人胚胎干细胞系与人孤雌胚胎干细胞系建立的研究
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摘要
目的胚胎干细胞是一类来源于哺乳动物原始生殖细胞和囊胚内细胞团的全能细胞,主要具有两方面显著特征：一是具有体外高度增殖和自我更新的能力,二是可被定向诱导分化成体内各种细胞类型。孤雌激活来源的人胚胎干细胞的基因型与卵子提供者的基因型完全相同,由其诱导分化的细胞用于机体移植完全不会引起免疫排斥。因此,人胚胎干细胞尤其是人孤雌胚胎干细胞具有巨大的医学研究价值与应用前景,如再生医学、人类发育生物学和细胞治疗等。本实验旨在建立稳定的、标准化的人胚胎干细胞体外培养方法,并且在保证人胚胎干细胞长期稳定传代培养的基础上,比较不同饲养层对维持人胚胎干细胞生长的作用影响,从而优化人胚胎干细胞的培养条件。与此同时,对培养体系进行改良,利用废弃卵母细胞获得的孤雌囊胚在改良的完全人源性培养条件下建立最接近临床标准的人孤雌胚胎干细胞系,为临床级人孤雌胚胎干细胞研究与应用奠定基础。
对象与方法本实验收集天津市中心妇产科医院生殖医学中心试管婴儿治疗患者的不成熟卵母细胞和移植后剩余无冻存价值的废弃胚胎,征得患者夫妇双方同意并签署知情同意书后用于本实验。应用人包皮成纤维细胞作为饲养层,85%KnockOut DMEM、15%KnockOut血清替代物、4ng/mlbFGF培养基,建立人胚胎干细胞系,并且对其特异性标志物SSEA-3、SSEA-4、SSEA-1、TRA-1-60、 TRA-1-81、转录因子Oct-4,碱性磷酸酶,体内或体外分化能力作全面的鉴定。同时,对小鼠胚胎成纤维细胞、人包皮成纤维细胞、人胚胎肺组织成纤维细胞三种饲养层的形态、性状、维持人胚胎干细胞生长能力如干细胞平均克隆形成数量、未分化克隆率进行比较,筛选出本实验室条件下最适合的人胚胎干细胞饲养层,并且应用这种饲养层细胞建立人孤雌胚胎干细胞系。应用不成熟人卵母细胞作为研究对象,体外培养成熟后,采用电脉冲联合6-DMAP孤雌激活及自发激活获得孤雌囊胚,同时改良人孤雌胚胎干细胞的培养体系,完全应用人源性培养基、人源性饲养层细胞,并且在操作中尽量减少异源性因素干扰,使建立的人孤雌胚胎干细胞系最大可能接近临床级应用标准。
结果利用本实验室建立的人胚胎干细胞培养方法和改良的同源性培养体系,分别建立了人胚胎干细胞系H-TJ1、H-TJ2和人孤雌胚胎干细胞系P-TJ,体外长期扩增均超过30代,经鉴定分别保持人胚胎干细胞和人孤雌胚胎干细胞的特性。接种在三种饲养层上的人胚胎干细胞均可以维持克隆样生长,生物学鉴定均符合人胚胎干细胞标准,但不同饲养层上克隆的形态差异大。相比于两种人来源饲养层,小鼠胚胎成纤维细胞作为经典的饲养层可以获得最多的人胚胎干细胞克隆,而作为同源性饲养层细胞,人包皮成纤维细胞饲养层明显优于另两种饲养层细胞,能很好地维持人胚胎干细胞增殖和未分化状态。
结论本研究成功建立了人胚胎干细胞和人孤雌胚胎干细胞培养体系,应用废弃胚胎和经体外成熟的M1、GV期卵母细胞分离得到了人胚胎干细胞系和人孤雌胚胎干细胞系,并且维持其人胚胎干细胞特性和人孤雌胚胎干细胞特性。人包皮成纤维细胞作为同源性饲养层细胞,不仅可以避免异源性污染并且可以维持人胚胎干细胞系在体外长期稳定传代。经过改良完全同源性的培养体系可以良好地维持人孤雌胚胎干细胞的性状和长期传代,本研究建立的人孤雌胚胎干细胞系接近临床级人胚胎干细胞系标准,为人胚胎干细胞系真正用于临床研究和应用打下了基础。
Objective Embryonic stem cells are plouripotent cells derived from human primordial germ cells and inner cell mass of mammalian blastocyst. They are characterized by high proliferative and self renewal potential and ability to differentiate into many kinds of functional cell types. Parthenogenetic ES cells are derived from the ICM of a parthenogenetic blastocyst have developed from a single MⅡ oocyte and they only contain the maternal genome. As a result, these cells were regarded as tremendous potential resources for regenerative medicine, human developmental biology and cell-based therapies. The main goal of this study is to establish a stable and standard culture protocol of human ES cells in our own faculty. We support the prolonged undifferentiated growth of hES cells. To this end, the effects of three types of feeder cells for human ES maintenance were analyzed, in order to optimize the culture condition. At the same time, we also try to establish the clinical human embryonic stem cells line using the optimal culture condition and immature oocytes resources which could facilitate studies of therapeutic cloning for research and clinical applications.
