人胚胎干细胞向神经上皮祖细胞和多巴胺能神经元诱导分化的研究
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摘要
人胚胎干细胞(human embryonic stem cells, hESCs)从植入前囊胚的内细胞团(inner cell mass, ICM)分离而来,具有多向分化潜能和无限自我更新能力,是细胞替代治疗潜在的种子细胞来源,也为人类胚胎发育机制和基因功能研究及药物筛选等提供了新的材料。神经退行性病由于神经细胞的缺失或死亡后机体无法再生出新的神经细胞替代原有细胞的功能,如帕金森病、阿尔茨海默病和亨廷顿病等,发病率高,病程缓慢,临床上尚无有效的治疗手段,hESCs来源的神经细胞替代治疗可能会为这些难治性疾病提供新的治疗前景。
hESCs作为种子细胞要发挥作用,首先要建立将其定向诱导分化为特定细胞的诱导方法,在体外高效地诱导产生高纯度的特定神经细胞,并具备相应的生物学功能。目前,hESCs诱导为神经上皮细胞和神经元的方法各有千秋,但是相互之间缺乏系统比较。本研究采用本所建系的多株hESCs进行体外诱导,建立了一个简单、高效、无血清、无动物细胞污染的神经上皮祖细胞(neuroepithelial progenitors,NEP)和多巴胺能神经元(dopaminergic neurons, DAN)的诱导体系,为研究神经系统的早期发育机制和帕金森病细胞替代治疗提供相应的技术平台和研究材料。
论文第一章的研究内容是建立简单高效的hESCs向神经上皮祖细胞诱导体系,比较发现人胚胎成纤维细胞(human embryonic fibroblasts, HEF)诱导体系与小鼠胚胎成纤维细胞(mouse embryonicfibroblasts, MEF)体系一样,可以在无血清培养条件下使hESCs直接贴壁诱导,产生具有典型rosette结构、表达Musashi, Nestin, Pax6等特异性抗原的NEP；且HEF体系的诱导效率更高。与悬浮诱导方法相比,直接贴壁诱导方法的效率更高。诱导培养基中添加或不添加外源性的成纤维细胞生长因子2(fibroblast growth factor 2, FGF2),对于NEP的诱导效率没有影响。采用无饲养层(feeder free, FF)可直接诱导出表达Musashi, Nestin和Pax6的NEP,及34%表达SSEA4的细胞。HEF和FF诱导体系下骨形成蛋白(bone morphogenetic protein, BMP)信号通路相关基因的检测显示,FF体系中BMP4高表达,而HEF中几无表达,提示HEF体系低BMP信号通路活性更有利于NEP的诱导发生。
论文第二章的研究内容是建立HEF和FF体系产生的NEP的增殖扩增和向DAN诱导分化的体系。FGF2可以促进神经球和贴壁的NEP的增殖扩增,且其促进作用与FGF2呈浓度依赖性关系。NEP可在体外传6代以上,并维持相应NEP标记及向神经元和胶质细胞分化的潜能。在区域特化因子成纤维细胞生长因子8(fibroblast growth factor8,FGF8)和音猬因子(sonic hedgehog, SHH)的作用下,NEP可诱导分化为表达Otx2和Enl的中脑DAN前体,并进一步分化为酪氨酸羟化酶(tyrosine hydroxylase, TH)阳性的DAN。HEF和FF诱导分化终末阶段的产生的Tuj1和TH阳性细胞间没有差异,且没有SSEA4阳性细胞残留,说明两种体系下产生DAN的效率相同。FF体系不依赖于饲养层细胞,有利于神经发育生物学的研究及将来神经细胞诱导体系的产业化。
论文第三章的研究内容是研究孤雌人胚胎干细胞(parthenogenetic human embryonic stem cells, phESCs)向NEP和DAN诱导分化的效率和安全性问题。和正常人胚胎干细胞(normal human embryonic stem cells, nhESCs)一样,phESCs可产生的Enl,TH和Tuj1阳性细胞,提示phESCs具备相同的向DAN分化的能力。NEP的比较染色体杂交检测未发现基因组染色体拷贝数的改变。印记基因H19, IGF2和Snrpn表达的检测发现,NEP基本维持了印记基因的表达模式。由此认为,phESCs具备向NEP和DAN分化的能力,体外分化中维持了其染色体和印记基因表达的稳定性,可以作为细胞替代治疗的来源。
本研究通过上述结果证实,HEF体系是hESCs向NEP分化的高效诱导体系；FGF和BMP信号通路对于NEP的诱导起关键作用；FF体系获得的NEP可分化为DAN; phESCs具备正常的神经细胞分化潜能。我们建立的NEP和DAN诱导体系,为hESCs的临床和基础研究奠定了基础,为未来的神经细胞替代治疗提供了可能。
Human embryonic stem cells (hESCs), isolated from the inner cell mass (ICM) of preimplanted blastocyst, possessing the ability of pluripotency and unlimited self-renewal, were considered as potential seed cell sources for cell replacement therapy, as well as novel materials for researches on human embryo developmental mechanism, gene functional assays and pharmacological screening. Neurodegenerative diseases were caused by lack or death of neural cells which could not be effectively regenerated by human body to replace the original ones, such as Parkinson Disease, Alzheimer Disease and Huntington Disease. These diseases, with a high rate of incidence and long period of process, lacked effective clinical treatments; hESCs derived neural cells-based replacement therapy might provide new prospects for these refractory diseases.
