人胚胎干细胞体内分化途径获取神经干细胞及DNA甲基化酶Dnmt3a和Dnmt3b表达下调对人胚胎干细胞的影响
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摘要
人类胚胎干细胞(human embryonic stem cells,hESCs)来源于早期胚胎、具有自我更新和分化发育为三个胚层组织潜能的多能性细胞。hESCs在多个领域,如细胞治疗、组织工程、发育生物学、基因功能研究、药物筛选等领域展示了巨大的应用前景。
第一章:人胚胎干细胞株HSF6培养。
目的:探索和建立人胚胎干细胞株HSF6的培养方法。
方法:从ICR胎鼠(E13.5)中分离胚胎成纤维细胞(mouse embryonicfibroblast,MEF),检测丝裂霉素C处理或γ射线照射后MEF的生长状态并作为hESCs饲养层;将hESCs培养于含10ng/mL碱性成纤维细胞生长因子的KO-DMEM培养基中,采用hESCs的酶消化法传代和机械法(巴氏德管制作的传代工具)传代;并对培养的HSF6进行碱性磷酸酶染色、表面标记检测、体外拟胚体(embryoid body,EB)分化能力检测等特征鉴定。
结果:第3-5代的MEF经10μg/mL丝裂霉素C处理1-3小时或经3000Radγ射线照射后能抑制增殖。酶消化法传代后hESCs克隆大小不均,分化克隆容易残留和扩增;机械法传代的hESCs克隆大小较均匀,分化克隆残留较少或培养中容易被剔除,但机械法传代过程繁琐、操作时间较长、工作量大;培养的HSF6能维持未分化状态。
结论:①建立了ICR胎鼠(E13.5)MEF的分离和培养方法,10μg/mL丝裂霉素C处理1-3小时,或3000Radγ射线照射后可作为HSF6的饲养层;②酶消化法和机械法均可用于hESCs的传代。
第二章:hESCs体内分化途径获取神经干细胞。
目的:从hESCs畸胎瘤中分离人神经干细胞(neural progenitor cells,NPC)。
方法:将hESCs注射到SCID鼠体内分化为畸胎瘤,采用贴壁培养法从中分离人NPC;免疫组织化学法检测NPC标记—巢蛋白(nestin);检测NPC分化能力,对分化细胞检测神经元标记—β微管蛋白Ⅲ(Neuron-specific classⅢbeta-tubulin,TuJⅠ)和胶质细胞标记—胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)。
结果:hESCs注射SCID鼠后5-8周后可分化为畸胎瘤,经过贴壁培养和连续传代从中分离到NPC,免疫组织化学检测nestin呈阳性,能分化为神经元和神经胶质细胞,分别表达TuJⅠ和GFAP。
结论:①利用贴壁培养法可从hESCs畸胎瘤中分离到NPC;②“hESCs-SCID鼠-畸胎瘤”模型为细胞组织工程、发育分化研究提供了一个新的方法。
第三章:Dnmt3a和Dnmt3b表达下调对hESCs的影响。
目的:研究DNA新生甲基化酶Dnmt3a(DNA methyltransferase 3a)和Dnmt3b(DNA methyltransferase 3b)表达下调后对hESCs的影响。
方法:观察Dnmt3a与Dnmt3b表达下调后hESCs的生长特性;免疫荧光检测hESCs表面标记;RT-PCR检测维持胚胎干细胞自我更新和维持未分化的相关基因;检测hESCs体外EB分化情况,在不同分化时间行AKP染色和RT-PCR检测三个胚层基因表达情况;检测hESCs在SCID鼠体内分化能力。
结果:Dnmt3a和Dnmt3b表达下调后hESCs:在MEF上呈克隆式生长,表面标记检测呈hESC特征,表达Oct3/4、Sox2和Nanog;EB分化障碍、AKP消退延迟,Dnmt3b下调后hESCs分化时神经外胚层标记基因提前出现;Dnmt3a和Dnmt3b表达下调后hESCs在SCID小鼠体内未形成畸胎瘤。
结论:①Dnmt3a和Dnmt3b表达下调后不影响hESCs未分化状态的维持,推测Dnmt3a和Dnmt3b不是hESCs未分化状态维持所必需的;②Dnmt3a和Dnmt3b表达下调导致hESCs分化缺陷,推测Dnmt3a和Dnmt3b是hESCs正常分化所必需的。
Human embryonic stem cells(hESCs) were derived from human early blastocysts,were able to be expanded long-term in vitro with stable karyotype,and were able to self-renewal and differentiate into all cells types of all three germ layers.The hESCs provided great promise for regenerative medicine and provided valuable tools for researching early human embryonic development and differentiation,gene functions and for evaluating of pharmacologic action.Based on introducing hESCs stain-HSF6,following studies were carried out.
Chapter 1 Study on culturing system of hESCs-HSF6.
Aims:to study and establish culturing system of hESCs-HSF6.
Methods:MEF cells were derivated from embryos of 13.5-days-pregency ICR mice.The MEF were treated with 10μg/mL mitomycin C or 3 000 Radγ-ray.The hESCs were maintained on the irradiated MEF in KO-DMEM with daily medium changes.The hESCs were passaged using two methods:enzymatical method with 1mg/mL collagenaseⅣand lmg/mL dispase and machanical method with Pasteur glass pipets.The characterizations of hESCs were carried out using immunostaining and differentiating.
Results:the passage 3 to 5 MEF did not proliferate after being treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray.The enzyme-treated expansion rapidly produced greater amounts of hESCs within a limited time frame.However,the cell clumps varied in size,and there was a probability that both the differentiated and undifferentiated cells would be transferred.The mechanical method was laborious and time-consuming,but the technique permitted efficient transfer of undifferentiated hESCs and results in similar clump sizes.In cases in which there were differentiated colonies,the combination of two methods allows mass production of hESCs by excluding differentiated colonies from passage by manual selection before enzyme treatment.Results of immunostaining and differentiaon showed that the hESCs could self-renewal and maintain undifferetiation in the culture system.
Conclusions:①ICR-MEF were derivated successfully from embryos of 13.5-days-pregency ICR mice;it was efficient to make feeder for hESCs-HSF6 with passage 3 to 5 ICR-MEF treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray MEF;②Both mechanical and enzymatic transfer methods for hESCs depent on experimental purpose. The mechanical transfer method was good for maintenance of hESC lines.
Chapter 2 Derivation of NPC from teratomas of hESCs.
Aims:to differentiate hESCs into teratomas in vivo and to derivate neural progenitor cells(NPC) from the teratomas.
Methods:hESCs clumps were harvested and were injected into SCID-beige mice.The mice were sacrificed and teratomas were got and identified.NPC were derivated from teratomas through plating cells onto polyornithine-(PO) and fibronectin(FN)-coated dishes in serum-free medium DMEM/F12 supplemented with B27 supplement,and 10ng/ml bFGF.The NPC were fixed for immunostaining with mouse monoclonal anti-nestin.The NPC were induced to differentiate by withdrawing bFGF and the differenctiated cells were fixed for immunostaining with mouse monoclonal anti-TuJⅠand mouse monoclonal anti-GFAP.
Results:The hESCs grafted into SCID mice consistently developed into teratomas after 5- to 8-week.They were analyzed histologically and contained representative tissues of the three germ layers.NPCs were derived successfully from the teratomas of hESCs through consecutive passages and culture in PO/FN-coated plate.Using antibodies against neural stem cell marker nestin,the cells were detected nestin positive.The NPC could differentiate into neurons and astrocytes.Using antibodies against neuronal markerβⅢtubulin(TuJⅠantigen) and astroglial marker glial fibrillary acidic protein(GFAP),it was found that TuJⅠ-positive or GFAP-positive cells were presented in these differentiated teratoma-derived NPCs.
Conclusions:①NPCs were derived successfully from the teratomas of hESCs through culture in PO/FN-coated plate;②the model of "hESCs-SCID mice-teratoma" provided a new approach or tool for cell-based therapy and reseach of differentiation or development.
Chapter 3 Effects of knockdown of Dnmt3a and Dnmt3b on hESCs.
Aims:to study on effects of knockdown of DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b(Dnmt3b) on hESCs.
Methods:The morphous and growth of hESCs were observed after the Dnmt3a and Dnmt3b were knockdown.Immunostaining with makers of hESCs were used to detect their characterizations.Reverse transcription PCR(RT-PCR) was used to detect Oct3/4,Nanog and Sox2 that were correlated to hESCs self-renewal.Potentials of differentiation of hESCs after being knockdown Dnmt3a and Dnmt3b were examined:in vitro be induced into embryoid body(EB) and in vivo be induced into teratoma.
Results:The hESCs could maintain self-renewal and undifferentiation after being knockdown Dnmt3a and Dnmt3b,and they grow colonly on MEF similar to wild type hESCs.Results of immunostaining showed that they still were SSEA-4-,TRA-1-60-,TRA-1-81- and OCT3/4-positive. They still expressed Oct3/4,Nanog and Sox2.The AKP staining of EB showd that they were difficult to differentiate into embryo bodies(EB) in vitro and knockdown of Dnmt3b could cause neuroectoderm emerge ahead of schedule in EBs.Both hESCs of knockdown of Dnmt3a and Dnmt3b could not differentiate into teratoma in SCID mice.
