人iPS细胞株的建立与ABCG2在人多能分化干细胞中缺失的意义探讨
发布时间：2018-08-15 12:55
【摘要】：人胚胎干细胞(embryonic stem cell, ESC)由于自身所具有的多向分化潜能和无限增殖的能力使它拥有广阔的基础及临床应用前景。但是,人ES细胞建系过程中需要破坏受精卵或囊胚,因此,伦理问题成为ES细胞研究无法逾越的障碍；此外,从ES细胞中获得的功能细胞对患者来说属于异体细胞,细胞治疗时的免疫排斥问题严重阻碍了它的临床研究进展。在这种背景下,通过在分化的体细胞中表达特定的几个转录因子,以诱导体细胞的重编程而获得的可不断自我更新且具有多向分化潜能的诱导性多能干细胞(induced pluripotent stem cell, iPSC)诞生了,由于它具有跟成体细胞完全相同的遗传背景,又不涉及伦理道德问题,因此在应用潜能方面显示出无比优越性。但是,iPS细胞是否真正具备与ES细胞相同的分化能力和分化效率,是否能够为将来的临床移植提供具备相应功能的供体材料,近年来一直是干细胞领域中所被高度关注的焦点。 干细胞中的旁系群体(side population, SP)也一直是干细胞研究的热点之一。对于众多干/祖细胞而言,一个非常显著的共性就是跨膜蛋白ATP绑定结构(ATP-Binding cassette, ABC)超家族成员之一,半转运子ABCG2的表达。它们介导着包括细胞毒性药物、类固醇、多肽、磷脂等在内的多类物质的转运。ABCG2对荧光染料Hoechst33342的特异性排除,被认为是干细胞中SP分选的分子基础,因此被用做这类细胞的特异性标记。然而ABCG2是否在人ES细胞和iPS细胞中表达?干细胞SP分选的“黄金法则”是否也同样适用于这些细胞呢? 带着这些问题,开始了我们的课题研究。整个课题是由三大研究部分所构成：第一部分-“人诱导多能干细胞(iPSCs)的建立”,第二部分-“人诱导多能干细胞(iPSCs)的多项分化和第三部分-“剖析在人胚胎干细胞(hESCs)和诱导多能分化干细胞(iPSCs)中膜转运子ABCG2的表达与意义”。 第一部分主要为iPS实验技术平台的建立。我们分别采用慢病毒转染系统和逆转录病毒转染系统,将不同的转录因子组合(Oct4,Sox2, c-Myc和K1f4或Oct4, Sox2, Nonog, c-Myc, Klf4和Lin28)导入人胎肺成纤维细胞(IMR-90)中,成功将其诱导成为具有完全相同遗传背景的iPS细胞株。经相关检测,发现这些iPS细胞均表达特异性多能分化标记,且能在小鼠体内形成包含三个胚层的畸胎瘤,提示它们具备跟ES细胞相似的多向分化潜能。 第二部分主要为包括造血、神经以及滋养层细胞在内的iPS细胞体外诱导分化。我们根据在人ES细胞的培养和分化的经验,分别将我们所建立的iPS细胞系分化成造血细胞、神经上皮细胞、各类终末神经元以及滋养层细胞,并特别比较了人的ES细胞和iPS细胞的神经分化效率。结果表明,我们所得到的iPS细胞均具备向上述各类细胞分化的能力,而且经功能学检测,发现将其诱导生成的运动神经元被激发后可产生与ES细胞来源运动神经元相似的动作电位,提示这类神经元是具备相关感触和传导功能的,有望用于将来的临床神经退行性病变的治疗,但是不同iPS细胞系在神经分化的效率上存在着差异,其深层次的机制目前尚不可知。 第三部分是一项发现导向型课题。我们在对人ES细胞及iPS细胞的研究过程中发现了一个非常奇特的现象,那就是区别于其他类型的干/祖细胞,人的ES细胞和iPS细胞胞核能够被荧光染料Hoechst所染色,通过进一步实验证明这种现象是由于干细胞SP的分选标记一ABCG2在人ES细胞和iPS细胞中的缺失所导致的。并且恰恰相反,通过常规方法Hoechst染色对人ES细胞进行流式细胞术分选,得到拒染Hoechst的“SP”细胞实际上是多能分化基因已经下调的分化细胞,证明经典的干细胞“SP”的定义与分选并不适用于人的多能分化干细胞(ES细胞和iPS细胞)。而有趣的是,来源于人ES细胞的滋养层细胞和神经祖细胞却能够表达ABCG2因而可以拒染Hoechst。与之相对,我们在小鼠的ES细胞中检测到ABCG2的表达以及对Hoechst的拒染,而在小鼠的滋养层细胞中却未发现其表达。在人ES细胞中强行表达ABCG2则可以显著增强它们对荧光染料Hoechst和化疗药物米托葸醌(mitoxanthrone, MTX)的抵抗性,并且大部分人ES细胞在撤去生长因bFGF(3天)后仍能维持其自我更新(self renew)状态。我们在表达ABCG2的人ES细胞中还发现磷酸化的AKT表达水平显著升高,提示ABCG2可能通过激活FGF信号通路的重要分支PI3K/AKT而降低人ES细胞对于子bFGF的依赖性,但是这其中的具体机制还有待进一步探讨。 综上所述,我们成功地通过不同的病毒转染技术建立了iPS技术平台,并进一步将所建立的iPS细胞系分化成造血干/祖细胞,神经上皮细胞和区域神经元,以及滋养层细胞,比较它们神经分化能力的差异并进行部分功能检测,初步探讨其做为不同细胞种类的移植候选物的可能性。同时我们首次系统性地阐述了干细胞SP分选标记ABCG2在人多能分化干细胞(ES细胞和iPS细胞)中的缺失,对于重新认识干细胞中的旁系群体、ABCG2与干细胞多能性的相关性、ES细胞的自我更新与物种差异有着积极意义。
