维持人胚胎干细胞不分化的鼠饲养层细胞标准化操作的建立与残留的丝裂霉素C对人胚胎干细胞基因组稳定性影响的研究
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摘要
人胚胎干细胞(human embryonic stem cells,hES cells)是一种从人类早期胚胎的内细胞团中分离出来的多能性干细胞。一方面,它能在体外特定的环境下保持不分化的状态并且无限扩增。另一方面,在特殊的诱导条件下,它又能分化成特定的组织细胞,如神经细胞,胰岛素分泌细胞,心肌细胞和造血细胞等,成为神经退行性疾病,糖尿病,心肌梗塞和血液疾病等疑难疾病的细胞替代治疗的重要细胞来源。
上世纪60年代开始到90年代末,小鼠,灵长类等哺乳动物的胚胎干细胞陆续建系成功。在此基础上,1998年,美国Thomson等建立了第一个人胚胎干细胞系,于是,研究开发这种具有巨大临床应用前景的生物资源,立即成为生物医学界最具吸引力的热门领域。但是,由于人胚胎干细胞取自人类早期发育胚胎,因此受到伦理道德方面的质疑,研究也一度受到限制,发展受到制约。2009年初,美国食品药品监督管理部门(Food and drug administration,FDA)批准了第一例利用人胚胎干细胞治疗脊髓损伤的临床治疗试验,随着这一临床治疗试验的获批,人胚胎干细胞的研究将又一次成为生物医学领域新的热潮,而相应的伦理规范也将进一步完善,为胚胎干细胞的研究提供依据与支持。
本课题组所在的研究所从2001年开始人胚胎干细胞的建系和培养工作,并于2002年成功建立人胚胎干细胞系。但是在课题研究之初,维持人胚胎干细胞体外培养的环境是很不稳定的,出现大量分化,甚至大量死亡的现象。人胚胎干细胞体外培养的体系较其它细胞严苛和复杂,需要事先制备一层有丝分裂灭活的饲养层细胞作为支持,然后才能进行传代。传代后的培养需每天更换添加了各种因子的培养基,直到六天后下一个传代日的到来再重复上述过程。一般来说,这一层有丝分裂灭活的饲养层细胞是鼠胚胎成纤维细胞,它取材比较方便,不存在伦理学争议,但是操作步骤繁杂,而且这种细胞分裂次数有限,每个批次的细胞用完都必需重新制备。本课题的研究就是从标准化鼠源性饲养层细胞的制备入手,分析造成人胚胎干细胞体外培养环境不稳定的因素,探讨人胚胎干细胞体外培养的安全性问题,为人胚胎干细胞临床应用提供安全稳定的种子细胞。本文共包括两大部分:
第一部分:标准化鼠胚胎成纤维细胞(mouse embryonic fibroblasts,mEFs)作为饲养层细胞(feeder cells)的操作程序。利用Microsoft office中的Access软件,建立小型管理数据库。
第二部分:探讨丝裂霉素C对人胚胎干细胞体外培养的毒性影响。试图揭示人胚胎干细胞体外核型变化以及DNA损伤修复应答缺陷的机制。
Human embryonic stem cells(hES cells) are pluripotent cells derived from inner cell mass of blastocyst at early stage of human embryo. On one hand,hES cells can maintain undifferentiated state under certain circumstance.On the other hand,it can be induced into large amounts of specific somatic cells including neural progenitor cells,cardiomyocytes, blood cells and insulin secreting cells in vitro for transplantation and to treat diseases such as neuronal degenerative disorders,cardiac infarcts, blood diseases and diabetes.Stem cell therapy contains the potential to revolutionize traditional medicine.
From 1960s to the second half of 1990s,mouse embryonic stem cells(mES cells) and Primate embryonic stem cells(pES cells) was established successfully.On the base of researches on this,Thomoson and his colleagues reported the first derivation of human embryonic stem cell (hES cell) line in 1998.Since then,it had become one of the hottest and most attractive fields in biomedicine to exploit this human being biological resource with great prospect for widespread clinical applications.
However,hES cells were derived form blastocyst at early stage of human embryo,there were a lot of debates on its ethics and brought restriction on its researches and developments.At the beginning of 2009, US Food and drug administration(FDA) approved first human trials of hES cell research on spinal cord injury.Along with the approval of first human trial,researches on hES cells will be one of the hottest fields in biomedicine again,as well as the criterion will be established to support the researches.