Conclusions:①Dnmt3a and Dnmt3b may be not essential for hESC maintaining self-renewal and undifferentiation;②Dnmt3a and Dnmt3b may be essential for hESC differentiation normally.
第一章:人胚胎干细胞株HSF6培养。
目的:探索和建立人胚胎干细胞株HSF6的培养方法。
方法:从ICR胎鼠(E13.5)中分离胚胎成纤维细胞(mouse embryonicfibroblast,MEF),检测丝裂霉素C处理或γ射线照射后MEF的生长状态并作为hESCs饲养层;将hESCs培养于含10ng/mL碱性成纤维细胞生长因子的KO-DMEM培养基中,采用hESCs的酶消化法传代和机械法(巴氏德管制作的传代工具)传代;并对培养的HSF6进行碱性磷酸酶染色、表面标记检测、体外拟胚体(embryoid body,EB)分化能力检测等特征鉴定。
结果:第3-5代的MEF经10μg/mL丝裂霉素C处理1-3小时或经3000Radγ射线照射后能抑制增殖。酶消化法传代后hESCs克隆大小不均,分化克隆容易残留和扩增;机械法传代的hESCs克隆大小较均匀,分化克隆残留较少或培养中容易被剔除,但机械法传代过程繁琐、操作时间较长、工作量大;培养的HSF6能维持未分化状态。
结论:①建立了ICR胎鼠(E13.5)MEF的分离和培养方法,10μg/mL丝裂霉素C处理1-3小时,或3000Radγ射线照射后可作为HSF6的饲养层;②酶消化法和机械法均可用于hESCs的传代。
第二章:hESCs体内分化途径获取神经干细胞。
目的:从hESCs畸胎瘤中分离人神经干细胞(neural progenitor cells,NPC)。
方法:将hESCs注射到SCID鼠体内分化为畸胎瘤,采用贴壁培养法从中分离人NPC;免疫组织化学法检测NPC标记—巢蛋白(nestin);检测NPC分化能力,对分化细胞检测神经元标记—β微管蛋白Ⅲ(Neuron-specific classⅢbeta-tubulin,TuJⅠ)和胶质细胞标记—胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)。
结果:hESCs注射SCID鼠后5-8周后可分化为畸胎瘤,经过贴壁培养和连续传代从中分离到NPC,免疫组织化学检测nestin呈阳性,能分化为神经元和神经胶质细胞,分别表达TuJⅠ和GFAP。
结论:①利用贴壁培养法可从hESCs畸胎瘤中分离到NPC;②“hESCs-SCID鼠-畸胎瘤”模型为细胞组织工程、发育分化研究提供了一个新的方法。
第三章:Dnmt3a和Dnmt3b表达下调对hESCs的影响。
目的:研究DNA新生甲基化酶Dnmt3a(DNA methyltransferase 3a)和Dnmt3b(DNA methyltransferase 3b)表达下调后对hESCs的影响。
方法:观察Dnmt3a与Dnmt3b表达下调后hESCs的生长特性;免疫荧光检测hESCs表面标记;RT-PCR检测维持胚胎干细胞自我更新和维持未分化的相关基因;检测hESCs体外EB分化情况,在不同分化时间行AKP染色和RT-PCR检测三个胚层基因表达情况;检测hESCs在SCID鼠体内分化能力。
结果:Dnmt3a和Dnmt3b表达下调后hESCs:在MEF上呈克隆式生长,表面标记检测呈hESC特征,表达Oct3/4、Sox2和Nanog;EB分化障碍、AKP消退延迟,Dnmt3b下调后hESCs分化时神经外胚层标记基因提前出现;Dnmt3a和Dnmt3b表达下调后hESCs在SCID小鼠体内未形成畸胎瘤。
结论:①Dnmt3a和Dnmt3b表达下调后不影响hESCs未分化状态的维持,推测Dnmt3a和Dnmt3b不是hESCs未分化状态维持所必需的;②Dnmt3a和Dnmt3b表达下调导致hESCs分化缺陷,推测Dnmt3a和Dnmt3b是hESCs正常分化所必需的。
Human embryonic stem cells(hESCs) were derived from human early blastocysts,were able to be expanded long-term in vitro with stable karyotype,and were able to self-renewal and differentiate into all cells types of all three germ layers.The hESCs provided great promise for regenerative medicine and provided valuable tools for researching early human embryonic development and differentiation,gene functions and for evaluating of pharmacologic action.Based on introducing hESCs stain-HSF6,following studies were carried out.
Chapter 1 Study on culturing system of hESCs-HSF6.
Aims:to study and establish culturing system of hESCs-HSF6.
Methods:MEF cells were derivated from embryos of 13.5-days-pregency ICR mice.The MEF were treated with 10μg/mL mitomycin C or 3 000 Radγ-ray.The hESCs were maintained on the irradiated MEF in KO-DMEM with daily medium changes.The hESCs were passaged using two methods:enzymatical method with 1mg/mL collagenaseⅣand lmg/mL dispase and machanical method with Pasteur glass pipets.The characterizations of hESCs were carried out using immunostaining and differentiating.
Results:the passage 3 to 5 MEF did not proliferate after being treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray.The enzyme-treated expansion rapidly produced greater amounts of hESCs within a limited time frame.However,the cell clumps varied in size,and there was a probability that both the differentiated and undifferentiated cells would be transferred.The mechanical method was laborious and time-consuming,but the technique permitted efficient transfer of undifferentiated hESCs and results in similar clump sizes.In cases in which there were differentiated colonies,the combination of two methods allows mass production of hESCs by excluding differentiated colonies from passage by manual selection before enzyme treatment.Results of immunostaining and differentiaon showed that the hESCs could self-renewal and maintain undifferetiation in the culture system.
Conclusions:①ICR-MEF were derivated successfully from embryos of 13.5-days-pregency ICR mice;it was efficient to make feeder for hESCs-HSF6 with passage 3 to 5 ICR-MEF treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray MEF;②Both mechanical and enzymatic transfer methods for hESCs depent on experimental purpose. The mechanical transfer method was good for maintenance of hESC lines.
Chapter 2 Derivation of NPC from teratomas of hESCs.
Aims:to differentiate hESCs into teratomas in vivo and to derivate neural progenitor cells(NPC) from the teratomas.
Methods:hESCs clumps were harvested and were injected into SCID-beige mice.The mice were sacrificed and teratomas were got and identified.NPC were derivated from teratomas through plating cells onto polyornithine-(PO) and fibronectin(FN)-coated dishes in serum-free medium DMEM/F12 supplemented with B27 supplement,and 10ng/ml bFGF.The NPC were fixed for immunostaining with mouse monoclonal anti-nestin.The NPC were induced to differentiate by withdrawing bFGF and the differenctiated cells were fixed for immunostaining with mouse monoclonal anti-TuJⅠand mouse monoclonal anti-GFAP.
Results:The hESCs grafted into SCID mice consistently developed into teratomas after 5- to 8-week.They were analyzed histologically and contained representative tissues of the three germ layers.NPCs were derived successfully from the teratomas of hESCs through consecutive passages and culture in PO/FN-coated plate.Using antibodies against neural stem cell marker nestin,the cells were detected nestin positive.The NPC could differentiate into neurons and astrocytes.Using antibodies against neuronal markerβⅢtubulin(TuJⅠantigen) and astroglial marker glial fibrillary acidic protein(GFAP),it was found that TuJⅠ-positive or GFAP-positive cells were presented in these differentiated teratoma-derived NPCs.
Conclusions:①NPCs were derived successfully from the teratomas of hESCs through culture in PO/FN-coated plate;②the model of "hESCs-SCID mice-teratoma" provided a new approach or tool for cell-based therapy and reseach of differentiation or development.
Chapter 3 Effects of knockdown of Dnmt3a and Dnmt3b on hESCs.
Aims:to study on effects of knockdown of DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b(Dnmt3b) on hESCs.
Methods:The morphous and growth of hESCs were observed after the Dnmt3a and Dnmt3b were knockdown.Immunostaining with makers of hESCs were used to detect their characterizations.Reverse transcription PCR(RT-PCR) was used to detect Oct3/4,Nanog and Sox2 that were correlated to hESCs self-renewal.Potentials of differentiation of hESCs after being knockdown Dnmt3a and Dnmt3b were examined:in vitro be induced into embryoid body(EB) and in vivo be induced into teratoma.
Results:The hESCs could maintain self-renewal and undifferentiation after being knockdown Dnmt3a and Dnmt3b,and they grow colonly on MEF similar to wild type hESCs.Results of immunostaining showed that they still were SSEA-4-,TRA-1-60-,TRA-1-81- and OCT3/4-positive. They still expressed Oct3/4,Nanog and Sox2.The AKP staining of EB showd that they were difficult to differentiate into embryo bodies(EB) in vitro and knockdown of Dnmt3b could cause neuroectoderm emerge ahead of schedule in EBs.Both hESCs of knockdown of Dnmt3a and Dnmt3b could not differentiate into teratoma in SCID mice.
Conclusions:①Dnmt3a and Dnmt3b may be not essential for hESC maintaining self-renewal and undifferentiation;②Dnmt3a and Dnmt3b may be essential for hESC differentiation normally.