Materials and methods The donated immature oocytes and discarded embryos were collected from the Center for Reproductive Medicine, Tianjin Central Hospital for Obstetrics and Gynecology. The egg donors was clearly informed of all the study details and they signed detailed informed consent documents voluntarily. The culture condition of human ES cells is seeding the human ES cells on Mitomycin C treated human fibroblasts feeder layers. The medium contains85%KnockOut DMEM,15%KnockOut serum replacement,4ng/mL bFGF. We established human ES cells using the human feeder layers and the medium. The primary antibodies included SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81and AP and ability of differentiation in Vitro and in Vivo was identified. Then, human ES cells were seeded on mouse embryonic fibroblasts, human foreskin fibroblasts and human embryonic pulmonary fibroblasts cells respectively. The proliferation of human ES cells, such as morphology of colonies, colony number, the ratio of undifferentiated colonies and characterization of the human embryonic stem cell line were observed. Furthermore, based on the experience from human ES cell culture, using the human oocytes activated by the parthenogenetic activation, we did the basic research for establishment and qualification of human parthenogenetic embryonic stem cells. Meanwhile, we improved the culture system in order to facilitate the further study and application of such cells in research and clinical settings.
Results With our culture method, human ES cell (H-TJ1&H-TJ2) and human parthenogenetic embryonic stem cell (P-TJ) form colonies, and express cell markers that characterize undifferentiated primate ES cells. All of the three feeder layers can ensure human ES colonies formed, maintain pluripotency, yet the morphology of colonies on different feeder layers varies obviously. There were more AP positive colonies and more cell quantity on mouse embryonic fibroblasts. As homologous feeder cells, the cloning efficiency on the human foreskin fibroblasts feeder layers was enough to enable to assure cell survival, and the differentiation rates were low enough to enable prolonged undifferentiated culture without continuous removal of the differentiated colonies from the culture. Human foreskin fibroblast feeder layer was better than the other two kinds of feeder cells.
Conclusions We successfully established the culture system of human embryonic stem cells and human parthenogenetic embryonic stem cells. We also isolated human embryonic stem cells lines and human parthenogenetic embryonic stem cells line using the discarded embryos and in vitro maturation of MⅡ, GV oocytes, and maintained their characteristics of human embryonic stem cells and human parthenogenetic embryonic stem cells characteristics. Human foreskin fibroblasts could maintain the characters of the human embryonic stem cells and the long-term cultivation as homologous feeder cells. The modified cultural system could maintain human parthenogenetic embryonic stem cells long-term cultivation. The human parthenogenetic embryonic stem cells is close to clinical grade human embryonic stem cell line standards. This should facilitate studies of therapeutic and laid the foundation for the application.
对象与方法本实验收集天津市中心妇产科医院生殖医学中心试管婴儿治疗患者的不成熟卵母细胞和移植后剩余无冻存价值的废弃胚胎,征得患者夫妇双方同意并签署知情同意书后用于本实验。应用人包皮成纤维细胞作为饲养层,85%KnockOut DMEM、15%KnockOut血清替代物、4ng/mlbFGF培养基,建立人胚胎干细胞系,并且对其特异性标志物SSEA-3、SSEA-4、SSEA-1、TRA-1-60、 TRA-1-81、转录因子Oct-4,碱性磷酸酶,体内或体外分化能力作全面的鉴定。同时,对小鼠胚胎成纤维细胞、人包皮成纤维细胞、人胚胎肺组织成纤维细胞三种饲养层的形态、性状、维持人胚胎干细胞生长能力如干细胞平均克隆形成数量、未分化克隆率进行比较,筛选出本实验室条件下最适合的人胚胎干细胞饲养层,并且应用这种饲养层细胞建立人孤雌胚胎干细胞系。应用不成熟人卵母细胞作为研究对象,体外培养成熟后,采用电脉冲联合6-DMAP孤雌激活及自发激活获得孤雌囊胚,同时改良人孤雌胚胎干细胞的培养体系,完全应用人源性培养基、人源性饲养层细胞,并且在操作中尽量减少异源性因素干扰,使建立的人孤雌胚胎干细胞系最大可能接近临床级应用标准。
结果利用本实验室建立的人胚胎干细胞培养方法和改良的同源性培养体系,分别建立了人胚胎干细胞系H-TJ1、H-TJ2和人孤雌胚胎干细胞系P-TJ,体外长期扩增均超过30代,经鉴定分别保持人胚胎干细胞和人孤雌胚胎干细胞的特性。接种在三种饲养层上的人胚胎干细胞均可以维持克隆样生长,生物学鉴定均符合人胚胎干细胞标准,但不同饲养层上克隆的形态差异大。相比于两种人来源饲养层,小鼠胚胎成纤维细胞作为经典的饲养层可以获得最多的人胚胎干细胞克隆,而作为同源性饲养层细胞,人包皮成纤维细胞饲养层明显优于另两种饲养层细胞,能很好地维持人胚胎干细胞增殖和未分化状态。
结论本研究成功建立了人胚胎干细胞和人孤雌胚胎干细胞培养体系,应用废弃胚胎和经体外成熟的M1、GV期卵母细胞分离得到了人胚胎干细胞系和人孤雌胚胎干细胞系,并且维持其人胚胎干细胞特性和人孤雌胚胎干细胞特性。人包皮成纤维细胞作为同源性饲养层细胞,不仅可以避免异源性污染并且可以维持人胚胎干细胞系在体外长期稳定传代。经过改良完全同源性的培养体系可以良好地维持人孤雌胚胎干细胞的性状和长期传代,本研究建立的人孤雌胚胎干细胞系接近临床级人胚胎干细胞系标准,为人胚胎干细胞系真正用于临床研究和应用打下了基础。