Our institute initiated the work of establishment and culturing of hES cells in 2001,and successfully established new lines in 2002.But at the beginning of hES cell research,the environment of culturing hES cells was instable,cells differentiated largely,some times even died. Routine culturing of hES cells is rigorous and complex than other cells. The culturing system consisted of a mitomitcally inactivated feeder cell layer and hES cell medium supplemented with kinds of factors.Firstly, we should prepare the feeder cell layer.Secondly,change feeder cell medium into hES cell medium after feeder cells attached to the dish. Finally,passage hES cell clones by cutting them into small pieces and change hES cell medium every day till next passage day.
Usually,the feeder cell layer is made of mouse embryonic fibroblasts(mEFs).It is easily to get and no ethic problem.But the preparation process is complex,and the number of cell division was limited,so we need derive new batches after ran out of old ones.This shudy based on variables during derivation of mEFs,preparation of mouse feeder cells and the environment ourside hES cells,and focus on the potential safety risk of hES culturing in vitro that may effect the clinical application in order to offer stable and safety hES cells.The thesis included 2 parts as follows:
1.Standardize the preparation process of mEF as feeder cells, established database for the administration of routine use of feeder cell by using Access in Microsoft Office.
2.Discuss the toxicity of mitomycin C(MMC) on the culturing of hES cells in vitro and try to explain the mechanisms underlying karyontype changes in hES cells and DNA damage response defect of hES cells.
上世纪60年代开始到90年代末,小鼠,灵长类等哺乳动物的胚胎干细胞陆续建系成功。在此基础上,1998年,美国Thomson等建立了第一个人胚胎干细胞系,于是,研究开发这种具有巨大临床应用前景的生物资源,立即成为生物医学界最具吸引力的热门领域。但是,由于人胚胎干细胞取自人类早期发育胚胎,因此受到伦理道德方面的质疑,研究也一度受到限制,发展受到制约。2009年初,美国食品药品监督管理部门(Food and drug administration,FDA)批准了第一例利用人胚胎干细胞治疗脊髓损伤的临床治疗试验,随着这一临床治疗试验的获批,人胚胎干细胞的研究将又一次成为生物医学领域新的热潮,而相应的伦理规范也将进一步完善,为胚胎干细胞的研究提供依据与支持。
本课题组所在的研究所从2001年开始人胚胎干细胞的建系和培养工作,并于2002年成功建立人胚胎干细胞系。但是在课题研究之初,维持人胚胎干细胞体外培养的环境是很不稳定的,出现大量分化,甚至大量死亡的现象。人胚胎干细胞体外培养的体系较其它细胞严苛和复杂,需要事先制备一层有丝分裂灭活的饲养层细胞作为支持,然后才能进行传代。传代后的培养需每天更换添加了各种因子的培养基,直到六天后下一个传代日的到来再重复上述过程。一般来说,这一层有丝分裂灭活的饲养层细胞是鼠胚胎成纤维细胞,它取材比较方便,不存在伦理学争议,但是操作步骤繁杂,而且这种细胞分裂次数有限,每个批次的细胞用完都必需重新制备。本课题的研究就是从标准化鼠源性饲养层细胞的制备入手,分析造成人胚胎干细胞体外培养环境不稳定的因素,探讨人胚胎干细胞体外培养的安全性问题,为人胚胎干细胞临床应用提供安全稳定的种子细胞。本文共包括两大部分:
第一部分:标准化鼠胚胎成纤维细胞(mouse embryonic fibroblasts,mEFs)作为饲养层细胞(feeder cells)的操作程序。利用Microsoft office中的Access软件,建立小型管理数据库。
第二部分:探讨丝裂霉素C对人胚胎干细胞体外培养的毒性影响。试图揭示人胚胎干细胞体外核型变化以及DNA损伤修复应答缺陷的机制。
Human embryonic stem cells(hES cells) are pluripotent cells derived from inner cell mass of blastocyst at early stage of human embryo. On one hand,hES cells can maintain undifferentiated state under certain circumstance.On the other hand,it can be induced into large amounts of specific somatic cells including neural progenitor cells,cardiomyocytes, blood cells and insulin secreting cells in vitro for transplantation and to treat diseases such as neuronal degenerative disorders,cardiac infarcts, blood diseases and diabetes.Stem cell therapy contains the potential to revolutionize traditional medicine.