引文
[1] Evans MJ and Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154-156
[2] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratomcaricinoma stem cells. Proc. Natl.Acad. Sci. USA 1981;78:7634-7638
[3] Thomson JA, Itskovitz-Eldor J, Sshapiro SS, et al. Embryonic stem cell lines derived from human blastocyst. Science 1998;282:1145-1147
[4] Shamblott MJ, Wang SP, Bugg EM, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA.1998,95:13726-13731
[5] Park SP, Lee YJ , Lee KS, et al. Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers. Hum. Reprod.2004;19:676-684
[6] Richards M, Tan S, Fong CY, et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.Stem Cells 2003;21(5):546-556
[7] Lee JB, Lee JE, Park JH, et al. Establishment and maintenance of human embryonics tem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol. Reprod. 2005;72(1):42-49
[8] Itskovitz-Eldor J, Schuldiner M, Karsenti D, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 2000;6:88-95
[9] Schuldiner M, Yanuka O, Itskovitz-Eldor J, et al. From the cover: effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2000;97:11307—11312
[10] Fraichard A, Chassande O, Bilbaut G, et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell. Sci. 1995;108: 3181-3188
[11] Perrier AL, Tabar V, Barberi T, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2004;101:12543-12548
[12] Rolletschek A, Chang H, Guan K, et al. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.Mech Dev 2001;105:93-104
[13]Carpenter MK,Inokuma MS,Denham J,et al.Enrichment of neurons and neural precursors from human embryonic stem cells.Exp.Neurol.2001;172:383-397
[14]Ng HH and Bird A.DNA methylation and chromatin modification.Curr.Opin.Genet.Dev.1999;9:158-163
[15]Robertson KD,Uzvolgyi E,Liang G,et al.The human DNA methyltransferases (DNMTs) 1,3a and 3b:coordinate mRNA expression in normal tissues and overexpression in tumors.Nucl.Acids Res.1999;27:2291-2298
[16]Chen T,Ueda Y,Dodge J,et al.Establishment and Maintenance of Genomic Methylation Patterns in Mouse Embryonic Stem Cells by Dnmt3a and Dnmt3b.Mol.Cell.Biol.2003;23(16):5594-5605
[17]Li E,Bestor TH,Jaenisch R.Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell 1992;12:915-926
[18]Kaneda M,Okano M,Hata K,et al.Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting.Nature 2004;429:900-903
[19]Tsumura A,Hayakawa T,Kumaki Y,et al.Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1,Dnmt3a and Dnmt3b.Genes to Cells 2006;11:805-814
[20]Reubinoff BE,Peva MF,Fong CY,et al.Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro.Nat.Biotechnol.2000;18:399-404
[21]Heins N,Englund MC,Sjoblom C,et al.Derivation,characterization,and differentiation of human embryonic stem cells.Stem Cells 2004;22:367-376
[22]刘民,李柏青.小鼠胚胎成纤维细胞的分离、扩增和抑制.中国实验动物学杂志.2002;12(4):215-219
[23]张怡,赵连三,汪成孝,等.小鼠胚胎成纤维细胞的分离与培养.四川大学学报(医学版).2003;34(2):344-346
[24]招霞,孙海翔,胡娅莉,等.ICR小鼠胚胎成纤维细胞的培养及饲养层的制作.医学研究生学报.2006;19:36-39
[25]张海锋,单伟,秦书俭.不同分离方法对鼠胚胎成纤维细胞生长影响的比较.中国组织工程研究与临床康复 2007;11:2001-2004
[26]王瑞琼,鄢远,黄坚峰.荧光法研究丝裂霉素C与DNA的作用机制.化学学 报.1999:57:171-175
[27]余树民,王晗,窦忠英.丝裂霉素C处理后鼠胚成纤维细胞活力分析.西北农林科技大学学报(自然科学版).2007;35(5):1-5
[28]黄奔,蒙超衡,石德顺,等.小鼠胎儿成纤维细胞饲养层制备的研究.实验研究.2005:5:16-18
[29]陈红,钱坤,张苏明,等.昆明白小鼠胎鼠成纤维细胞不同接种密度对人胚胎干细胞的影响.中国组织工程研究与临床康复.2007:11:443-446
[30]Sperger JM,Chen X,Draper JS,et al.Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors.Proc.Natl.Acad.Sci.USA 2003;100(23):13350-13355
[31]Beattie GM,Lopez AD,Bucay N,et al.Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers.Stem Cells 2005;23(4):489-495
[32]James D,Levine AJ,Besser D,et al.YGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.Development 2005;132(6):1273-1282
[33]Xie CQ,Lin G,Luo KL,et al.Newly expressed proteins of mouse embryonic fibroblasts irradiated to be inactive.Biochem.Biophys.Res.Commun.2004;315(3):581-588
[34]Noboru S,Laurent M,Leandros S,et al.Maintenance of pluripotency in human and mouse embryonic stem cell through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat.Med.2004;10(1):55-63
[35]Martin JM,Muotri A,Gage F,et al.Human embryonic stem cells express an immunogenic nonhuman sialic acid,Nat.Med.2005;11:228-232
[36]Oh SK,Kim HS,Park YB,et al.Methods for expansion of human embryonic stem ceils.Stem Cells 2005;23;605-609
[37]Buzzard JJ,Gough NM,Crook JM,et al.Karyotype of human ES cells during extended culture.Nat.Biotechnol.2004;22(4):381-382
[38]Draper JS,Smith K,Gokhale P,et al.Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.Nat.Biotechnol.2004;22(1):53-54
[39]Sen A,Kallos MS and Behie LA.Passaging protocols for mammalian neural stem cells in suspension bioreactors.Biotechnol.Progr.2002;18:337-345
[40]Shen Y,Chow J,Wang Z,et al.Abnormal CpG island methylation occurs during in vitro differentiation of human embryonic stem cells.Hum.Mol.Genet. 2006;15:2623-2635
[41]Dono R.Fibroblast growth factors as regulators of central nervous system development and function.Am J Physiol Regul Integr Comp Physiol,2003;284:867-881
[42]D'Amour KA,Agulnick AD,Eliazer S,et al.Efficient differentiation of human embryonic stem cells to definitive endoderm.Nat.Biotechnol.2005;23:1534-1541
[43]Andrews PW,Damjanov I.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2.Differentiation in vivo and in vitro.Lab Invest.1984;50:147-162
[44]Skotheim RI,Monni O,Mousses S,et al.New insights into testicular germ cell tumorigenesis from gene expression profiling.Cancer Res.2002;62:2359-2364
[45]Nelson PT,Kondziolka D,Weeksler L.Clonal human(hNT) neuron grafts for stroke therapy:neuropathology in a patient 27 months after implantation.Am.J.Pathol.2002;160:1201-1206
[46]Richards M,Fong CY,Chan WK,et al.Bongso.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat.Biotechnol.2002;20:933-936
[47]Xu C,Inokuma MS,Denham J,et al.Feeder-free growth of undifferentiated.human embryonic stem cells.Nat.Biotechnol.2001;19:971-974
[48]Richards M,Tan S,Fong CY,et al.Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.Stem Cells 2003;21:546-556
[49]Draper JS,Smith K,Gokhale P,et al.Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.Nat.Biotechnol.2004;22:53-54
[50]Levenberg S,Huang NF,Lavik E,et al.Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds.Proc.Natl.Acad.Sci.USA 2003;100:12741-12746
[51]Shin S,Sun Y,Liu Y,et al.Whole genome analysis of human neural stem cells derived from embryonic stem cells and stem and progenitor cells isolated from fetal tissue.Stem Cells 2007;25:1298-1306
[52]孔慧娟,孙莹璞.人胚胎干细胞保持未分化状态调控机制.同外医学计划生育.生殖健康分册2006:25:124-127
[53]Rosner MH,Vigano MA,Ozato K,et al.A Pou-domain transcription factor in early stem cells and germ cells of the mammalian embryo.Nature 1990;345:686-692
[54]Daheron L,Opitz SL,Zaehres H,et al.LIF/STAT3 signaling fails to maintain self-renew of human embryonic stem cells.Stem Cells,2004;22(5):770-778
[55]Niwa H,Miyazaki JI,Smith AG.Quantitative expression of Oct-3/4 defines differentiation,dedifferentiation or self-renewal of ES cells.Nat.Genet.2000;24(3):372-376
[56]Bhatlacharya B,Miura T,Brandenberger R,et al.Gene expression in human embryonic stem cell lines:unique molecular signature.Blood 2004;103:2956-2964
[57]Botquin V,Hess H,Fuhrmann G,et al.New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2.Genes Dev.1998;12:2073-2090
[58]Mitsui K,Yokuzawa Y,Itoh H,et al.The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.Cell 2003;113:631-642
[59]Chambers I,Colby D,Roberson M,et al.Functional expression cloning of Nanog a pluripotency sustaining factor in embryonic stem cells.Cell 2003;113:643-655
[60]Chambers I,Colby D,Robertson M,et al.Functional expression cloning of Nanog,a pluripotency sustaining factor in embryonic stem cells.Cell,2003;113(5):643-655
[61]Rodda DJ,Chew JL,Lim LH,et al.Transcriptional regulation of Nanog by OCT4 and SOX2.J.Biol.Chem.2005;280(26):24731-24734
[62]Pan G,Li J,Zhou Y,et al.A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal.FASEB 2006;20:1730-1732
[63]Yakahashi K and Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell 2006;126:663-676
[64]金志刚,刘莉,谢治慧,等.干细胞命运决定的分子调控网络.生命科学.2007;19(4):364-371
[65]Boyer LA,Lee TI,Cole MF,et al.Core transcriptional regulatory circuitry in human embryonic stem cells.Cell,2005;122(6):947-956
[66]Boer B,Kopp J,Mallanna S,et al.Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes. Nucl. Acids Res. 2007;42:1 — 14
[67] Hattorj N, Nishino K, Ko YG, et al. Epigenetic control of mouse Oct-4 gene expression in ES cells and TS cells. J. Biol. Chem. 2004;279:17063—17069