Objective Embryonic stem cells are plouripotent cells derived from human primordial germ cells and inner cell mass of mammalian blastocyst. They are characterized by high proliferative and self renewal potential and ability to differentiate into many kinds of functional cell types. Parthenogenetic ES cells are derived from the ICM of a parthenogenetic blastocyst have developed from a single MⅡ oocyte and they only contain the maternal genome. As a result, these cells were regarded as tremendous potential resources for regenerative medicine, human developmental biology and cell-based therapies. The main goal of this study is to establish a stable and standard culture protocol of human ES cells in our own faculty. We support the prolonged undifferentiated growth of hES cells. To this end, the effects of three types of feeder cells for human ES maintenance were analyzed, in order to optimize the culture condition. At the same time, we also try to establish the clinical human embryonic stem cells line using the optimal culture condition and immature oocytes resources which could facilitate studies of therapeutic cloning for research and clinical applications.
Materials and methods The donated immature oocytes and discarded embryos were collected from the Center for Reproductive Medicine, Tianjin Central Hospital for Obstetrics and Gynecology. The egg donors was clearly informed of all the study details and they signed detailed informed consent documents voluntarily. The culture condition of human ES cells is seeding the human ES cells on Mitomycin C treated human fibroblasts feeder layers. The medium contains85%KnockOut DMEM,15%KnockOut serum replacement,4ng/mL bFGF. We established human ES cells using the human feeder layers and the medium. The primary antibodies included SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81and AP and ability of differentiation in Vitro and in Vivo was identified. Then, human ES cells were seeded on mouse embryonic fibroblasts, human foreskin fibroblasts and human embryonic pulmonary fibroblasts cells respectively. The proliferation of human ES cells, such as morphology of colonies, colony number, the ratio of undifferentiated colonies and characterization of the human embryonic stem cell line were observed. Furthermore, based on the experience from human ES cell culture, using the human oocytes activated by the parthenogenetic activation, we did the basic research for establishment and qualification of human parthenogenetic embryonic stem cells. Meanwhile, we improved the culture system in order to facilitate the further study and application of such cells in research and clinical settings.
Results With our culture method, human ES cell (H-TJ1&H-TJ2) and human parthenogenetic embryonic stem cell (P-TJ) form colonies, and express cell markers that characterize undifferentiated primate ES cells. All of the three feeder layers can ensure human ES colonies formed, maintain pluripotency, yet the morphology of colonies on different feeder layers varies obviously. There were more AP positive colonies and more cell quantity on mouse embryonic fibroblasts. As homologous feeder cells, the cloning efficiency on the human foreskin fibroblasts feeder layers was enough to enable to assure cell survival, and the differentiation rates were low enough to enable prolonged undifferentiated culture without continuous removal of the differentiated colonies from the culture. Human foreskin fibroblast feeder layer was better than the other two kinds of feeder cells.
Conclusions We successfully established the culture system of human embryonic stem cells and human parthenogenetic embryonic stem cells. We also isolated human embryonic stem cells lines and human parthenogenetic embryonic stem cells line using the discarded embryos and in vitro maturation of MⅡ, GV oocytes, and maintained their characteristics of human embryonic stem cells and human parthenogenetic embryonic stem cells characteristics. Human foreskin fibroblasts could maintain the characters of the human embryonic stem cells and the long-term cultivation as homologous feeder cells. The modified cultural system could maintain human parthenogenetic embryonic stem cells long-term cultivation. The human parthenogenetic embryonic stem cells is close to clinical grade human embryonic stem cell line standards. This should facilitate studies of therapeutic and laid the foundation for the application.
引文
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