[Abstract]:Human embryonic stem cell (ESC) has a broad foundation and clinical application prospects because of its multi-directional differentiation potential and unlimited proliferation ability. However, the establishment of human ES cell lines requires the destruction of fertilized eggs or blastocysts, so ethical issues become an insurmountable obstacle to ES cell research. Functional cells obtained in cells are allogeneic to patients, and immunological rejection during cell therapy seriously hampers its clinical advances. In this context, the self-renewal and abundance of these cells can be achieved by inducing reprogramming of somatic cells by expressing specific transcription factors in differentiated somatic cells Induced pluripotent stem cells (iPSC) with differentiated potentials were born. Because of their identical genetic background with adult cells and no ethical issues, iPSC has shown tremendous superiority in application potential. However, whether iPS cells really have the same differentiation ability as ES cells or not Differentiation efficiency and the ability to provide functional donor materials for future clinical transplantation have been the focus of attention in the field of stem cells in recent years.
Side population (SP) in stem cells has also been one of the hotspots in stem cell research. For many stem/progenitor cells, one of the most remarkable similarities is the expression of ABCG2, a semitransporter, a member of the ATP-Binding cassette (ABC) superfamily. The specific exclusion of Hoechst 33342 by ABCG2 is considered to be the molecular basis for SP sorting in stem cells and is therefore used as a specific marker for such cells. The same applies to these cells?
With these problems, we began our research. The whole project is composed of three parts: the first part - "the establishment of human induced pluripotent stem cells (iPSCs)," the second part - "the multiple differentiation of human induced pluripotent stem cells (iPSCs) and the third part -"the analysis of human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs)". Expression and significance of ABCG2 in cell membrane (iPSCs).
The first part is the establishment of an experimental platform for iPS. Different combinations of transcription factors (Oct4, Sox2, c-Myc and K1f4 or Oct4, Sox2, Nonog, c-Myc, Klf4 and Lin28) were successfully induced into human fetal lung fibroblasts (IMR-90) using lentiviral and retroviral transfection systems. These iPS cells expressed specific pluripotent markers and could form teratomas containing three embryonic layers in mice, suggesting that they had similar pluripotent potential to ES cells.