[68] Gidekel S and Bergman Y. A unique developmental pattern of oct-3/4 DNA methylation is controlled by a cis-demodification element. J. Biol. Chem.2002;277(37):34521-34530
[69] Okano M, Bell DW, Haber DA, et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell 1999;99:247—257
[70] Lei, H, Oh SP, Okano M, et al. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 1996; 122:3195—3205
[1]Evans MJ and Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature 1981;292:154-156
[2]Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratomcaricinoma stem cells.Proc.Natl.Acad.Sci.USA 1981;78:7634-7638
[3]Thomson JA,Itskovitz-Eldor J,Sshapiro SS,et al.Embryonic stem cell lines derived from human blastocyst.Science 1998;282:1145-1147
[4]Shamblott MJ,Axelman J,Wang SP,et al.Derivation of pluripotent stem cells from cultured human primordial germ cells.Proc.Natl.Acad.Sci.USA.1998;95:13726-13731
[5]Thomas KR and Capecchi MR.Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.Cell 1987;51:503-512
[6]Alvarez-Dolado M,Pardal R,Garcia-Vardugo JM,et al.Fusion of bone-marrow-derived cells with Purkinje neurons,cardiomyoctys.Nature 2003;425:968-973
[7]Wilmut I,Schnieke AE,McWhir J,et al.Viable offspring derived from fetal and adult mammalian cells.Nature 1997;385:810-813
[8] Amit M, Carpenter MK, Inokuma MS, et al. Clonally derived human embryonic stem cells lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 2000;227:271-278
[9] Gearhart JD and Mintz B. Contact-mediated myogenesis and increased acetycholinesterase activity in primary cultures of mouse teratocarcinoma cells. Proc. Natl. Acad. Sci. USA 1974;71:1734-1738
[10]Resnick JL, Bixler LS, Cheng L, et al. Long-term proliferation of mouse primordial germ cells in culture. Nature 1992;359:550-551
[11]Nichols J, Zevnik B, Anastassiadis K, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95: 379-391
[12]Pesce M, Anastassiadis K, and Scholer HR. Oct-4: lessons of totipotency from embryonic stem cells. Cells Tissues Organs 1999;165:144-152
[13]Anna M, Wobus D and Kenneth RB. Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy. Physiol. Rev. 2005;85:635-678
[14] Chambers I, Colby D, Robertson M, et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643 -655
[15] Williams RL,Hilton DJ, Pease S, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684-687
[16]Niwa H, Burdon T, Chambers I, et al. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 1998;12:2048-2060
[17]Boeuf H, Hauss C, Graeve FD, et al. Leukemia inhibitory factor- dependent transcriptional activation in embryonic stem cells. J. Cell Biol. 1997;138:1207-1217
[18]Ying QL, Nichols J, Chambers I, et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281 -292
[19]Brandenberger R, Wei H, Zhang S, et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat. Biotechnol. 2004;22:707-716
[20] Abeyta MJ, Clark AT, Rodriguez RT, et al. Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum. Mol. Genet. 2004; 13:601-608
[21] Richards M, Tan SP, Tan JH, et al. The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 2004;22:51-64
[22] Itskovitz-Eldor J, Schuldiner M, Karsenti D, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 2000;6:88-95
[23] Schuldiner M, Yanuka O, Itskovitz-Eldor J, et al. From the cover: effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA2000;97: 11307-11312
[24]Bagutti C, Wobus AM, Fassler R, et al. Differentiation of embryonal stem cells into keratinocytes: comparison of wildtype and beta 1 integrin-deficient cells.Dev.Biol. 1996;179:184-196
[25]Coraux C, Hilmi C, Rouleau M, et al. Reconstituted skin from murine embryonic stem cells. Curr. Biol. 2003;13:849-853
[26]Fraichard A, Chassande O, Bilbaut G, et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 1995;108:3181-3188
[27]Rolletschek A, Chang H, Guan K, et al. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.Mech.Dev. 2001;105:93-104
[28] Carpenter MK, Inokuma MS, Denham J, et al. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp. Neurol. 2001;172:383-397
[29]Nakano T, Kodama H, and Honjo T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 1994;265:1098-1101
[30]Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 2001;108:407-414
[31]Reppel M, Boettinger C, and Hescheler J. Beta-adrenergic and muscarinic modulation of human embryonic stem cell-derived cardio-myocytes. Cell Physiol.Biochem. 2004; 14:187-196
[32]Dani C, Smith AG, Dessolin S, et al. Differentiation of embryonic stem cells into adipocytes in vitro. J. Cell Sci. 1997;110: 1279-1285
[33] Kramer J, Hegert C, Guan K, et al. Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech. Dev. 2000;92:193-205
[34]Rohwedel J, Maltsev V, Bober E, et al. Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev.Biol. 1994;164: 87-101
[35]Chinzei R, Tanaka Y, Shimizu-Saito K, et al. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes.Hepatology 2002;36:22-29
[36]Leon-Quinto T, Jones J, Skoudy A, et al. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia 2004;47:1442-1451
[37]Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulinproducing cells.Proc. Natl. Acad. Sci. USA 2003;100:998-1003
[38]Hubner K, Fuhrmann G, Christenson LK, et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003;300: 1251 - 1256
[39]Toyooka Y, Tsunekawa N, Akasu R, et al. Embryonic stem cells can form germ cells in vitro. Proc. Natl. Acad. Sci. USA2003;100:11457-11462
[40]Geijsen N, Horoschak M, Kim K, et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2003;427:148-154
[41]Cecconi F, Meyer BI. Gene trap: a way to identify novel genes and unravel their biological function. FEBS 2000;480:63-71
[42]Blobel GA, Simon MC, and Orkin SH. Rescue of GATA-1-deficient embryonic stem cells by heterologous GATA-binding proteins. Mol. Cell. Biol. 1995;15:626-633
[43]Perrier AL, Tabar V, Barberi T, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2004;101:12543-12548
[44]Segev H, Fishman B, Ziskind A, et al. Differentiation of human embryonic stem cells into insulin-producing clusters.Stem Cells 2004;22:265-274
[45]Klug MG, Soonpaa MH, Koh GY, et al. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin.Invest. 1996;98:216-224
[46] Levenberg S, Huang NF, Lavik E, et al. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc. Natl. Acad. Sci. USA 2003; 100:12741-12746
[47]Davila JC, Cezar GG, Thiede M, et al. Use and application of stem cells in toxicology. Toxicol. Sci. 2004;79:214-223
[48]Wobus AM, Wallukat G, and Hescheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2- channel blockers.Differentiation 1991;48: 173-182
[49]Kim JH, Auerbach JM, Rodriguez-Gomez JA, et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease.Nature 2002;418: 50-56
[50] Richards M, Fong CY, Chan WK, et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat. Biotechnol. 2002;20:933-936
[51] Martin JM, Muotri A, Gage F, et al. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 2005;112:28-232
[52]Xu C, Inokuma MS, Denham J, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 2001;19:971-974
[53] Richards M, Tan S, Fong CY, et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.Stem Cells 2003;21:546-556
[54]Pruszuk J, Sonntag KC, Aung MH, et al. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cell Express,published online 2007; dol:10,1634/stem cells.2006-0744
[55]Drukker M, Katz G, Urbach A, et al. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2002;99:9864-9869
[56]Zwaka TP and Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003;3: 319-321
[57] Lanza RP, Cibelli JB, and West MD. Human therapeutic cloning. Nat. Med.1999;5: 975-977
[58] Chen Y, He ZX, Liu A, et al. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Res. 2003;13:251-63
[1]Jan Tesarik,Carmen Mendoza,and Ermanno Greco.Paternal effects acting during the first cell cycle of human preimplantation development after ICSI.Hum.Reprod.2002;17:184-189
[2]陈大元.受精生物学—受精机制与生殖工程.科学出版社.2000;北京
[3]Palermo G.,Joris H,Devroey P,et al.Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte.Lancet 1992:340:17-18
[4]Balakier H.Fertilization and early embryology Tripronuclear human zygotes:the first cell cycle and subsequent development.Hum.Reprod.1993;8:1892-1897
[5]Feng H and Hershlag A.Fertilization Abnormalities Following Human In Vitro Fertilization and Intracytoplasmic Sperm Injection.Microsc.Res.Yechniq.2003;61:358-361
