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differentiation of embryonic stem cells into corneal epithelium

Science China(Life Sciences) 2005年第5期48卷 471-480页

作者： WANG Zhichong GE Jian HUANG Bing GAO Qianying LIU Bingqian WANG Linghua YU Ling FAN Zhigang LU Xiaoming LIU Jingbo Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510060 China

Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and di-vided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any in-ducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immuno-histochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula oc-cludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.

关键词： embryonic stem cells, differentiation, corneal epithelium, microenvironment, superficial corneoscleral limbal stroma.

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Genetic analysis and mapping of gene fzp(t)controlling spikelet differentiation in rice

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Science China(Life Sciences) 2003年第3期46卷 328-334页

作者：段远霖李维明吴为人潘润森周元昌祁建民林荔辉陈志伟毛大梅刘华清张丹凤薛勇彪 College of Crops Fujian Agricultural & Forestry University Fuzhou 350002 China Academy of Agricultural Science Fujian Province Fuzhou 350000 China Institute of Genetics & Developmental Biology Chinese Academy of Sciences Beijing 100080 China

A mutant of spikelet differentiation in rice called frizzle panicle (fzp) was discovered in the progeny of a cross between Oryza sativa ssp. indica cv. V20B and cv. Hua1B. The mutant ex-hibits normal plant morphology but has apparently fewer tillers. The most striking change in fzp is that its spikelet differentiation is completely blocked, with unlimited subsequent rachis branches generated from the positions where spikelets normally develop in wild-type plants. Genetic analy-sis suggests that fzp is controlled by a single recessive gene, which is temporarily named fzp (t). Based on its mutant phenotype, fzp (t) represents a key gene controlling spikelet differentiation. Some F2 mutant plants derived from various genetic background appeared as the middle type? suggesting that the action of fzp (t) is influenced by the presence of redundant, modifier or interac-tive genes. By using simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) method, fzp (t) gene was mapped in the terminal region of the long arm of chromosome 7, with RM172 and RM248 on one side, 3.2 cM and 6.4 cM from fzp (t), and RM18 and RM234 on the other side, 23.1 cM and 26.3 cM from fzp(t), respectively. These results will facilitate the positional cloning and function studies of the gene.

关键词： rice (O. sativa L), spikelet differentiation, fzp, genetics, gene mapping.

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cDNA cloning and function analysis of two novel erythroid differentiation related genes

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Science China(Life Sciences) 2001年第1期44卷 99-105页

作者：王鑫王敦成陈兴胡美茹王建安黎燕郭宁沈倍奋 Beijing Institute of Basic Medical Sciences Beijing 100850 China

Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen λ-gtll human cDNA expression library of fetal liver in order to obtain the relevant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' functions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a protein characterized by a leu-cine-zipper domain. Both of them were expressed differentially in K562 cells and hemin-induced K562 cells. The evidence suggested that both of them were involved in erythroid differentiation. We investigated the expression pattern of EDRF1 and EDRF2 by RT-PCR technique. The results of RT-PCR suggested that EDRF1 and EDRF2 might play a critical role in early stage of organ development and histological differentiation. EDRF1 and EDRF2 might start the program of erythroid development, and also regulate the development of erythroid tissue and the expression of globin gene at different stage of the development.

关键词： erythroid differentiation, HS2, cDNA cloning, leucine zipper domain, RT-PCR.

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In vitro differentiation of mouse ES cells into hepatocytes with coagulation factors VIII and IX expression profiles

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Science China(Life Sciences) 2006年第3期49卷 259-264页

作者： MENG Ying HUANG Shaoliang MIN Jun GUO Zhongmin Center for Stem Cell Research the Second Affiliated Hospital of Sun Yat-sen University Guangzhou 510120 China Center for Stem Cell Research the Second Affiliated Hospital of Sun Yat-sen University Guangzhou 510120 China Department of Pediatrics the Second Affiliated Hospital of Sun Yat-sen University Guangzhou 510120 China Center for Stem Cell Research the Second Affiliated Hospital of Sun Yat-sen University Guangzhou 510120 China Department of Surgery the Second Affiliated Hospital of Sun Yat-sen University Guangzhou 510120 China Experimental Animal Center Sun Yat-sen Medical College Sun Yat-sen University Guangzhou 510089 China

Coagulation factors II, V, VII, VIII, IX and X are produced by *** factors VIII and IX deficiencies, which result in hemophilia A and B, have the potential torespond to cellular replacement therapy. Embryonic stem (ES) cells provide a unique source fortherapeutic applications. Here, E14 mouse ES cells have been induced into hepatocytes in *** revealed that ES-derived hepatic-like cells were round or polyhedral shaped with distinctboundary of individual cells, and some arranged in trabeculae. These cells expressed endodermal- orliver-specific mRNA---transthyretin (TTR), alpha 1-anti-trypsin (AAT), alpha-fetoprotein (AFP),albumin (ALB), glucose-6-phoshpatase (G6P) and tyrosine aminotransferase (TAT). Approximately(85.1±0.5)% of the ES-derived cells was stained positive green with ICG uptake. These cells werealso stained magenta as a result of PAS reaction. In this paper, expression of coagulation factorsVIII and IX mRNA in the ES-derived cells is documented. Therefore, ES cells might be developed assubstitute donor cells for the therapy of coagulation factor deficiencies.