From 1960s to the second half of 1990s,mouse embryonic stem cells(mES cells) and Primate embryonic stem cells(pES cells) was established successfully.On the base of researches on this,Thomoson and his colleagues reported the first derivation of human embryonic stem cell (hES cell) line in 1998.Since then,it had become one of the hottest and most attractive fields in biomedicine to exploit this human being biological resource with great prospect for widespread clinical applications.
However,hES cells were derived form blastocyst at early stage of human embryo,there were a lot of debates on its ethics and brought restriction on its researches and developments.At the beginning of 2009, US Food and drug administration(FDA) approved first human trials of hES cell research on spinal cord injury.Along with the approval of first human trial,researches on hES cells will be one of the hottest fields in biomedicine again,as well as the criterion will be established to support the researches.
Our institute initiated the work of establishment and culturing of hES cells in 2001,and successfully established new lines in 2002.But at the beginning of hES cell research,the environment of culturing hES cells was instable,cells differentiated largely,some times even died. Routine culturing of hES cells is rigorous and complex than other cells. The culturing system consisted of a mitomitcally inactivated feeder cell layer and hES cell medium supplemented with kinds of factors.Firstly, we should prepare the feeder cell layer.Secondly,change feeder cell medium into hES cell medium after feeder cells attached to the dish. Finally,passage hES cell clones by cutting them into small pieces and change hES cell medium every day till next passage day.
Usually,the feeder cell layer is made of mouse embryonic fibroblasts(mEFs).It is easily to get and no ethic problem.But the preparation process is complex,and the number of cell division was limited,so we need derive new batches after ran out of old ones.This shudy based on variables during derivation of mEFs,preparation of mouse feeder cells and the environment ourside hES cells,and focus on the potential safety risk of hES culturing in vitro that may effect the clinical application in order to offer stable and safety hES cells.The thesis included 2 parts as follows:
1.Standardize the preparation process of mEF as feeder cells, established database for the administration of routine use of feeder cell by using Access in Microsoft Office.
2.Discuss the toxicity of mitomycin C(MMC) on the culturing of hES cells in vitro and try to explain the mechanisms underlying karyontype changes in hES cells and DNA damage response defect of hES cells.
引文
Baker DEC,Harrison NJ,Maltby E et al.Adaptation to culture of human embryonic stem cells and oncogenesis in vivo.Nat.biotechnol(2007) 25,207-215.
Buzzard JJ,Gough NM,Crook JM et al.Karyotype of human ES cells during extended culture.Nat.biotechnol.(2004) 22,381-382.
Cobo F,Stacey GN,Hunt C et al.Microbiological control in stem cell banks:approaches to standardisation.Appl Microbiol Biotechnol(2005) 68,456-466
Cowan CA,Klimanskaya I,McMahon J et al.Derivation of embryonic stem-cell lines from human blastocysts.N.Engl.J.Med.(2004) 350,1353-1356.
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
Evans MJ and Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature(1981) 292,154-156.
Frankfurt OS and Krishan A.Enzyme-linked immunosorbent assay(ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening.J.Immunol.Methods(2001) 253,133-144.
Hata T,Hoshi T,Kanamori K et al.Mitomycin,a new antibiotic from Stretomyces.Antibiot.Ser.A.(1956) 9,141-146.
Heng BC,Cao T,Liu H et al.Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells.Cell Biochem Funct.(2005) 23,141-146.
Heng BC,Liu H,Cao T.Feeder cell density-a key parameter in human embryonic stem cell culture.In Vitro Cell,Dev.Biol.—Animal(2004) 40,255-257.
Hoffman LM and Carpenter MK.Characterization and culture of human embryonic stem cells.Nat.biotechnoL(2005) 23,699-708.
Jeffreys AJ,Wilson V,Thein SL.Hypervariable 'minisatellite' regions in human DNA.Nature(1985) 314,67-73.
Klein CA,and Schmidt-Kittler O.Comparative genomic hybridization,loss of heterozygosity,and DNA sequence analysis of single cells.Proc.Natl.Acad.Sci.USA(1999) 96,4497-4452.
Lobrich M and Jeggo PA.The impact of a negligent G2/M checkpoint on genomic instability and cancer induction.Nat.Rev.Cancer.(2007)7,861-869.
Maitra A,Arking DE,Shivapurkar N et.al.Genomic alterations in cultured human embryonic stem cells.Nature Genetics(2005)37,1099-1103.
Martin GR,Evans MJ.Differentiation of Clonal Lines of Teratocarcinoma Cells:Formation of Embryoid Bodies in Vitro.Proc.Natl Acad.Sci.USA(1975) 72,1441-1445.
Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc.Natl.Acad.Sci.USA(1981)78,7634-7638.
Mitalipova MM,Rao RR,Hover DM et al.Preserving the genetic integrity of human embryonic stem cells.Nat.biotecthnol.(2005)23,19-20.
Nakamura Y,Leppert M,O'Connell P et al.Variable number of tandem repeat(VNTR)markers for human gene mapping.Science.(1987)235,1616-1622.
Nozal MJ,Bernal JL,Martin MT et al.LC-ESI-MSD fast determination of residual mitomycin C in hen aqueous humour after corneal refractive surgery.J.Pharm.Biomed.Anal(2006)40,100-104.
Puck TT and Marcus PI.A rapid method for viable cell titration and clone production with Hela cells in tissue culture:the use of X-ittadiated cells to supply conditioning factors.Proc Natl Acad Sci.USA(1955)41,432-437.
Reubinoff B,Pera MF,Fong CY et al.Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro.Nat.Biotechnol.(2000) 15,399-404.
Richards M,Fong Chin Yee,Woon Khiong Chan et al.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat.biotechnol.(2002) 20,933-936
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.
Robertson,E.J.(1987).Embryo-derived stem cell lines.Teratocarcinoma and embryonic stem cells-a practical approach.(Washington DC:IRL Press).
Prudhomme M,Attaiech L,Sanchez Get al.Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae.Science 2006;313,89-92
Thomson JA,Itskovitz-Eldor J,Shapiro SS et al.Embryonic stem cell lines derived from human blastocysts.Science(1998) 282,1145-1147
Tomasz M,and Palom Y.The mitomysin bioreductive antitumor agent:cross linking and alkylation of DNA as the molecular basis of their activity.Pharmacol.Ther.(1997)76,73-78.
Xie CQ,Lin G,Luo KL et al.Newly expressed proteins of mouse embryonic fibroblast irradiated to be inactive.Biochem.Biophys.Res.Commun.(2004) 315,581-588.
Xu CH,Inokuma MS,Denham J et al.Feeder-free growth of undifferentiated human embryonic stem cells.Nat.biotechnol.(2001) 19,971-974
Yang S,Lin G,Tan YQ et al.Tumor progression of culture-adapted human embryonic stem cells during long-term culture.Genes Chromosomes Cancer.(2008).47,665-679.
Yao SY,Chen S,Clark J et al.Long-term self-renewal and directed differentiation of
human embryonic stem cells in chemically defined conditions.Proc.Natl.Acad.Sci.USA.(2006) 103,6906-6912.
Zheng HY,Wang X,Warren AJ,et al.Nucleotide excision repair- and polymerase η-mediated error-prone removal of mitomycin-C interstrand cross-links.Mol.Cell.Biol.(2003) 23,754-761.
Zhou D and Lu GX.Mitomycin C cause chromosome change of human embryonic stem cells.Cell Research.(2008) 18s,124
Zhou J,Ou-Yang Q,Li J,et al.Human Feeder Ceils Support Establishment and Definitive Endoderm Differentiation of Human Embryonic Stem Cells.Stem Cells Dev.(2008) 17,737-750.
黄念君.染色体畸变试验中CHL细胞系/剂量法则的应用。遗传.(1984)6,26-28
Buzzard JJ,Gough NM,Crook JM et al.Karyotype of human ES cells during extended culture.Nat.biotechnol.(2004) 22,381-382.
Cobo F,Stacey GN,Hunt C et al.Microbiological control in stem cell banks:approaches to standardisation.Appl Microbiol Biotechnol(2005) 68,456-466
Cowan CA,Klimanskaya I,McMahon J et al.Derivation of embryonic stem-cell lines from human blastocysts.N.Engl.J.Med.(2004) 350,1353-1356.
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
Evans MJ and Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature(1981) 292,154-156.
Frankfurt OS and Krishan A.Enzyme-linked immunosorbent assay(ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening.J.Immunol.Methods(2001) 253,133-144.
Hata T,Hoshi T,Kanamori K et al.Mitomycin,a new antibiotic from Stretomyces.Antibiot.Ser.A.(1956) 9,141-146.
Heng BC,Cao T,Liu H et al.Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells.Cell Biochem Funct.(2005) 23,141-146.
Heng BC,Liu H,Cao T.Feeder cell density-a key parameter in human embryonic stem cell culture.In Vitro Cell,Dev.Biol.—Animal(2004) 40,255-257.