[6]Yu SL,Lee RKK,Su JT,et al.Distinction between paternal and maternal contributions to the tripronucleus in human zygotes obtained after in vitro fertilization.Taiwanese J.Obstet.Gynecol.2006;45:313-316
[7]Grossmann M,Calafell JM,Brandy N,et al.Origin of tripronucleate zygotes after intracytoplasmic sperm injection.Hum.Reprod.1997;12:2762-2765
[8]Egozcue S,Blanco J,Vidal F,et al.Diploid sperm and the origin of triploidy.Hum.Reprod.2002;17:5-7
[9]Funaki K,Mikamo K.Giant diploid oocytes as a cause of digynic triploidy in mammals.Cytogenet.Cell Genet.1980;28:158-68
[10]Lubna P,Toth TL,Leykin L,et al.High incidence of triploidy in in-vitro fertilized oocytes from a patient with a previous history of recurrent gestational trophoblastic disease.Hum.Reprod.1996;11:1529-1532
[11] Egli D, Rosains J, Birkhoff G, et al. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature 2007;447:679-686
[12]Yie SM, Collins JA, Daya S, et al. Polyploidy and failed fertilization in in-vitro fertilization are related to patient's age and gamete quality. Hum. Reprod.1996;11:614-617
[13] Wolf DP, Byrd W, Dandekar P, et al. Sperm concentration and the fertilization of human eggs in vitro. Biol. Reprod. 1984;31:837-848
[14] Lowe B, Osborn JC, Fothergill DJ, et al. Factors associated with accidental fractures of the zona pellucida and multipronuclear human oocytes following in-vitro fertilization. Hum. Reprod. 1988;3(7):901-904
[15]Pieters M, Dumoulin JCM, Ignoul-Vanvuchelen RCM, et al. Triploidy after in vitro fertilization: cytogenetic analysis of human zygotes and embryos. J. Assist.Reprod. Gen. 1992;9:68-76
[16]Rosenbusch B, Schneider M and Sterzik K. Chromosomal analysis of mutipronuclear zygotes obtained after partial zona dissection of the oocytes. Mol.Hum. Reprod. 1998;4:1065-1070
[17]Rosenbusch B, Schneider M and Sterzik K. The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization. Hum. Reprod.1997;12:2257-2262
[18]Rosenbusch BE, Schneider M, and Hanf V. Tetraploidy and partial endoreduplication in a tripronuclear zygote obtained after intracytoplasmic sperm injection. Fertil. Steril. 1998; 69:344-346
[19]Rosenbusch B, Schneider M and Sterzik K. Triploidy caused by endoreduplication in a human zygote obtained after in-vitro fertilization. Hum.Reprod. 1997;12:1059-1061
[20]Staessen C and Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum. Reprod. 1997;12:321-327
[21] Palermo SM, Colombero LT, Cohen J, et al. Genetics of abnormal human fertilization. Hum. Reprod. 1995;10 (Suppl 1):120-127
[22]Macas E, Imthurn B, Roselli M, et al. Chromosome analysis of single- and multipronucleated human zygotes proceeded after the intracytoplasmic sperm injection procedure. J. Assist. Reprod. Gen. 1996;13:345-350
[23]Macas E, Zweife C, and Imthurn B. Numerical chromosome anomalies detected in paternally derived pronuclei of tripronuclear zygotes after intracytoplasmic sperm injection. Fertil. Steril. 2006;85:1753-1760
[24]Macas E, Imthurn B, Rosselli M, et al. The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection.Hum.Reprod. 1996;11:2496-2501
[25] Yan J, Chen Z, Feng HL. Aneuploid analysis of tripronuclear zygotes derived from in vitro fertilization and intracytoplasmic sperm injection in humans. Fertil.Steril. 2005;83:1485-1488
[26]Rosenbusch B, Schneider M, Kreienberg R, et al. Cytogenetic analysis of human zygotes displaying three pronuclei and on polar body after intracyoplasmic sperm injection. Hum. Reprod. 2001;16:2362~2367
[27]颜军昊,陈子江,李媛,胡美京.体外受精后多元核受精卵的移植价值.生殖与避孕. 2005;25:537-541
[28]Munne S, Sandalinas M, Escudero T, et al. Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect. Reprod.Biomed. Online. 2002;4:223-232
[29]Hinchcliffe EH and Sluder G. "It Takes Two to Tango": understanding how centrosome duplication is regulated throughout the cell cycle Genes & Development 2001;15:1167-1181
[30]Andrew W. Murray. Recycling the cell cycle: cyclins revisited. Cell 2004;116:221 -234
[31]Matsumoto Y and Mailer JL. A centrosomal localization signal in cyclin Erequired for Cdk2-independent S phase entry. Science 2004;306:885-888
[32] Edward H. Hinchcliffe, Miller FJ, et al. Requirement of a centrosomal activity for cell cycle progression through G1 into S phase. Science 2001;291:1547-1550
[33]Schetten G. The centrosome and its mode of inheritance the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev.Biol. 1994;165:299-335
[34]Simerly C, Wu GJ, Zoran S, et al. The paternal inheritance of the centrosome the cells microtubule-organizing center in humans and the implications for infertility.Nat.Med. 1995;1(1):47-52
[35]Simerly C, Zoran SS, Payne C, et al. Biparental inheritance of y-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm. Mol. Biol. Cell. 1999;10:2955-2969
[36]Rawe VY, Terada Y, Nakamura S, et al. A pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage. Hum.Reprod. 2002;17:2344-2349
[37]Sathanathan AH, Kola I, Osborne J, et al. Centrioles in the beginning of human development. Proc. Natl. Acad. Sci. USA 1991;88:4806-4810
[38] Terada Y, Simerly CR, Hewitson L, et al. Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm. Biol. Reprod. 2000;62:557-563
[39]Nakamura S, Terada Y, Horiuchi T, et al. Human Sperm Aster Formation and Pronuclear Decondensation in Bovine Eggs Following Intracytoplasmic Sperm Injection Using a Piezo-Driven Pipette: A Novel Assay for Human Sperm Centrosomal Function. Biol. Reprod. 2001;65:1359-1363
[40] Asch R, Simerly C, Ord T, et al. The stages at which human fertilization arrests:microtubule and chromosome configurations in inseminated oocytes which failed to complete fertilization and development in humans. Mol. Hum. Reprod.1995;1:239-248
[41] Kola I, Trounson A, Dawson G, et al. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotype analysis of embryos. Biol. Reprod.1987;37:395-401
[42]Plachot M, Veiga A, Montagut J, et al. Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multi-centric study. Hum.Reprod. 1988;3:627-635
[43]Chatzimeletiou K, Morrison EE, Prapas N, et al. Spindle abnormalities in normally developing and arrested human preimplantation embryos in vitro identified by confocal laser scanning microscopy. Hum. Reprod. 2005;20:672-682
[44]Briggs R and King TJ. Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. Proc. Natl. Acad. Sci. USA 1952;38:455-463
[45]Illmensee K, Hoppe PC. Nuclear transplantation in mouse musculus:developmental potential of nuclei from preimplantation embryos. Cell 1981;23:9-16
[46]McGrath JS, Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 1983,220:1300-1302
[47] Campbell KH, Mc Whir J, Ritchie WA, et al. Sheep cloned by nuclear transfer from a cultured cell line. Nature 1996;380:64-67
[48] Chan A, Dominko T, Luetjens CM, et al. Clonal Propagation of Primate Offspring by Embryo Splitting. Science 2000:287:317-319
[49]Rawlins RG, Binor Z, Radwanska E, et al. Microsurgical enucleation of tripronuclear human zygotes. Fertil. Steril.1988;50:266-272
[50]Modlinski JA. Haploid mouse embryos obtained by microsurgical removal of one pronucleus. J. Embryol. Exp. Morphol.1975;33:897-905
[51]Kimura Y and Yanagimachi R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring.Development 1995;121:2397-2405
[52]Kimura Y and Yanagimachi R. Intracytoplasmic sperm injection in the mouse.Biology of Reproduction 1995;52:709-720
[53] Malter HE and Cohen J. Embryonic development after microsurgical repair of polyspermic human zygotes. Fertil. Steril. 1989;52:373-380
[54]Wiker S, Miter W, Wright G, et al. Recognition of Paternal Pronuclei in Human Zygotes. Journal of in Vitro Fertilization and Embryo Transfer 1990;7:33-37
[55] Gordon JW, Grunfeld L, Garissi GJ, et al. Successful microsurgical removal of a pronucleus from tripronuclear human zygotes. Fertil. Steril. 1989;52:367-372
[56] Palermo G, MunneA S and Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum. Reprod. 1994;9:1220—1225
[57]Ivakhnenko V, Cieslak J and Verlinsky Y. A microsurgical technique for enucleation of multipronuclear human zygotes. Hum. Reprod. 2000;15:911-916
[58]Katteral S and Chen C. Normal birth after microsurgical enucleation of tripronuclear human zygotes: Case report. Hum. Reprod. 2003;18:1319-1322
[59]Escriba MJ, Martin J, Rubio C, et al. Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos. Fertil. Steril.2006;86:1601-1607
[2] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratomcaricinoma stem cells. Proc. Natl.Acad. Sci. USA 1981;78:7634-7638
