人胚胎干细胞定向诱导分化为肝细胞的实验研究
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摘要
终末期肝衰竭仍然是困扰人类健康的一类顽疾,目前,它的治疗主要依赖于原位肝移植(orthotopic liver transplantation,OLT),但是供体的极度短缺、移植及手术本身相关的死亡、终生服用免疫抑制剂及其所致的致死性并发症等一系列问题极大地限制了这一治疗手段的广泛开展。细胞移植与生物人工肝(bioartificial liver,BAL)作为终末期肝病的替代、补充治疗方法,不仅为患者寻求合适的供肝来源争取到宝贵的时机,甚至可以帮助患者平安渡过危险期,通过肝再生而自然恢复。令人遗憾的是,细胞来源受限、数量不足以及功能欠缺等仍是制约其进一步发展的瓶颈问题。具有高度自我更新和发育全能性的人胚胎干细胞(embryonic stem cells,ES cells)在适宜的环境中可以向各种组织细胞分化,有望为生物人工肝、肝细胞移植、甚至肝组织工程等提供理想的种子细胞。然而,影响人ES细胞向肝细胞有效分化的因素及其调控机制、诱导分化效率及分化细胞的功能状态等均存在许多不确定性,ES细胞应用于临床尚有许多难以逾越的障碍。本研究探讨了诱导人ES细胞分化为具有合成、代谢、分泌及贮存等功能的肝细胞的方法,并通过遗传修饰建立稳定转染pAlb-EGFP的人ES细胞株,提供了纯化目的细胞的手段,为人ES细胞在以细胞治疗和基因治疗为基础的肝脏再生医学中的应用奠定了基础。
本研究主要包括三部分内容。
一、细胞因子联合胞外基质诱导人ES细胞向肝系细胞分化我们采用以小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)作为饲养层,并摸索了两种传代方法(机械法和胶原酶法),建立了标准的人ES细胞体外培养体系。
为进一步探讨人ES细胞肝向分化的潜能,我们在培养初期先将人ES细胞悬浮培养形成包含三胚层结构的拟胚体(embryoid body,EB),然后将其接种到预先包被有Ⅰ型胶原的培养板上,以含有地塞米松、胰岛素等的条件培养液进行肝系诱导。形态学、细胞表型变化及功能检测等多方面结果表明,诱导后的部分细胞基本符合肝细胞的特征,证明人ES细胞具有向肝细胞样细胞分化的潜能。
二、分阶段富集联合转基因基质细胞共培养诱导人ES细胞向肝脏细胞分化
在前述试验研究中,我们证实了人ES细胞能够在特定环境中向肝细胞样细胞分化,但事实上分化的结果仍存在一定争议。因为与成体干细胞有所不同,ES细胞具有更为特异的全能分化潜能,可以向内、中、外三个胚层乃至胚外组织进行分化,而脏壁内胚层(visceral endoderm)可以表达许多与肝细胞相似的基因,但它只能够参与形成胚外卵黄囊结构,并不能分化发育成肝脏或胰脏等实质性器官,这为获得真正意义上的肝细胞造成障碍。为此,本研究根据人ES细胞全能分化的特点及肝发育机制,设计了阶段性富集联合转基因基质细胞共培养的新策略。首先形成包含了外层的原始内胚层和内层的原始外胚层的早期发育的简单EB,在低血清的条件下,以高浓度的activin A诱导其向定型内胚层的分化。RT-PCR和免疫荧光结果显示,activin A作用3d的细胞高表达(>80%)内胚层标志SOX17,而同时脏壁内胚层的标志α-甲胎蛋白(alpha-fetoprotein,AFP)表达缺失,证明所获细胞为定型内胚层细胞。为进一步探讨定型内胚层细胞向肝细胞分化的潜能与机制,我们利用转基因基质细胞共培养系统模拟肝发育时的微环境。共培养11d后,形态学、细胞表型变化及功能检测等多方面结果表明,定型内胚层细胞可以在bFGF和胎肝基质细胞共同创造的环境中向肝系分化,并在HGF、OSM和Dex的作用下进一步分化成熟,这一研究为获得大量真正意义上的肝细胞开创了新的出路。
三、建立pAlb-EGFP遗传修饰的人ES细胞株
如前所述,新的诱导策略排除了相似细胞的干扰,但所得细胞仍为一混合体,其致瘤、致癌等不可预知的风险不容忽视。在本部分研究中,我们拟通过基因修饰人ES细胞,从而为今后的应用研究提供纯化目的细胞的手段。白蛋白(albumin,Alb)在肝脏发育早期及成体肝脏中均有所表达,是识别肝细胞的一个较为特异的标志。我们设计将含Alb启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的质粒pAlb-EGFP转入人ES细胞,由于该载体中含有一段neo抗性基因,所以可以通过G418条件培养进行抗性筛选。通过条件培养基杀灭试验我们首先确定了G418筛选的最佳浓度(200μg/ml)。在此基础上,我们利用EXGen 500转染试剂进行转染并实施抗性筛选,PCR结果证实筛选到的阳性克隆内确实含有Alb-EGFP这段序列,证实为稳定转染,这一研究可能为日后的应用增添新的纯化肝细胞的工具。
人ES细胞向成熟的、有生理功能的肝细胞分化,为肝脏疾病的细胞治疗提供了新的选择。对于我们这样一个病毒性肝炎及各种原因导致的终末期肝病高发的国家更具有特殊的意义。具有发育全能性的人ES细胞将为生物人工肝、肝(干/祖)细胞移植和以肝(干/祖)细胞为载体的基因转移治疗等提供优良的种子细胞,借助核移植技术和现有的ES细胞库等资源有望在肝脏疾病的治疗中发挥更大的作用,而现代生物科学及其相关技术在该领域中的应用势必将加快其在临床移植修复治疗中的应用进程。
Orthotopic liver transplantation (OLT) remains the most successful treatment for many cases of end-stage liver disease. However, the efficiency of liver transplantation is limited by the shortage of available donor organs, risk of rejection, infections, and other complications caused by the lifelong immunosuppression. Liver cell therapies, including hepatocyte transplantation and bioartificial liver (BAL), are considered promising new approaches to treatment of patients with end-stage liver disease. Unfortunately, The major limitation of these approaches is the availability of human livers as a source of whole liver transplantation or for suspensions of parenchymal cells. With their potential to develop into virtually any cell type, human embryonic stem cells (hES cells) might be an ideal source of donor liver cells for cell therapies aimed at restoring or