The second part is the induction and differentiation of iPS cells, including hematopoietic, neurotrophoblastic and neurotrophoblast cells in vitro. Based on our experience in the culture and differentiation of human ES cells, we differentiated our iPS cell lines into hematopoietic, neuroepithelial, terminal neurons and trophoblast cells, and compared human cells in particular. The results showed that all the iPS cells we obtained possessed the ability to differentiate into the above-mentioned types of cells, and functional tests showed that the motor neurons induced by these cells could produce action potentials similar to those of ES-derived motor neurons. It is expected to be used in the treatment of neurodegenerative diseases in the future because of its related sensory and conductive functions. However, there are differences in the efficiency of neural differentiation among different iPS cell lines, and the underlying mechanism is still unknown.
The third part is a discovery-oriented project. We have found a very strange phenomenon in the study of human ES cells and iPS cells, which is different from other types of stem/progenitor cells. The nuclei of human ES cells and iPS cells can be stained by fluorescent dye Hoechst. Further experiments show that this phenomenon is due to The absence of ABCG2 in human ES cells and iPS cells, a marker for stem cell SP sorting, was caused by the absence of ABCG2. On the contrary, Hoechst staining was used for flow cytometry sorting of human ES cells to obtain Hoechst-resistant "SP" cells, which were actually pluripotent differentiated cells whose genes had been down-regulated. The definition and sorting of "P" does not apply to human pluripotent stem cells (ES cells and iPS cells). Interestingly, trophoblast cells and neural progenitor cells derived from human ES cells can express ABCG2 and therefore can resist Hoechst. In contrast, we detected the expression of ABCG2 and the resistance to Hoechst in mouse ES cells. The expression of ABCG2 in human ES cells significantly enhanced their resistance to fluorescent dye Hoechst and the chemotherapeutic drug mitoxanthrone (MTX), and most human ES cells maintained their self-renew state after the removal of bFGF (3 days). We also found a significant increase in phosphorylated AKT expression in human ES cells expressing ABCG2, suggesting that ABCG2 may reduce the dependence of human ES cells on bFGF by activating PI3K/AKT, an important branch of the FGF signaling pathway, but the specific mechanism remains to be further explored.
To sum up, we have successfully established an iPS technology platform through different virus transfection techniques, and further differentiated the established iPS cell lines into hematopoietic stem/progenitor cells, neuroepithelial cells, regional neurons, and trophoblast cells, and compared their differences in neural differentiation ability and carried out some functional tests to preliminarily explore their role. For the first time, we systematically elucidate the deletion of stem cell SP sorting marker ABCG2 in human pluripotent stem cells (ES cells and iPS cells), the relationship between ABCG2 and stem cell pluripotency, the self-renewal and species of ES cells for the purpose of reconsidering the collateral population in stem cells. The difference is of positive significance.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
本文编号：2184267
[Abstract]:Human embryonic stem cell (ESC) has a broad foundation and clinical application prospects because of its multi-directional differentiation potential and unlimited proliferation ability. However, the establishment of human ES cell lines requires the destruction of fertilized eggs or blastocysts, so ethical issues become an insurmountable obstacle to ES cell research. Functional cells obtained in cells are allogeneic to patients, and immunological rejection during cell therapy seriously hampers its clinical advances. In this context, the self-renewal and abundance of these cells can be achieved by inducing reprogramming of somatic cells by expressing specific transcription factors in differentiated somatic cells Induced pluripotent stem cells (iPSC) with differentiated potentials were born. Because of their identical genetic background with adult cells and no ethical issues, iPSC has shown tremendous superiority in application potential. However, whether iPS cells really have the same differentiation ability as ES cells or not Differentiation efficiency and the ability to provide functional donor materials for future clinical transplantation have been the focus of attention in the field of stem cells in recent years.