[3] Thomson JA, Itskovitz-Eldor J, Sshapiro SS, et al. Embryonic stem cell lines derived from human blastocyst. Science 1998;282:1145-1147
[4] Shamblott MJ, Wang SP, Bugg EM, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. USA.1998,95:13726-13731
[5] Park SP, Lee YJ , Lee KS, et al. Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers. Hum. Reprod.2004;19:676-684
[6] Richards M, Tan S, Fong CY, et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.Stem Cells 2003;21(5):546-556
[7] Lee JB, Lee JE, Park JH, et al. Establishment and maintenance of human embryonics tem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol. Reprod. 2005;72(1):42-49
[8] Itskovitz-Eldor J, Schuldiner M, Karsenti D, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 2000;6:88-95
[9] Schuldiner M, Yanuka O, Itskovitz-Eldor J, et al. From the cover: effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2000;97:11307—11312
[10] Fraichard A, Chassande O, Bilbaut G, et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell. Sci. 1995;108: 3181-3188
[11] Perrier AL, Tabar V, Barberi T, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2004;101:12543-12548
[12] Rolletschek A, Chang H, Guan K, et al. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.Mech Dev 2001;105:93-104
[13]Carpenter MK,Inokuma MS,Denham J,et al.Enrichment of neurons and neural precursors from human embryonic stem cells.Exp.Neurol.2001;172:383-397
[14]Ng HH and Bird A.DNA methylation and chromatin modification.Curr.Opin.Genet.Dev.1999;9:158-163
[15]Robertson KD,Uzvolgyi E,Liang G,et al.The human DNA methyltransferases (DNMTs) 1,3a and 3b:coordinate mRNA expression in normal tissues and overexpression in tumors.Nucl.Acids Res.1999;27:2291-2298
[16]Chen T,Ueda Y,Dodge J,et al.Establishment and Maintenance of Genomic Methylation Patterns in Mouse Embryonic Stem Cells by Dnmt3a and Dnmt3b.Mol.Cell.Biol.2003;23(16):5594-5605
[17]Li E,Bestor TH,Jaenisch R.Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell 1992;12:915-926
[18]Kaneda M,Okano M,Hata K,et al.Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting.Nature 2004;429:900-903
[19]Tsumura A,Hayakawa T,Kumaki Y,et al.Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1,Dnmt3a and Dnmt3b.Genes to Cells 2006;11:805-814
[20]Reubinoff BE,Peva MF,Fong CY,et al.Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro.Nat.Biotechnol.2000;18:399-404
[21]Heins N,Englund MC,Sjoblom C,et al.Derivation,characterization,and differentiation of human embryonic stem cells.Stem Cells 2004;22:367-376
[22]刘民,李柏青.小鼠胚胎成纤维细胞的分离、扩增和抑制.中国实验动物学杂志.2002;12(4):215-219
[23]张怡,赵连三,汪成孝,等.小鼠胚胎成纤维细胞的分离与培养.四川大学学报(医学版).2003;34(2):344-346
[24]招霞,孙海翔,胡娅莉,等.ICR小鼠胚胎成纤维细胞的培养及饲养层的制作.医学研究生学报.2006;19:36-39
[25]张海锋,单伟,秦书俭.不同分离方法对鼠胚胎成纤维细胞生长影响的比较.中国组织工程研究与临床康复 2007;11:2001-2004
[26]王瑞琼,鄢远,黄坚峰.荧光法研究丝裂霉素C与DNA的作用机制.化学学 报.1999:57:171-175
[27]余树民,王晗,窦忠英.丝裂霉素C处理后鼠胚成纤维细胞活力分析.西北农林科技大学学报(自然科学版).2007;35(5):1-5
[28]黄奔,蒙超衡,石德顺,等.小鼠胎儿成纤维细胞饲养层制备的研究.实验研究.2005:5:16-18
[29]陈红,钱坤,张苏明,等.昆明白小鼠胎鼠成纤维细胞不同接种密度对人胚胎干细胞的影响.中国组织工程研究与临床康复.2007:11:443-446
[30]Sperger JM,Chen X,Draper JS,et al.Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors.Proc.Natl.Acad.Sci.USA 2003;100(23):13350-13355
[31]Beattie GM,Lopez AD,Bucay N,et al.Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers.Stem Cells 2005;23(4):489-495
[32]James D,Levine AJ,Besser D,et al.YGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.Development 2005;132(6):1273-1282
[33]Xie CQ,Lin G,Luo KL,et al.Newly expressed proteins of mouse embryonic fibroblasts irradiated to be inactive.Biochem.Biophys.Res.Commun.2004;315(3):581-588
[34]Noboru S,Laurent M,Leandros S,et al.Maintenance of pluripotency in human and mouse embryonic stem cell through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat.Med.2004;10(1):55-63
[35]Martin JM,Muotri A,Gage F,et al.Human embryonic stem cells express an immunogenic nonhuman sialic acid,Nat.Med.2005;11:228-232
[36]Oh SK,Kim HS,Park YB,et al.Methods for expansion of human embryonic stem ceils.Stem Cells 2005;23;605-609
[37]Buzzard JJ,Gough NM,Crook JM,et al.Karyotype of human ES cells during extended culture.Nat.Biotechnol.2004;22(4):381-382
[38]Draper JS,Smith K,Gokhale P,et al.Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.Nat.Biotechnol.2004;22(1):53-54
[39]Sen A,Kallos MS and Behie LA.Passaging protocols for mammalian neural stem cells in suspension bioreactors.Biotechnol.Progr.2002;18:337-345
[40]Shen Y,Chow J,Wang Z,et al.Abnormal CpG island methylation occurs during in vitro differentiation of human embryonic stem cells.Hum.Mol.Genet. 2006;15:2623-2635
[41]Dono R.Fibroblast growth factors as regulators of central nervous system development and function.Am J Physiol Regul Integr Comp Physiol,2003;284:867-881
[42]D'Amour KA,Agulnick AD,Eliazer S,et al.Efficient differentiation of human embryonic stem cells to definitive endoderm.Nat.Biotechnol.2005;23:1534-1541
[43]Andrews PW,Damjanov I.Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2.Differentiation in vivo and in vitro.Lab Invest.1984;50:147-162
[44]Skotheim RI,Monni O,Mousses S,et al.New insights into testicular germ cell tumorigenesis from gene expression profiling.Cancer Res.2002;62:2359-2364
[45]Nelson PT,Kondziolka D,Weeksler L.Clonal human(hNT) neuron grafts for stroke therapy:neuropathology in a patient 27 months after implantation.Am.J.Pathol.2002;160:1201-1206
[46]Richards M,Fong CY,Chan WK,et al.Bongso.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat.Biotechnol.2002;20:933-936
[47]Xu C,Inokuma MS,Denham J,et al.Feeder-free growth of undifferentiated.human embryonic stem cells.Nat.Biotechnol.2001;19:971-974
[48]Richards M,Tan S,Fong CY,et al.Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.Stem Cells 2003;21:546-556
[49]Draper JS,Smith K,Gokhale P,et al.Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.Nat.Biotechnol.2004;22:53-54
[50]Levenberg S,Huang NF,Lavik E,et al.Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds.Proc.Natl.Acad.Sci.USA 2003;100:12741-12746
[51]Shin S,Sun Y,Liu Y,et al.Whole genome analysis of human neural stem cells derived from embryonic stem cells and stem and progenitor cells isolated from fetal tissue.Stem Cells 2007;25:1298-1306
[52]孔慧娟,孙莹璞.人胚胎干细胞保持未分化状态调控机制.同外医学计划生育.生殖健康分册2006:25:124-127
[53]Rosner MH,Vigano MA,Ozato K,et al.A Pou-domain transcription factor in early stem cells and germ cells of the mammalian embryo.Nature 1990;345:686-692
[54]Daheron L,Opitz SL,Zaehres H,et al.LIF/STAT3 signaling fails to maintain self-renew of human embryonic stem cells.Stem Cells,2004;22(5):770-778
[55]Niwa H,Miyazaki JI,Smith AG.Quantitative expression of Oct-3/4 defines differentiation,dedifferentiation or self-renewal of ES cells.Nat.Genet.2000;24(3):372-376
[56]Bhatlacharya B,Miura T,Brandenberger R,et al.Gene expression in human embryonic stem cell lines:unique molecular signature.Blood 2004;103:2956-2964
[57]Botquin V,Hess H,Fuhrmann G,et al.New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2.Genes Dev.1998;12:2073-2090
[58]Mitsui K,Yokuzawa Y,Itoh H,et al.The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.Cell 2003;113:631-642
[59]Chambers I,Colby D,Roberson M,et al.Functional expression cloning of Nanog a pluripotency sustaining factor in embryonic stem cells.Cell 2003;113:643-655
[60]Chambers I,Colby D,Robertson M,et al.Functional expression cloning of Nanog,a pluripotency sustaining factor in embryonic stem cells.Cell,2003;113(5):643-655
[61]Rodda DJ,Chew JL,Lim LH,et al.Transcriptional regulation of Nanog by OCT4 and SOX2.J.Biol.Chem.2005;280(26):24731-24734
[62]Pan G,Li J,Zhou Y,et al.A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal.FASEB 2006;20:1730-1732
[63]Yakahashi K and Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell 2006;126:663-676
[64]金志刚,刘莉,谢治慧,等.干细胞命运决定的分子调控网络.生命科学.2007;19(4):364-371
[65]Boyer LA,Lee TI,Cole MF,et al.Core transcriptional regulatory circuitry in human embryonic stem cells.Cell,2005;122(6):947-956
[66]Boer B,Kopp J,Mallanna S,et al.Elevating the levels of Sox2 in embryonal carcinoma cells and embryonic stem cells inhibits the expression of Sox2:Oct-3/4 target genes. Nucl. Acids Res. 2007;42:1 — 14
[67] Hattorj N, Nishino K, Ko YG, et al. Epigenetic control of mouse Oct-4 gene expression in ES cells and TS cells. J. Biol. Chem. 2004;279:17063—17069
