong Province Guangzhou Guangdong 510600 China Experimental Animal Center Sun Yat-sen University Guangzhou Guangdong 510080 China

Objective To investigate whether germ like cells isolated from embryoid body formed by mouse embryonic stem cells could survive and initiate spermatogenesis in seminiferous tubules of adult mice. Methods SSEA-1＋ cells were isolated from embryoid bodies prepared from mouse EGFP-ES cells, after retinoic acid treatment, the cells were detected for the expression of alkaline phosphatase, Rnh2, stella, fragilis, Tex14, Sry, Hsp90-a, Stra8 and integrin a6, and then, the cells were transplanted into seminiferous tubules of busulfan-treated adult mice. Results Six days after retinoic acid treatment, alkaline phosphatase expressing cells could still be found in embryoid body （EB） derived cells, indicating the existence of retinoic acid-resistant primordial germ cells. When the SSEA-1＋ cells isolated from embryoid bodies were stimulated with retinoic acid for 6 days, some of these cells expressed cell markers of Hsp90-a, Stra8 and integrin a6, resembling the expression profile of spermatogonial stem cells. Forty-five days after cell transplantation, a little amount of GFP-expressing cells attached to the basement membrane of seminiferous tubule and formed small colonies; Three months later, these cells started amplification in the form of cell chains with varied length, and moving towards the lumen of the seminiferous tubules. Five months after the transplantation, multilayered cell mass was found in seminiferous tubules of two, out of four recipient mice. There was no GFP-expressing cells existed in non-cell-transplanted seminiferous tubules. Conclusion In our study, although full-termed spermatogenesis was not observed in all of the recipients, the results did indicate that the embryoid body contains germ like cells, and these cells can survive and initiate amplification in seminiferous tubules of adult mouse.

关键词： germ like cells embryonic stem cell embryoid body seminiferous tubule

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Efficient production of chimeric mice from embryonic stem cells injected into 4-to 8-cell and blastocyst embryos

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Journal of Animal Science and Biotechnology 2013年第4期4卷 264-270页

作者： Minhua Hu Hengxi Wei Jingfeng Zhang Yinshan Bai Fenglei Gao Li Li Shouquan Zhang Agricultural Animal Genomics and Molecular Breeding Key Lab of Guangdong Province College of Animal Science South China Agricultural University

Background： Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem （ES） cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES ceils into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator （PMM）, and trace the fate of the injected ES cells. Results： We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly. Conclusions： These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.

关键词： Chimeric mice embryonic stem cell Microinjection

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Research on Isolation and Clone of embryonic stem cell-Like in Bovine

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Agricultural Sciences in China 2002年第4期1卷 450-458页

作者： AN Li-long, YANG Qi, XIAO Mei, FENG Xiu-Liang, YANG Chun-rong, LEI An-min, GAO Zhi-min, DOU Zhong-ying and QIU Huai( Agriculture College of Zhanjiang Ocean University, Zhanjiang 524008 , P. R . China Key laboratory of AnimalReproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China , Northwestern Scienceand Technology University of Agriculture and Forestry, yangling 712100 , P.R. China) Agriculture College of Zhanjiang Ocean University Zhanjiang 524008 P. R. China Key laboratory of Animal Reproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China Northwestern Science and Technology University of Agriculture and Forestry Yangling 712100 P.R. China

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.

关键词： Bovine Murine embryonic stem cell Inner cell mass Isolation Clone

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embryonic stem cell——Patentable Subject Matter?

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China's Foreign Trade 2003年第1期 55-55页

Recently a special seminar was held to discuss the definition of the method of cloning human, patentability of the embryonic stem cell and adult stem cell and the therapeutic cloning method, the applicable and scientific guidelines to be referred in examining the emerging science and technology. The participants were examiners from the Chemical Examination Department I of the State Intellectual Property Office(SIPO).

关键词： Office Patentable Subject Matter cell embryonic stem cell

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The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores

The derivation of two additional human embryonic stem cell l...