关键词： ES cell, differentiation, hepatocyte, coagulation factor.

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Non-contemporaneous variations and Holder's principle

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Science China(Physics,Mechanics & Astronomy) 2003年第5期46卷 449-459页

作者：梁立孚胡海昌刘石泉 Space Engineering Department of Harbin Engineering University Harbin 150001 China Chinese Academy of Space Technology Institute of Spacecraft System Engineering Beijing 100086 China China Sanjiang Space Industry Group Corporation Wuhan 430015 China

In the process of deducing the Holder principle, a key step is to use the concept of non-contemporaneous variations. In this paper, whether starting from analytic method or from graphic solution method, the authors prove that the expression formula of non-contemporaneous variations is incorrect when the variable functions have zero-order nearness degree, and obtain a new expression. From the view of calculus of variations and differential calculus, the non-contemporaneous variations are studied. The study result shows that the concept of non-contemporaneous variations is a combination of the concept of variations and the concept of differentiation. The authors prove that the new expression is correct and obtain an equivalent expression of it. By means of this equivalent expression, this paper proves that the above expression formula of non-contemporaneous variations is correct when the variable functions have one-order nearness degree. Further study shows that, in the process of deducing Holder's principle, there is an implicit expression. Whether starting from analytic method or from graphic solution method, the authors discovered that the implicit expression of non-contemporaneous variations is incorrect when the variable functions have zero-order nearness degree and have one-order nearness degree. This paper proves that the implicit expression of non-contemporaneous variations is correct when the variable functions have two-order nearness degree. Further study shows that Holder's principle is tenable when the variable functions have two-order nearness degree.

关键词： differentiation, variation, non-contemporaneous variations, non-holonomic systems.

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A Simple Shear Model for the Asymmetry in the Paleoproterozoic Fold Belt in Liaodong Peninsula

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Global Geology 2001年第1期6卷 26-33页

作者： Cui Yingchun, Liu Junlai, Ma Lijie and Guan Huimei (Dept. Earth Sci., Jilin University, Changchun 130061 P.R. China) Dept. Earth Sci. Jilin University Changchun 130061 P.R. China

Several aspects of the Liaodong Paleoproterozioc fold belt, including its components, tectonic, metamorphic and magmatic characteristics are discussed in the paper, with emphasis on the asymmetry of the fold belt and differences between the northern lineal-fold belt and the southern dome-fold belt. It is suggested that the fold belt is one magmatic passive margin, instead of a rift zone or the classical passive continental margin. The formation and evolution of the fold belt is dis- cussed with a simple shear model, which may best explain the co-existence of contemporaneous tectonic facies with different characteristics, the volcanic and plutonic activities, sedimentary formations and their geological attributes etc.

关键词： Liaodong, differentiation, magmatic passive continental margin, simple shear model

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Antisense EDRF1 gene inhibited GATA-1transcription factor DNA-binding activity in K562 cells

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Science China(Life Sciences) 2002年第3期45卷 289-297页

作者：王敦成沈倍奋马淑华王升启 Beijing Institute of Basic Medical Sciences Beijing 100850 China Beijing Institute of Radiation Medicine Sciences Beijing 100850 China

Our previous studies showed that EDRF1 influenced expression of a-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid proliferation and differentiation by affecting the interaction between GATA-1 and its cis-elements.

关键词： erythroid differentiation, EMSA, transcription factor, cytokines, microarray.

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Neural precursors derived from human embryonic stem cells

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Science China(Life Sciences) 2005年第3期48卷 295-299页

作者： Peng Hongmei 1,2 & Chen Gui’an 1 1. Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100083, China 2. Department of Obstetrics and Gynecology, General Hospital of PLA, Beijing 100853, China Department of Obstetrics and Gynecology Peking University Third Hospital Beijing100083 China Department of Obstetrics and Gynecology General Hospital of PLA Beijing100853 China Department of Obstetrics and Gynecology Peking University Third Hospital Beijing100083 China

Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells. hES cells (Line PKU-1 and Line PKU-2) were cultured in sus- pension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs). The EBs were then cultured in N2 medium containing bFGF in poly-L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2―3 short processes of the spreading out- growth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4―5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing β-tubulin isotypeIII, GABA, serotonin and synaptophysin. Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oli- godendrocytes expressing O4. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS) in vitro.

关键词： human embryonic stem cell, differentiation, neural precursor.

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