[68] Gidekel S and Bergman Y. A unique developmental pattern of oct-3/4 DNA methylation is controlled by a cis-demodification element. J. Biol. Chem.2002;277(37):34521-34530
[69] Okano M, Bell DW, Haber DA, et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell 1999;99:247—257
[70] Lei, H, Oh SP, Okano M, et al. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 1996; 122:3195—3205
[1]Evans MJ and Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature 1981;292:154-156
[2]Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratomcaricinoma stem cells.Proc.Natl.Acad.Sci.USA 1981;78:7634-7638
[3]Thomson JA,Itskovitz-Eldor J,Sshapiro SS,et al.Embryonic stem cell lines derived from human blastocyst.Science 1998;282:1145-1147
[4]Shamblott MJ,Axelman J,Wang SP,et al.Derivation of pluripotent stem cells from cultured human primordial germ cells.Proc.Natl.Acad.Sci.USA.1998;95:13726-13731
[5]Thomas KR and Capecchi MR.Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.Cell 1987;51:503-512
[6]Alvarez-Dolado M,Pardal R,Garcia-Vardugo JM,et al.Fusion of bone-marrow-derived cells with Purkinje neurons,cardiomyoctys.Nature 2003;425:968-973
[7]Wilmut I,Schnieke AE,McWhir J,et al.Viable offspring derived from fetal and adult mammalian cells.Nature 1997;385:810-813
[8] Amit M, Carpenter MK, Inokuma MS, et al. Clonally derived human embryonic stem cells lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 2000;227:271-278
[9] Gearhart JD and Mintz B. Contact-mediated myogenesis and increased acetycholinesterase activity in primary cultures of mouse teratocarcinoma cells. Proc. Natl. Acad. Sci. USA 1974;71:1734-1738
[10]Resnick JL, Bixler LS, Cheng L, et al. Long-term proliferation of mouse primordial germ cells in culture. Nature 1992;359:550-551
[11]Nichols J, Zevnik B, Anastassiadis K, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95: 379-391
[12]Pesce M, Anastassiadis K, and Scholer HR. Oct-4: lessons of totipotency from embryonic stem cells. Cells Tissues Organs 1999;165:144-152
[13]Anna M, Wobus D and Kenneth RB. Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy. Physiol. Rev. 2005;85:635-678
[14] Chambers I, Colby D, Robertson M, et al. Functional expression cloning of nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643 -655
[15] Williams RL,Hilton DJ, Pease S, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 1988;336:684-687
[16]Niwa H, Burdon T, Chambers I, et al. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 1998;12:2048-2060
[17]Boeuf H, Hauss C, Graeve FD, et al. Leukemia inhibitory factor- dependent transcriptional activation in embryonic stem cells. J. Cell Biol. 1997;138:1207-1217
[18]Ying QL, Nichols J, Chambers I, et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281 -292
[19]Brandenberger R, Wei H, Zhang S, et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat. Biotechnol. 2004;22:707-716
[20] Abeyta MJ, Clark AT, Rodriguez RT, et al. Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum. Mol. Genet. 2004; 13:601-608
[21] Richards M, Tan SP, Tan JH, et al. The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 2004;22:51-64
[22] Itskovitz-Eldor J, Schuldiner M, Karsenti D, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 2000;6:88-95
[23] Schuldiner M, Yanuka O, Itskovitz-Eldor J, et al. From the cover: effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA2000;97: 11307-11312
[24]Bagutti C, Wobus AM, Fassler R, et al. Differentiation of embryonal stem cells into keratinocytes: comparison of wildtype and beta 1 integrin-deficient cells.Dev.Biol. 1996;179:184-196
[25]Coraux C, Hilmi C, Rouleau M, et al. Reconstituted skin from murine embryonic stem cells. Curr. Biol. 2003;13:849-853
[26]Fraichard A, Chassande O, Bilbaut G, et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J. Cell Sci. 1995;108:3181-3188
[27]Rolletschek A, Chang H, Guan K, et al. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors.Mech.Dev. 2001;105:93-104
[28] Carpenter MK, Inokuma MS, Denham J, et al. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp. Neurol. 2001;172:383-397
[29]Nakano T, Kodama H, and Honjo T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 1994;265:1098-1101
[30]Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 2001;108:407-414
[31]Reppel M, Boettinger C, and Hescheler J. Beta-adrenergic and muscarinic modulation of human embryonic stem cell-derived cardio-myocytes. Cell Physiol.Biochem. 2004; 14:187-196
[32]Dani C, Smith AG, Dessolin S, et al. Differentiation of embryonic stem cells into adipocytes in vitro. J. Cell Sci. 1997;110: 1279-1285
[33] Kramer J, Hegert C, Guan K, et al. Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech. Dev. 2000;92:193-205
[34]Rohwedel J, Maltsev V, Bober E, et al. Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev.Biol. 1994;164: 87-101
[35]Chinzei R, Tanaka Y, Shimizu-Saito K, et al. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes.Hepatology 2002;36:22-29
[36]Leon-Quinto T, Jones J, Skoudy A, et al. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia 2004;47:1442-1451
[37]Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulinproducing cells.Proc. Natl. Acad. Sci. USA 2003;100:998-1003
[38]Hubner K, Fuhrmann G, Christenson LK, et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003;300: 1251 - 1256
[39]Toyooka Y, Tsunekawa N, Akasu R, et al. Embryonic stem cells can form germ cells in vitro. Proc. Natl. Acad. Sci. USA2003;100:11457-11462
[40]Geijsen N, Horoschak M, Kim K, et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2003;427:148-154
[41]Cecconi F, Meyer BI. Gene trap: a way to identify novel genes and unravel their biological function. FEBS 2000;480:63-71
[42]Blobel GA, Simon MC, and Orkin SH. Rescue of GATA-1-deficient embryonic stem cells by heterologous GATA-binding proteins. Mol. Cell. Biol. 1995;15:626-633
[43]Perrier AL, Tabar V, Barberi T, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2004;101:12543-12548
[44]Segev H, Fishman B, Ziskind A, et al. Differentiation of human embryonic stem cells into insulin-producing clusters.Stem Cells 2004;22:265-274
[45]Klug MG, Soonpaa MH, Koh GY, et al. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin.Invest. 1996;98:216-224
[46] Levenberg S, Huang NF, Lavik E, et al. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc. Natl. Acad. Sci. USA 2003; 100:12741-12746
[47]Davila JC, Cezar GG, Thiede M, et al. Use and application of stem cells in toxicology. Toxicol. Sci. 2004;79:214-223
[48]Wobus AM, Wallukat G, and Hescheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2- channel blockers.Differentiation 1991;48: 173-182
[49]Kim JH, Auerbach JM, Rodriguez-Gomez JA, et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease.Nature 2002;418: 50-56
[50] Richards M, Fong CY, Chan WK, et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat. Biotechnol. 2002;20:933-936
[51] Martin JM, Muotri A, Gage F, et al. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 2005;112:28-232
[52]Xu C, Inokuma MS, Denham J, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 2001;19:971-974
[53] Richards M, Tan S, Fong CY, et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells.Stem Cells 2003;21:546-556
[54]Pruszuk J, Sonntag KC, Aung MH, et al. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cell Express,published online 2007; dol:10,1634/stem cells.2006-0744
[55]Drukker M, Katz G, Urbach A, et al. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc. Natl. Acad. Sci. USA 2002;99:9864-9869
[56]Zwaka TP and Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003;3: 319-321
[57] Lanza RP, Cibelli JB, and West MD. Human therapeutic cloning. Nat. Med.1999;5: 975-977
[58] Chen Y, He ZX, Liu A, et al. Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes. Cell Res. 2003;13:251-63
[1]Jan Tesarik,Carmen Mendoza,and Ermanno Greco.Paternal effects acting during the first cell cycle of human preimplantation development after ICSI.Hum.Reprod.2002;17:184-189
[2]陈大元.受精生物学—受精机制与生殖工程.科学出版社.2000;北京
[3]Palermo G.,Joris H,Devroey P,et al.Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte.Lancet 1992:340:17-18
[4]Balakier H.Fertilization and early embryology Tripronuclear human zygotes:the first cell cycle and subsequent development.Hum.Reprod.1993;8:1892-1897
[5]Feng H and Hershlag A.Fertilization Abnormalities Following Human In Vitro Fertilization and Intracytoplasmic Sperm Injection.Microsc.Res.Yechniq.2003;61:358-361