substituting lost hepatic mass in diseased livers. Nevertheless, there are lots of doubts and uncertainties in the influencing factors and control agents of effectively inducing hES cells differentiation, the efficiency of hES cells’differentiation into hepatocytes, and differentiated cells functional state, etc., and there are many obstacles in application of them clinically. In this study, we investigated the feasibility and methods of differentiation of hES cells into functional hepatocytes, and established a hES cell strain stably transfected by pAlb-EGFP. The studies offered a foundation of gene and stem cell engineering-based regenerative medicine for end-stage liver diseases.
Our study mainly includes three parts.
1. Differentiation of hES cells into hepatocytes by supplementing soluble growth factors and extracellular matrix.
We established a set of standard culture method of hES cells on a feeder layer ofγ-irradiated mouse embryonic fibroblast (MEF) cells.
To investigated the differentiated potential of hES cells into the hepatocytes, Firstly, embryoid bodies (EBs) were generated to enhance cell-cell interactions and form the three dimensional structure approaching the embryonic environment. After 7 days, they were inoculated on tissue culture plates coated with collagen type I, and cultured in media containing dexamethasone and insulin. Our results show that the induced cells exhibit similar morphology, phenotype, and functions to hepatocytes.
2. Differentiation of hES cells into hepatocytes by co-culture with transgenic stromal cells.
In part 1, we demonstrated that hES cells could differentiate into hepatocyte-like cells. However, similar with previously employed methods, this strategy does not exclude the extraembryonic endoderm differentiation of ES cells, which makes the hepatic identities of the differentiated cells controversial. This is especially important for hES cells differentiation, as hES cells tend to differentiate toward extraembryonic endoderm cells, which also express most of the hepatocyte markers, such asα-fetoprotein, albumin and transthyretin. According to the potential of hES cells to develop into virtually any cell type and the mechanism of liver development, a novel strategy has been developed to derive hepatocyes from hES cells using 4 sequential inducing steps lasting 16 days. Firstly, embryoid bodies were generated by growing hES cells in suspension for 2 days; secondly, embryoid bodies were lineage restricted to definitive endoderm with 3 days of treatment with human recombination activin A; thirdly, cells were differentiated further by co-culturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourthly, treating them for 6 days with soluble signals comprised of hFLSCs-derived bFGF, hepatocyte growth factor, Oncostatin M, and dexamethasone. After treatment of activin A for 3d, more than 80% of the differentiated cells showed expression of a definitive endoderm marker, SOX17 but no AFP expression indicating that these cells were not visceral endoderm cells. Morphologic, phenotypic, and functional analysis proved that hES cells can differentiate into authentic hepatocytes in vitro.