In order to make hESCs function as seed cells, we should first establish methods to direct them to differentiate into committed specific cells, and to induce highly purified specific neurons in vitro with proper biological functions. Present methods for induction of hESCs into neural epithelium and neurons had their individual merits and drawbacks, but systematic comparisons between them had not been carried out yet. Our purpose of the study, using several self-established hESC lines, was to perform neural induction in vitro and establish a simple, efficient, serum free and animal cell free culture system for neuroepithelial progenitors (NEP) and dopaminergic neurons (DAN), and to provide technical platforms and research materials for early neural development researches as well as for cell replacement therapy for Parkinson Disease.
The first chapter introduced the establishment of simple and efficient inductive system of hESCs to NEP. Comparison between human embryonic fibroblasts (HEF) and mouse embryonic fibroblasts (MEF) found that both of them could induce hESCs into NEP with typical rosette structures and expression of Musashi, Nestin and Pax6 under serum free conditions, and inductive efficiency was even higher on HEF. Compared with the floating culture strategy, the direct adherent strategy proved to be more efficient. With or without addition of exogenetic fibroblast growth factor 2 (FGF2) in the culture medium, the inductive efficiency of NEP were not affected. Using feeder free (FF) culture system could directly induce rosette structures expressing Musashi, Nestin and Pax6, along with 34% SSEA4 positive cells. The assay of bone morphogenetic proteins (BMP) signalling pathway related gene expression in HEF and FF inductive systems found that FF system had higher expression of BMP4, while HEF system almost no expression, indicating lower BMP signalling activities in HEF system was more propitious for NEP induction.
The second chapter continued to establish systems for HEF and FF system derived NEP to proliferate and expand and differentiate into DAN. FGF2 was found to promote the proliferation and expansion of neurospheres and replated adherent NEP, the function of which had a dose-dependent relationship with FGF2. NEP could be culture for at least 6 passages maintaining the NEP related marks and potentials for neuronal and glial differentiation. With the neural pattern specification factors fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), NEP could be induced into Otx2 and En1 positive midbrain dopaminergic neuronal precursors, and further into tyrosine hydroxylase (TH) positive DAN. The proportion of Tuj1 and TH positive cells in HEF or FF induced terminally differentiated cells had no difference, and no SSEA4 positive cells remained, which indicated that efficiency of DAN production was similar in both systems. As FF system was independent on feeder cells, it could be more useful in the researches on nervous system development and industrialization of future neural cell differentiation system.
The third chapter focused on the efficiency and safety of parthenogenetic human embryonic stem cells (phESCs) inducted into NEP and DAN. PhESCs, as well as normal human embryonic stem cells (nhESCs), could produce En1, TH and Tuj1 positive cells, which indicated the similar ability of phESCs to differentiate into DAN. Comparative genome hybridization assay found no change of genome chromosome copies. Expression of imprinted genes, such as H19, IGF2 and Snrpn indicated that NEP had basically maintained the expression pattern of imprinted genes. Thus, phESCs with the ability to differentiate into NEP and DAN as well as to maintain the stability of chromosomes and expression of imprinted genes, could be served as sources for cell replacement therapy.
