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Inducing human induced pluripotent stem cell differentiation through embryoid bodies:A practical and stable approach

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World Journal of Stem Cells 2020年第1期12卷 25-34页

作者： Ning-Ning Guo Li-Ping Liu Yun-Wen Zheng Yu-Mei Li Institute of Regenerative Medicine Affiliated Hospital of Jiangsu UniversityJiangsu UniversityZhenjiang 212001Jiangsu ProvinceChina Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery University of Tsukuba Faculty of MedicineTsukubaIbaraki 305-8575Japan Yokohama City University School of Medicine YokohamaKanagawa 234-0006Japan Division of Regenerative Medicine Center for Stem Cell Biology and Regenerative MedicineThe Institute of Medical Sciencethe University of TokyoTokyo 108-8639Japan

Human induced pluripotent stem cells(hiPSCs)are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical *** are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell ***,significant limitations exist when using conventional flat culturing methods especially concerning cell expansion,differentiation efficiency,stability maintenance and multicellular 3D structure establishment,differentiation *** bodies(EBs),the multicellular aggregates spontaneously generated from iPSCs in the suspension system,might help to address these *** to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve,EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing,differentiation efficiency enhancement,ex vivo simulation,and organoid *** can potentially also be used in early prediction of iPSC differentiation *** improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs,critical factors including iPSC pluripotency maintenance,generation of uniform morphology using micro-pattern 3D culture systems,proper cellular density inoculation,and EB size control are discussed on the basis of both published data and our own laboratory ***,the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.

关键词： Induced pluripotent stem cells Suspension culture embryoid body Early prediction Committed differentiation Heterogeneity Three-dimensional culture Scaling-up Quality control

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The Defined siRNAs Suppress Nanog and Sox2 Expressions in Mouse ES Cells

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Agricultural Sciences in China 2011年第9期10卷 1475-1481页

作者： LEI Lei DOU Lin WANG Hua-yan College of Veterinary Medicine/Shaanxi Center for Stem Cell Engineering and Technology Northwest A＆F University Yangling 712100 P.R.China

Nanog, Oct4 and Sox2 are important transcription factors that are expressed in embryonic stem （ES） cells or embryonic carcinoma （EC） cells, but in most cases they are absent in somatic cells. These factors play a key role to maintain embryonic stem cell self-renew and pluripotency. Down-regulation of Nanog and Sox2 gene expression can change multiple gene expression pattems and signal transduction pathways, and will initiate ES cell differentiation. This study was designed to select the efficient small interfering RNA （siRNA） fragments that inhibit Nanog and Sox2 gene expression in mouse J1 ES cells and P19 EC cells. Among synthesized siRNAs we screened out the siRNA N301 for Nanog and siRNA $720 for Sox2, which not only down- regulated of Nanog and Sox2 gene expression, but also interfered embryoid bodies formation. Our study provided the defined siRNA fragments that could be used to investigate the epigenetic function of Nanog and Sox2 genes.

关键词： Nanog Sox2 siRNA embryonic stem cells embryoid body

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A novel strategy to derive iPS cells from porcine fibroblasts

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Science China(Life Sciences) 2011年第6期54卷 553-559页

作者： RUAN WeiMin HAN JianYong LI Pin CAO SuYing AN Yang LIM Bing LI Ning State Key Laboratory for Agrobiotechnology College of Biological SciencesChina Agricultural UniversityBeijing 100193China Stem Cell and Developmental Biology Genome Institute of Singapore138672Singapore

Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state by over-expressing four reprogramming *** technology has created an interest in deriving iPS cells from domesticated animals such as pigs,sheep and *** murine leukemia retrovirus vectors have been widely used to generate and study mouse iPS ***,this retrovirus system infects only mouse and rat cells,which limits its use in establishing iPS cells from other *** our study,we demonstrate a novel retrovirus strategy to efficiently generate porcine iPS cells from embryonic *** transfected four human reprogramming factors (Oct4,Sox2,Klf4 and Myc) into fibroblasts in one step by using a VSV-G envelope-coated pantropic retrovirus that was easily packaged by GP2-293 *** established six embryonic stem (ES)-like cell lines in human ES cell medium supplemented with *** showed a similar morphology to human ES cells with a high nuclei-cytoplasm ratio and phase-bright flat *** iPS cells could form embryoid bodies in vitro and differentiate into the three germ layers in vivo by forming teratomas in immunodeficient mice.

关键词： induced pluripotent stem cells Moloney murine leukemia retrovirus vectors embryoid body teratoma

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Practical choice for robust and efficient differentiation of human pluripotent stem cells

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World Journal of Stem Cells 2020年第8期12卷 752-760页

作者： Mei Fang Li-Ping Liu Hang Zhou Yu-Mei Li Yun-Wen Zheng Institute of Regenerative Medicine Affiliated Hospital of Jiangsu UniversityJiangsu UniversityZhenjiang 212001Jiangsu ProvinceChina School of Biotechnology and Heath Sciences Wuyi UniversityJiangmen 529020Guangdong ProvinceChina Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery University of Tsukuba Faculty of MedicineTsukubaIbaraki 305-8575Japan City University School of Medicine YokohamaKanagawa 234-0006Japan Division of Regenerative Medicine Center for Stem Cell Biology and Regenerative MedicineThe Institute of Medical Sciencethe University of TokyoTokyo 108-8639Japan