3. Establishment of hES cell strain stably transfected with pAlb-EGFP.
Although we have developed improvements on strategies to differentiate hES cells into liver fates, the induced cells still a complex, which might be a danger in future application. Albumin (Alb) is expressed both in embryonic and adult liver, which make it as a specifically hepatic marker. In this study, we aimed to generate a hES cell strain expressing enhance green fluorescent protein (EGFP) under the control of the Alb promoter. Because the pAlb-EGFP contained an SV40-driven neomycin selectable marker, which confers resistance to G418 antibiotic, we can select positive clone by G418 conditional culture. The results showed that 200μg/ml is the optimum G418 concentration for selection. Next, we transfected hES cells with pAlb-EGFP by EXGen 500 and selected positive clone with 200μg/ml G418. RT-PCR proved that there is an Alb-EGFP sequence in the gene of positive clone previously selected, which demonstrated the transfection is stable and may be applied in future.
Differentiation of hES cells into functional hepatocytes offer a novel method for cell therapies in liver failure. It is of great importance to China, a country with a high morbidity in liver disorders. hES cells, which have permanent self-renewal capability and developmental pluripotency, might be an ideal source of seed cells for BAL, cells transplantation and gene therapy based on liver (stem/progenitor) cells. More importantly, its application in liver dysfunction treatment will be enlarged by nuclear transfer and banking on hES cells. Furthermore, the induction of modern biotechnology will also make a contribution to its clinical application.
本研究主要包括三部分内容。
一、细胞因子联合胞外基质诱导人ES细胞向肝系细胞分化我们采用以小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)作为饲养层,并摸索了两种传代方法(机械法和胶原酶法),建立了标准的人ES细胞体外培养体系。
为进一步探讨人ES细胞肝向分化的潜能,我们在培养初期先将人ES细胞悬浮培养形成包含三胚层结构的拟胚体(embryoid body,EB),然后将其接种到预先包被有Ⅰ型胶原的培养板上,以含有地塞米松、胰岛素等的条件培养液进行肝系诱导。形态学、细胞表型变化及功能检测等多方面结果表明,诱导后的部分细胞基本符合肝细胞的特征,证明人ES细胞具有向肝细胞样细胞分化的潜能。
二、分阶段富集联合转基因基质细胞共培养诱导人ES细胞向肝脏细胞分化
在前述试验研究中,我们证实了人ES细胞能够在特定环境中向肝细胞样细胞分化,但事实上分化的结果仍存在一定争议。因为与成体干细胞有所不同,ES细胞具有更为特异的全能分化潜能,可以向内、中、外三个胚层乃至胚外组织进行分化,而脏壁内胚层(visceral endoderm)可以表达许多与肝细胞相似的基因,但它只能够参与形成胚外卵黄囊结构,并不能分化发育成肝脏或胰脏等实质性器官,这为获得真正意义上的肝细胞造成障碍。为此,本研究根据人ES细胞全能分化的特点及肝发育机制,设计了阶段性富集联合转基因基质细胞共培养的新策略。首先形成包含了外层的原始内胚层和内层的原始外胚层的早期发育的简单EB,在低血清的条件下,以高浓度的activin A诱导其向定型内胚层的分化。RT-PCR和免疫荧光结果显示,activin A作用3d的细胞高表达(>80%)内胚层标志SOX17,而同时脏壁内胚层的标志α-甲胎蛋白(alpha-fetoprotein,AFP)表达缺失,证明所获细胞为定型内胚层细胞。为进一步探讨定型内胚层细胞向肝细胞分化的潜能与机制,我们利用转基因基质细胞共培养系统模拟肝发育时的微环境。共培养11d后,形态学、细胞表型变化及功能检测等多方面结果表明,定型内胚层细胞可以在bFGF和胎肝基质细胞共同创造的环境中向肝系分化,并在HGF、OSM和Dex的作用下进一步分化成熟,这一研究为获得大量真正意义上的肝细胞开创了新的出路。
三、建立pAlb-EGFP遗传修饰的人ES细胞株
如前所述,新的诱导策略排除了相似细胞的干扰,但所得细胞仍为一混合体,其致瘤、致癌等不可预知的风险不容忽视。在本部分研究中,我们拟通过基因修饰人ES细胞,从而为今后的应用研究提供纯化目的细胞的手段。白蛋白(albumin,Alb)在肝脏发育早期及成体肝脏中均有所表达,是识别肝细胞的一个较为特异的标志。我们设计将含Alb启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的质粒pAlb-EGFP转入人ES细胞,由于该载体中含有一段neo抗性基因,所以可以通过G418条件培养进行抗性筛选。通过条件培养基杀灭试验我们首先确定了G418筛选的最佳浓度(200μg/ml)。在此基础上,我们利用EXGen 500转染试剂进行转染并实施抗性筛选,PCR结果证实筛选到的阳性克隆内确实含有Alb-EGFP这段序列,证实为稳定转染,这一研究可能为日后的应用增添新的纯化肝细胞的工具。
人ES细胞向成熟的、有生理功能的肝细胞分化,为肝脏疾病的细胞治疗提供了新的选择。对于我们这样一个病毒性肝炎及各种原因导致的终末期肝病高发的国家更具有特殊的意义。具有发育全能性的人ES细胞将为生物人工肝、肝(干/祖)细胞移植和以肝(干/祖)细胞为载体的基因转移治疗等提供优良的种子细胞,借助核移植技术和现有的ES细胞库等资源有望在肝脏疾病的治疗中发挥更大的作用,而现代生物科学及其相关技术在该领域中的应用势必将加快其在临床移植修复治疗中的应用进程。