With those results we concluded that HEF system was a highly efficient NEP inductive system for hESCs; and that FGF and BMP signalling pathways were essential for NEP induction; and that FF system derived NEP could produce DAN; and that phESCs possessed normal differentiating potentials for neural cells. These NEP and DAN inductive systems we established could provide basement for hESCs clinical and basic researches, as well as possibility for future neural cell replacement therapy.
hESCs作为种子细胞要发挥作用,首先要建立将其定向诱导分化为特定细胞的诱导方法,在体外高效地诱导产生高纯度的特定神经细胞,并具备相应的生物学功能。目前,hESCs诱导为神经上皮细胞和神经元的方法各有千秋,但是相互之间缺乏系统比较。本研究采用本所建系的多株hESCs进行体外诱导,建立了一个简单、高效、无血清、无动物细胞污染的神经上皮祖细胞(neuroepithelial progenitors,NEP)和多巴胺能神经元(dopaminergic neurons, DAN)的诱导体系,为研究神经系统的早期发育机制和帕金森病细胞替代治疗提供相应的技术平台和研究材料。
论文第一章的研究内容是建立简单高效的hESCs向神经上皮祖细胞诱导体系,比较发现人胚胎成纤维细胞(human embryonic fibroblasts, HEF)诱导体系与小鼠胚胎成纤维细胞(mouse embryonicfibroblasts, MEF)体系一样,可以在无血清培养条件下使hESCs直接贴壁诱导,产生具有典型rosette结构、表达Musashi, Nestin, Pax6等特异性抗原的NEP；且HEF体系的诱导效率更高。与悬浮诱导方法相比,直接贴壁诱导方法的效率更高。诱导培养基中添加或不添加外源性的成纤维细胞生长因子2(fibroblast growth factor 2, FGF2),对于NEP的诱导效率没有影响。采用无饲养层(feeder free, FF)可直接诱导出表达Musashi, Nestin和Pax6的NEP,及34%表达SSEA4的细胞。HEF和FF诱导体系下骨形成蛋白(bone morphogenetic protein, BMP)信号通路相关基因的检测显示,FF体系中BMP4高表达,而HEF中几无表达,提示HEF体系低BMP信号通路活性更有利于NEP的诱导发生。
论文第二章的研究内容是建立HEF和FF体系产生的NEP的增殖扩增和向DAN诱导分化的体系。FGF2可以促进神经球和贴壁的NEP的增殖扩增,且其促进作用与FGF2呈浓度依赖性关系。NEP可在体外传6代以上,并维持相应NEP标记及向神经元和胶质细胞分化的潜能。在区域特化因子成纤维细胞生长因子8(fibroblast growth factor8,FGF8)和音猬因子(sonic hedgehog, SHH)的作用下,NEP可诱导分化为表达Otx2和Enl的中脑DAN前体,并进一步分化为酪氨酸羟化酶(tyrosine hydroxylase, TH)阳性的DAN。HEF和FF诱导分化终末阶段的产生的Tuj1和TH阳性细胞间没有差异,且没有SSEA4阳性细胞残留,说明两种体系下产生DAN的效率相同。FF体系不依赖于饲养层细胞,有利于神经发育生物学的研究及将来神经细胞诱导体系的产业化。
论文第三章的研究内容是研究孤雌人胚胎干细胞(parthenogenetic human embryonic stem cells, phESCs)向NEP和DAN诱导分化的效率和安全性问题。和正常人胚胎干细胞(normal human embryonic stem cells, nhESCs)一样,phESCs可产生的Enl,TH和Tuj1阳性细胞,提示phESCs具备相同的向DAN分化的能力。NEP的比较染色体杂交检测未发现基因组染色体拷贝数的改变。印记基因H19, IGF2和Snrpn表达的检测发现,NEP基本维持了印记基因的表达模式。由此认为,phESCs具备向NEP和DAN分化的能力,体外分化中维持了其染色体和印记基因表达的稳定性,可以作为细胞替代治疗的来源。
本研究通过上述结果证实,HEF体系是hESCs向NEP分化的高效诱导体系；FGF和BMP信号通路对于NEP的诱导起关键作用；FF体系获得的NEP可分化为DAN; phESCs具备正常的神经细胞分化潜能。我们建立的NEP和DAN诱导体系,为hESCs的临床和基础研究奠定了基础,为未来的神经细胞替代治疗提供了可能。
Human embryonic stem cells (hESCs), isolated from the inner cell mass (ICM) of preimplanted blastocyst, possessing the ability of pluripotency and unlimited self-renewal, were considered as potential seed cell sources for cell replacement therapy, as well as novel materials for researches on human embryo developmental mechanism, gene functional assays and pharmacological screening. Neurodegenerative diseases were caused by lack or death of neural cells which could not be effectively regenerated by human body to replace the original ones, such as Parkinson Disease, Alzheimer Disease and Huntington Disease. These diseases, with a high rate of incidence and long period of process, lacked effective clinical treatments; hESCs derived neural cells-based replacement therapy might provide new prospects for these refractory diseases.