Side population (SP) in stem cells has also been one of the hotspots in stem cell research. For many stem/progenitor cells, one of the most remarkable similarities is the expression of ABCG2, a semitransporter, a member of the ATP-Binding cassette (ABC) superfamily. The specific exclusion of Hoechst 33342 by ABCG2 is considered to be the molecular basis for SP sorting in stem cells and is therefore used as a specific marker for such cells. The same applies to these cells?
With these problems, we began our research. The whole project is composed of three parts: the first part - "the establishment of human induced pluripotent stem cells (iPSCs)," the second part - "the multiple differentiation of human induced pluripotent stem cells (iPSCs) and the third part -"the analysis of human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs)". Expression and significance of ABCG2 in cell membrane (iPSCs).
The first part is the establishment of an experimental platform for iPS. Different combinations of transcription factors (Oct4, Sox2, c-Myc and K1f4 or Oct4, Sox2, Nonog, c-Myc, Klf4 and Lin28) were successfully induced into human fetal lung fibroblasts (IMR-90) using lentiviral and retroviral transfection systems. These iPS cells expressed specific pluripotent markers and could form teratomas containing three embryonic layers in mice, suggesting that they had similar pluripotent potential to ES cells.
The second part is the induction and differentiation of iPS cells, including hematopoietic, neurotrophoblastic and neurotrophoblast cells in vitro. Based on our experience in the culture and differentiation of human ES cells, we differentiated our iPS cell lines into hematopoietic, neuroepithelial, terminal neurons and trophoblast cells, and compared human cells in particular. The results showed that all the iPS cells we obtained possessed the ability to differentiate into the above-mentioned types of cells, and functional tests showed that the motor neurons induced by these cells could produce action potentials similar to those of ES-derived motor neurons. It is expected to be used in the treatment of neurodegenerative diseases in the future because of its related sensory and conductive functions. However, there are differences in the efficiency of neural differentiation among different iPS cell lines, and the underlying mechanism is still unknown.
The third part is a discovery-oriented project. We have found a very strange phenomenon in the study of human ES cells and iPS cells, which is different from other types of stem/progenitor cells. The nuclei of human ES cells and iPS cells can be stained by fluorescent dye Hoechst. Further experiments show that this phenomenon is due to The absence of ABCG2 in human ES cells and iPS cells, a marker for stem cell SP sorting, was caused by the absence of ABCG2. On the contrary, Hoechst staining was used for flow cytometry sorting of human ES cells to obtain Hoechst-resistant "SP" cells, which were actually pluripotent differentiated cells whose genes had been down-regulated. The definition and sorting of "P" does not apply to human pluripotent stem cells (ES cells and iPS cells). Interestingly, trophoblast cells and neural progenitor cells derived from human ES cells can express ABCG2 and therefore can resist Hoechst. In contrast, we detected the expression of ABCG2 and the resistance to Hoechst in mouse ES cells. The expression of ABCG2 in human ES cells significantly enhanced their resistance to fluorescent dye Hoechst and the chemotherapeutic drug mitoxanthrone (MTX), and most human ES cells maintained their self-renew state after the removal of bFGF (3 days). We also found a significant increase in phosphorylated AKT expression in human ES cells expressing ABCG2, suggesting that ABCG2 may reduce the dependence of human ES cells on bFGF by activating PI3K/AKT, an important branch of the FGF signaling pathway, but the specific mechanism remains to be further explored.
To sum up, we have successfully established an iPS technology platform through different virus transfection techniques, and further differentiated the established iPS cell lines into hematopoietic stem/progenitor cells, neuroepithelial cells, regional neurons, and trophoblast cells, and compared their differences in neural differentiation ability and carried out some functional tests to preliminarily explore their role. For the first time, we systematically elucidate the deletion of stem cell SP sorting marker ABCG2 in human pluripotent stem cells (ES cells and iPS cells), the relationship between ABCG2 and stem cell pluripotency, the self-renewal and species of ES cells for the purpose of reconsidering the collateral population in stem cells. The difference is of positive significance.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 徐大鹏;婴幼儿关节软骨细胞的体外培养和表型研究及逆向诱导分化初探[D];苏州大学;2013年
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