Hoffman LM and Carpenter MK.Characterization and culture of human embryonic stem cells.Nat.biotechnoL(2005) 23,699-708.
Jeffreys AJ,Wilson V,Thein SL.Hypervariable 'minisatellite' regions in human DNA.Nature(1985) 314,67-73.
Klein CA,and Schmidt-Kittler O.Comparative genomic hybridization,loss of heterozygosity,and DNA sequence analysis of single cells.Proc.Natl.Acad.Sci.USA(1999) 96,4497-4452.
Lobrich M and Jeggo PA.The impact of a negligent G2/M checkpoint on genomic instability and cancer induction.Nat.Rev.Cancer.(2007)7,861-869.
Maitra A,Arking DE,Shivapurkar N et.al.Genomic alterations in cultured human embryonic stem cells.Nature Genetics(2005)37,1099-1103.
Martin GR,Evans MJ.Differentiation of Clonal Lines of Teratocarcinoma Cells:Formation of Embryoid Bodies in Vitro.Proc.Natl Acad.Sci.USA(1975) 72,1441-1445.
Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc.Natl.Acad.Sci.USA(1981)78,7634-7638.
Mitalipova MM,Rao RR,Hover DM et al.Preserving the genetic integrity of human embryonic stem cells.Nat.biotecthnol.(2005)23,19-20.
Nakamura Y,Leppert M,O'Connell P et al.Variable number of tandem repeat(VNTR)markers for human gene mapping.Science.(1987)235,1616-1622.
Nozal MJ,Bernal JL,Martin MT et al.LC-ESI-MSD fast determination of residual mitomycin C in hen aqueous humour after corneal refractive surgery.J.Pharm.Biomed.Anal(2006)40,100-104.
Puck TT and Marcus PI.A rapid method for viable cell titration and clone production with Hela cells in tissue culture:the use of X-ittadiated cells to supply conditioning factors.Proc Natl Acad Sci.USA(1955)41,432-437.
Reubinoff B,Pera MF,Fong CY et al.Embryonic stem cell lines from human blastocysts:somatic differentiation in vitro.Nat.Biotechnol.(2000) 15,399-404.
Richards M,Fong Chin Yee,Woon Khiong Chan et al.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.Nat.biotechnol.(2002) 20,933-936
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.
Robertson,E.J.(1987).Embryo-derived stem cell lines.Teratocarcinoma and embryonic stem cells-a practical approach.(Washington DC:IRL Press).
Prudhomme M,Attaiech L,Sanchez Get al.Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae.Science 2006;313,89-92
Thomson JA,Itskovitz-Eldor J,Shapiro SS et al.Embryonic stem cell lines derived from human blastocysts.Science(1998) 282,1145-1147
Tomasz M,and Palom Y.The mitomysin bioreductive antitumor agent:cross linking and alkylation of DNA as the molecular basis of their activity.Pharmacol.Ther.(1997)76,73-78.
Xie CQ,Lin G,Luo KL et al.Newly expressed proteins of mouse embryonic fibroblast irradiated to be inactive.Biochem.Biophys.Res.Commun.(2004) 315,581-588.
Xu CH,Inokuma MS,Denham J et al.Feeder-free growth of undifferentiated human embryonic stem cells.Nat.biotechnol.(2001) 19,971-974
Yang S,Lin G,Tan YQ et al.Tumor progression of culture-adapted human embryonic stem cells during long-term culture.Genes Chromosomes Cancer.(2008).47,665-679.
Yao SY,Chen S,Clark J et al.Long-term self-renewal and directed differentiation of
human embryonic stem cells in chemically defined conditions.Proc.Natl.Acad.Sci.USA.(2006) 103,6906-6912.
Zheng HY,Wang X,Warren AJ,et al.Nucleotide excision repair- and polymerase η-mediated error-prone removal of mitomycin-C interstrand cross-links.Mol.Cell.Biol.(2003) 23,754-761.
Zhou D and Lu GX.Mitomycin C cause chromosome change of human embryonic stem cells.Cell Research.(2008) 18s,124
Zhou J,Ou-Yang Q,Li J,et al.Human Feeder Ceils Support Establishment and Definitive Endoderm Differentiation of Human Embryonic Stem Cells.Stem Cells Dev.(2008) 17,737-750.
黄念君.染色体畸变试验中CHL细胞系/剂量法则的应用。遗传.(1984)6,26-28
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