In order to make hESCs function as seed cells, we should first establish methods to direct them to differentiate into committed specific cells, and to induce highly purified specific neurons in vitro with proper biological functions. Present methods for induction of hESCs into neural epithelium and neurons had their individual merits and drawbacks, but systematic comparisons between them had not been carried out yet. Our purpose of the study, using several self-established hESC lines, was to perform neural induction in vitro and establish a simple, efficient, serum free and animal cell free culture system for neuroepithelial progenitors (NEP) and dopaminergic neurons (DAN), and to provide technical platforms and research materials for early neural development researches as well as for cell replacement therapy for Parkinson Disease.
The first chapter introduced the establishment of simple and efficient inductive system of hESCs to NEP. Comparison between human embryonic fibroblasts (HEF) and mouse embryonic fibroblasts (MEF) found that both of them could induce hESCs into NEP with typical rosette structures and expression of Musashi, Nestin and Pax6 under serum free conditions, and inductive efficiency was even higher on HEF. Compared with the floating culture strategy, the direct adherent strategy proved to be more efficient. With or without addition of exogenetic fibroblast growth factor 2 (FGF2) in the culture medium, the inductive efficiency of NEP were not affected. Using feeder free (FF) culture system could directly induce rosette structures expressing Musashi, Nestin and Pax6, along with 34% SSEA4 positive cells. The assay of bone morphogenetic proteins (BMP) signalling pathway related gene expression in HEF and FF inductive systems found that FF system had higher expression of BMP4, while HEF system almost no expression, indicating lower BMP signalling activities in HEF system was more propitious for NEP induction.
The second chapter continued to establish systems for HEF and FF system derived NEP to proliferate and expand and differentiate into DAN. FGF2 was found to promote the proliferation and expansion of neurospheres and replated adherent NEP, the function of which had a dose-dependent relationship with FGF2. NEP could be culture for at least 6 passages maintaining the NEP related marks and potentials for neuronal and glial differentiation. With the neural pattern specification factors fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), NEP could be induced into Otx2 and En1 positive midbrain dopaminergic neuronal precursors, and further into tyrosine hydroxylase (TH) positive DAN. The proportion of Tuj1 and TH positive cells in HEF or FF induced terminally differentiated cells had no difference, and no SSEA4 positive cells remained, which indicated that efficiency of DAN production was similar in both systems. As FF system was independent on feeder cells, it could be more useful in the researches on nervous system development and industrialization of future neural cell differentiation system.
The third chapter focused on the efficiency and safety of parthenogenetic human embryonic stem cells (phESCs) inducted into NEP and DAN. PhESCs, as well as normal human embryonic stem cells (nhESCs), could produce En1, TH and Tuj1 positive cells, which indicated the similar ability of phESCs to differentiate into DAN. Comparative genome hybridization assay found no change of genome chromosome copies. Expression of imprinted genes, such as H19, IGF2 and Snrpn indicated that NEP had basically maintained the expression pattern of imprinted genes. Thus, phESCs with the ability to differentiate into NEP and DAN as well as to maintain the stability of chromosomes and expression of imprinted genes, could be served as sources for cell replacement therapy.
With those results we concluded that HEF system was a highly efficient NEP inductive system for hESCs; and that FGF and BMP signalling pathways were essential for NEP induction; and that FF system derived NEP could produce DAN; and that phESCs possessed normal differentiating potentials for neural cells. These NEP and DAN inductive systems we established could provide basement for hESCs clinical and basic researches, as well as possibility for future neural cell replacement therapy.
引文
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