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中华医学会第九次全国物理医学与康复学学术会议

作者： CHEN Hong Rehabilitation Department,Tongji Hospital,Tongji medical college,Huazhong University of Science and Technology,Wuhan,430030

embryonic stem cells are derived from inner cell mass of blastocysts. They have infinite proliferative capability and can differentiate into all types of adult cells (heart cells,skin cells,neural cells and et al.) of the body under special conditions,therefore,providing unique opportunities for rehabilitation of diseases and disorders with no therapies or at best,par- tially effective ones such as cerebral ischemia,diabetes,heart attack and et al.However,immune rejection can lead to the failure of human embryonic stem cell (hES cell) transplantation.One approach to address the problem is to establish hES cell line banks that have cells of various HLA types.Due to the limited source of human embryos and to ethical reasons,the hES cell lines are not readily available.If the blastocysts that develop from those discarded D3 embryos with low morphological scores can be used as a source to establish hESC lines,the problem with availability of human embryos may be eased.This study was undertaken to determine whether discarded day 3 embryos with low morphological scores could develop into blastocysts and produce hES cell lines. Methods D3 embryos with low morphological scores were assessed according to Steer CV scoring system and cultured in sequential culture medium to blasto- cyst stage.Blastocysts were evaluated by Gardner’s scale and the relation between blastocyst formation rate and D3 score was investigated. (1) Trophectoderm cells were separated from the ICMs by immunosurgery and isolated ICMs were cultured for 5-8 days on mitomycin-treated mouse embryonic fibroblasts (MEF).Colonies derived from the ICMs were passed every 4-7 days and evaluated for cell surface markers including AKP,OCT-4,SSEA-4,SSEA-1, TRA-1-60,TRA-1-81,differentiation potentials and karyotypes. Results (1) A total of 19 blastocysts were obtained from 130 embryos (quality score＜ 16)and blastulation rate was 14.62%.Most of blastocysts cavitated or ex- panded on day 6.A total of 10.17% had full or expanding cavities and 3.85% embryos showed early

关键词： embryonic stem cell embryo blastocyst inner cell mass in vitro fertilization in vivo differentiation in vitro differentiation

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Fetal dorsal root ganglion, embryonic stem cell and adult neural stem cell survival, differentiation following inplantation into the adult scala tympani and estibulocochlear nerve

Fetal dorsal root ganglion, embryonic stem cell and adult ne...

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中国神经科学学会第六届学术会议暨学会成立十周年庆祝大会

作者： Christine Regala Jonas Frisén Mats Ulfendahl Petri Olivius Department of Clinical Neuroscience Department of Clinical Neuroscience Department of Otolaryngology Department of Clinical Neuroscience Department of Otolaryngology Center for Hearing and Communication Research

＜正＞Either sensory hair cell or spiral ganglion neuron plays critical role in hearing. Loss or degeneration of hair cell and/or spiral ganglion neuron causes peripheral sensorineural hearing loss. Unfortunately, neither of them can be regenerated after loss in mammalian. We implanted fetal dorsal root ganglion （DRG） neurons, embryonic stem cell （ESC） and adult neural stem cell （ANSC） into adult animal cochlea. DRG neuron, ESC and ANSC have either lacZ or GFP reporter genes when implanted. We found that DRG neurons, ESCs and ANSCs could survive, migrate and differentiate transplantation into various species （rat and guinea pig）. The results suggest that new potential treatment using DRG, ESC and ANSC might be the case in near future in clinical patient.

关键词： embryonic stem cell adult neural stem cell

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Regulation of endogenous retroviruses in murine embryonic stem cells and early embryos

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Journal of Molecular cell Biology 2023年第8期15卷 1-14页

作者： Xinyi Lu State Key Laboratoryof Medicinal Chemical Biology Nankai UniversityTianjin 300350China

Endogenous retroviruses(ERVs)are important components of transposable elements that constitute∼40%of the mouse genome.ERVs exhibit dynamic expression patterns during early embryonic development and are engaged in numerous biological processes.Therefore,ERV expression must be closely monitored in cells.Most studies have focused on the regulation of ERV expression in mouse embryonic stem cells(ESCs)and during early embryonic development.This review touches on the classification,expression,and functions of ERVs in mouse ESCs and early embryos and mainly discusses ERV modulation strategies from the perspectives of transcription,epigenetic modification,nucleosome/chromatin assembly,and post-transcriptional control.

关键词： endogenous retrovirus embryonic stem cell epigenetic regulation transcriptional regulation post-transcriptional regulation

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