[6]Yu SL,Lee RKK,Su JT,et al.Distinction between paternal and maternal contributions to the tripronucleus in human zygotes obtained after in vitro fertilization.Taiwanese J.Obstet.Gynecol.2006;45:313-316
[7]Grossmann M,Calafell JM,Brandy N,et al.Origin of tripronucleate zygotes after intracytoplasmic sperm injection.Hum.Reprod.1997;12:2762-2765
[8]Egozcue S,Blanco J,Vidal F,et al.Diploid sperm and the origin of triploidy.Hum.Reprod.2002;17:5-7
[9]Funaki K,Mikamo K.Giant diploid oocytes as a cause of digynic triploidy in mammals.Cytogenet.Cell Genet.1980;28:158-68
[10]Lubna P,Toth TL,Leykin L,et al.High incidence of triploidy in in-vitro fertilized oocytes from a patient with a previous history of recurrent gestational trophoblastic disease.Hum.Reprod.1996;11:1529-1532
[11] Egli D, Rosains J, Birkhoff G, et al. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature 2007;447:679-686
[12]Yie SM, Collins JA, Daya S, et al. Polyploidy and failed fertilization in in-vitro fertilization are related to patient's age and gamete quality. Hum. Reprod.1996;11:614-617
[13] Wolf DP, Byrd W, Dandekar P, et al. Sperm concentration and the fertilization of human eggs in vitro. Biol. Reprod. 1984;31:837-848
[14] Lowe B, Osborn JC, Fothergill DJ, et al. Factors associated with accidental fractures of the zona pellucida and multipronuclear human oocytes following in-vitro fertilization. Hum. Reprod. 1988;3(7):901-904
[15]Pieters M, Dumoulin JCM, Ignoul-Vanvuchelen RCM, et al. Triploidy after in vitro fertilization: cytogenetic analysis of human zygotes and embryos. J. Assist.Reprod. Gen. 1992;9:68-76
[16]Rosenbusch B, Schneider M and Sterzik K. Chromosomal analysis of mutipronuclear zygotes obtained after partial zona dissection of the oocytes. Mol.Hum. Reprod. 1998;4:1065-1070
[17]Rosenbusch B, Schneider M and Sterzik K. The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization. Hum. Reprod.1997;12:2257-2262
[18]Rosenbusch BE, Schneider M, and Hanf V. Tetraploidy and partial endoreduplication in a tripronuclear zygote obtained after intracytoplasmic sperm injection. Fertil. Steril. 1998; 69:344-346
[19]Rosenbusch B, Schneider M and Sterzik K. Triploidy caused by endoreduplication in a human zygote obtained after in-vitro fertilization. Hum.Reprod. 1997;12:1059-1061
[20]Staessen C and Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum. Reprod. 1997;12:321-327
[21] Palermo SM, Colombero LT, Cohen J, et al. Genetics of abnormal human fertilization. Hum. Reprod. 1995;10 (Suppl 1):120-127
[22]Macas E, Imthurn B, Roselli M, et al. Chromosome analysis of single- and multipronucleated human zygotes proceeded after the intracytoplasmic sperm injection procedure. J. Assist. Reprod. Gen. 1996;13:345-350
[23]Macas E, Zweife C, and Imthurn B. Numerical chromosome anomalies detected in paternally derived pronuclei of tripronuclear zygotes after intracytoplasmic sperm injection. Fertil. Steril. 2006;85:1753-1760
[24]Macas E, Imthurn B, Rosselli M, et al. The chromosomal complements of multipronuclear human zygotes resulting from intracytoplasmic sperm injection.Hum.Reprod. 1996;11:2496-2501
[25] Yan J, Chen Z, Feng HL. Aneuploid analysis of tripronuclear zygotes derived from in vitro fertilization and intracytoplasmic sperm injection in humans. Fertil.Steril. 2005;83:1485-1488
[26]Rosenbusch B, Schneider M, Kreienberg R, et al. Cytogenetic analysis of human zygotes displaying three pronuclei and on polar body after intracyoplasmic sperm injection. Hum. Reprod. 2001;16:2362~2367
[27]颜军昊,陈子江,李媛,胡美京.体外受精后多元核受精卵的移植价值.生殖与避孕. 2005;25:537-541
[28]Munne S, Sandalinas M, Escudero T, et al. Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect. Reprod.Biomed. Online. 2002;4:223-232
[29]Hinchcliffe EH and Sluder G. "It Takes Two to Tango": understanding how centrosome duplication is regulated throughout the cell cycle Genes & Development 2001;15:1167-1181
[30]Andrew W. Murray. Recycling the cell cycle: cyclins revisited. Cell 2004;116:221 -234
[31]Matsumoto Y and Mailer JL. A centrosomal localization signal in cyclin Erequired for Cdk2-independent S phase entry. Science 2004;306:885-888
[32] Edward H. Hinchcliffe, Miller FJ, et al. Requirement of a centrosomal activity for cell cycle progression through G1 into S phase. Science 2001;291:1547-1550
[33]Schetten G. The centrosome and its mode of inheritance the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev.Biol. 1994;165:299-335
[34]Simerly C, Wu GJ, Zoran S, et al. The paternal inheritance of the centrosome the cells microtubule-organizing center in humans and the implications for infertility.Nat.Med. 1995;1(1):47-52
[35]Simerly C, Zoran SS, Payne C, et al. Biparental inheritance of y-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm. Mol. Biol. Cell. 1999;10:2955-2969
[36]Rawe VY, Terada Y, Nakamura S, et al. A pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage. Hum.Reprod. 2002;17:2344-2349
[37]Sathanathan AH, Kola I, Osborne J, et al. Centrioles in the beginning of human development. Proc. Natl. Acad. Sci. USA 1991;88:4806-4810
[38] Terada Y, Simerly CR, Hewitson L, et al. Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm. Biol. Reprod. 2000;62:557-563
[39]Nakamura S, Terada Y, Horiuchi T, et al. Human Sperm Aster Formation and Pronuclear Decondensation in Bovine Eggs Following Intracytoplasmic Sperm Injection Using a Piezo-Driven Pipette: A Novel Assay for Human Sperm Centrosomal Function. Biol. Reprod. 2001;65:1359-1363
[40] Asch R, Simerly C, Ord T, et al. The stages at which human fertilization arrests:microtubule and chromosome configurations in inseminated oocytes which failed to complete fertilization and development in humans. Mol. Hum. Reprod.1995;1:239-248
[41] Kola I, Trounson A, Dawson G, et al. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotype analysis of embryos. Biol. Reprod.1987;37:395-401
[42]Plachot M, Veiga A, Montagut J, et al. Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multi-centric study. Hum.Reprod. 1988;3:627-635
[43]Chatzimeletiou K, Morrison EE, Prapas N, et al. Spindle abnormalities in normally developing and arrested human preimplantation embryos in vitro identified by confocal laser scanning microscopy. Hum. Reprod. 2005;20:672-682
[44]Briggs R and King TJ. Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. Proc. Natl. Acad. Sci. USA 1952;38:455-463
[45]Illmensee K, Hoppe PC. Nuclear transplantation in mouse musculus:developmental potential of nuclei from preimplantation embryos. Cell 1981;23:9-16
[46]McGrath JS, Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 1983,220:1300-1302
[47] Campbell KH, Mc Whir J, Ritchie WA, et al. Sheep cloned by nuclear transfer from a cultured cell line. Nature 1996;380:64-67
[48] Chan A, Dominko T, Luetjens CM, et al. Clonal Propagation of Primate Offspring by Embryo Splitting. Science 2000:287:317-319
[49]Rawlins RG, Binor Z, Radwanska E, et al. Microsurgical enucleation of tripronuclear human zygotes. Fertil. Steril.1988;50:266-272
[50]Modlinski JA. Haploid mouse embryos obtained by microsurgical removal of one pronucleus. J. Embryol. Exp. Morphol.1975;33:897-905
[51]Kimura Y and Yanagimachi R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring.Development 1995;121:2397-2405
[52]Kimura Y and Yanagimachi R. Intracytoplasmic sperm injection in the mouse.Biology of Reproduction 1995;52:709-720
[53] Malter HE and Cohen J. Embryonic development after microsurgical repair of polyspermic human zygotes. Fertil. Steril. 1989;52:373-380
[54]Wiker S, Miter W, Wright G, et al. Recognition of Paternal Pronuclei in Human Zygotes. Journal of in Vitro Fertilization and Embryo Transfer 1990;7:33-37
[55] Gordon JW, Grunfeld L, Garissi GJ, et al. Successful microsurgical removal of a pronucleus from tripronuclear human zygotes. Fertil. Steril. 1989;52:367-372
[56] Palermo G, MunneA S and Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum. Reprod. 1994;9:1220—1225
[57]Ivakhnenko V, Cieslak J and Verlinsky Y. A microsurgical technique for enucleation of multipronuclear human zygotes. Hum. Reprod. 2000;15:911-916
[58]Katteral S and Chen C. Normal birth after microsurgical enucleation of tripronuclear human zygotes: Case report. Hum. Reprod. 2003;18:1319-1322
[59]Escriba MJ, Martin J, Rubio C, et al. Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos. Fertil. Steril.2006;86:1601-1607