Human pluripotent stem cells(hPSCs)have the distinct advantage of being able to differentiate into cells of all three germ *** cells or tissues derived from hPSCs have many uses such as drug screening,disease modeling,and transplantation *** are currently a wide variety of differentiation methods ***,most of the existing differentiation methods are unreliable,with uneven differentiation efficiency and poor *** the same time,it is difficult to choose the optimal method when faced with so many differentiation schemes,and it is time-consuming and costly to explore a new differentiation ***,it is critical to design a robust and efficient method of *** this review article,we summarize a comprehensive approach in which hPSCs are differentiated into target cells or organoids including brain,liver,blood,melanocytes,and mesenchymal *** was accomplished by employing an embryoid body-based three-dimensional(3D)suspension culture system with multiple cells *** method has high stable differentiation efficiency compared to the conventional 2D culture and can meet the requirements of clinical ***,ex vivo co-culture models might be able to constitute organoids that are highly similar or mimic human organs for potential organ transplantation in the future.

关键词： Human pluripotent stem cells Three dimensional embryoid body Differentiation Efficient Three germ layers

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Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

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Neural Regeneration Research 2013年第4期8卷 293-300页

作者： Xingrong Yan Yanhong Yang Wei Liu Wenxin Geng Huichong Du Jihong Cui Xin Xie Jinlian Hua Shumin Yu Liwen Li Fulin Chen College of Life Sciences Northwest University Department of Obstetrics & Gynecology Tangdu Hospitalthe Fourth Military Medical University of Chinese PLA College of Veterinary Medicine Northwest A&F University College of Veterinary Medicine Sichuan Agricultural University

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stern cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, ~lll-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neuregenesis related genes （Sox-1, Nestin, GABA, Pax6, Zic5 and Pitxl） in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

关键词： neural regeneration stem cells parthenogenesis parthenogenetic embryonic stem cells embryonic stem cells neuronal cells karyotypes Oct4 differentiation embryoid body mice grants-supported paper photographs-containing paper neuroregeneration

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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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Study on Artificial Seeds of Plants

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Journal of Botanical Research 2020年第4期2卷 1-3页

作者： Han shan Yang Minran Chen Yanyi Biological chemical North China University of Science and TechnologyTangshanHebei063000China Bioinformatics North China University of Science and TechnologyTangshanHebei063000China

The establishment and development of artificial seed technology is to quickly reproduce excellent varieties or hybrids,which can be applied to hybrid generation seeds produced by the three-two line *** some varieties that are difficult to propagate with seeds or plant species with unstable genetic traits and poor fertility,artificial seed technology can also be used for mass *** particular,some new plants created through genetic engineering,such as somatic hybrids or transgenic plants,can be propagated or maintained by artificial seed *** addition,artificial seed technology can be used for the maintenance and rapid propagation of virus-free *** with ordinary test tube seedlings,artificial seeds have low cost,convenient transportation,and to a certain extent reduce vitrified *** particular,the production of artificial seeds does not occupy a large amount of *** can be produced all year ***,the research on artificial seeds has developed rapidly in the world.

关键词： embryoid body Artificial seed

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P1 plays an important role in regulation of FGF-4 expression through posttranslational modification of Sox-2

P1 plays an important role in regulation of FGF-4 expression...

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中国遗传学会“发育、遗传和疾病”研讨会

作者： Furong Gao Ying Jin Stem Cell Research Lab,Institute Of Health Sciences,Shanghai Institutes for Biological Sciences,CAS/Shanghai Jiaotong University School of Medicine

Fbroblast growth factor 4（FGF-4）is a signaling polypeptide essential in embryonic development,transcription factors Oct-4 and Sox-2 are known to regulate FGF-4 expression through binding co-operatively to HMG/POU cassettes of FGF-4 ***,it has been suggested that Oct-4 and Sox-2 are not sufficient for expression of *** further identify transcription factors and coactivators that participate in the expression of FGF-4,affinity chromatography was conducted using oligonucleotides containing HMG/POU cassettes fromFGF-4 enhancer to isolate potential Oct-4/Sox-2 interacting *** isolated proteins were analyzed with mass spectrometry and verified by Western blot analysis and chromatin immunoprecipitation（CHIP）assay..P1,a nuclear protein widely known for its catalytic activity that uniquely modifies target proteins,was ***-immunoprecipitation studies indicate a physical interaction between PI and Oct-4,P1 and Sox-2 .To study the role of P1 in the regulation of FGF-4 expression,we differentiated P1 knockout(P1)embryonic stem cells（ES cells）and wild type ES cells by spontaneous formation of embryoid body（EB）and retinoid acid（RA）*** found that expression of FGF-4 was lower in EB and differentiated cells derived from PI knockout(P1) ES cells than that derived from wild type ES *** with construct expressing PI in P1cells can rescue FGF-4 *** expression profile suggested that P1-/-ES cells easily differentiated into giant trophoblast ***,addition of FGF-4 can reverse this tendency,consisting with the function of FGF4 in maintaining self-renewal of trophobloast stem cells..To understand the molecular mechanism leading to the lower expression of FGF4 in P1-/- ES cells,protein level of Oct-4 and Sox2 was compared between wild type ES and P1-/- ES cells during ES cell ***,the protein level of Sox2 was found significantly higher in P1ES cell- derived EB than that in wild type ES

关键词： ES cells Oct-4 Sox-2 FGF-4 embryoid body differentiation

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