Orthotopic liver transplantation (OLT) remains the most successful treatment for many cases of end-stage liver disease. However, the efficiency of liver transplantation is limited by the shortage of available donor organs, risk of rejection, infections, and other complications caused by the lifelong immunosuppression. Liver cell therapies, including hepatocyte transplantation and bioartificial liver (BAL), are considered promising new approaches to treatment of patients with end-stage liver disease. Unfortunately, The major limitation of these approaches is the availability of human livers as a source of whole liver transplantation or for suspensions of parenchymal cells. With their potential to develop into virtually any cell type, human embryonic stem cells (hES cells) might be an ideal source of donor liver cells for cell therapies aimed at restoring or substituting lost hepatic mass in diseased livers. Nevertheless, there are lots of doubts and uncertainties in the influencing factors and control agents of effectively inducing hES cells differentiation, the efficiency of hES cells’differentiation into hepatocytes, and differentiated cells functional state, etc., and there are many obstacles in application of them clinically. In this study, we investigated the feasibility and methods of differentiation of hES cells into functional hepatocytes, and established a hES cell strain stably transfected by pAlb-EGFP. The studies offered a foundation of gene and stem cell engineering-based regenerative medicine for end-stage liver diseases.
Our study mainly includes three parts.
1. Differentiation of hES cells into hepatocytes by supplementing soluble growth factors and extracellular matrix.
We established a set of standard culture method of hES cells on a feeder layer ofγ-irradiated mouse embryonic fibroblast (MEF) cells.
To investigated the differentiated potential of hES cells into the hepatocytes, Firstly, embryoid bodies (EBs) were generated to enhance cell-cell interactions and form the three dimensional structure approaching the embryonic environment. After 7 days, they were inoculated on tissue culture plates coated with collagen type I, and cultured in media containing dexamethasone and insulin. Our results show that the induced cells exhibit similar morphology, phenotype, and functions to hepatocytes.
2. Differentiation of hES cells into hepatocytes by co-culture with transgenic stromal cells.
In part 1, we demonstrated that hES cells could differentiate into hepatocyte-like cells. However, similar with previously employed methods, this strategy does not exclude the extraembryonic endoderm differentiation of ES cells, which makes the hepatic identities of the differentiated cells controversial. This is especially important for hES cells differentiation, as hES cells tend to differentiate toward extraembryonic endoderm cells, which also express most of the hepatocyte markers, such asα-fetoprotein, albumin and transthyretin. According to the potential of hES cells to develop into virtually any cell type and the mechanism of liver development, a novel strategy has been developed to derive hepatocyes from hES cells using 4 sequential inducing steps lasting 16 days. Firstly, embryoid bodies were generated by growing hES cells in suspension for 2 days; secondly, embryoid bodies were lineage restricted to definitive endoderm with 3 days of treatment with human recombination activin A; thirdly, cells were differentiated further by co-culturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourthly, treating them for 6 days with soluble signals comprised of hFLSCs-derived bFGF, hepatocyte growth factor, Oncostatin M, and dexamethasone. After treatment of activin A for 3d, more than 80% of the differentiated cells showed expression of a definitive endoderm marker, SOX17 but no AFP expression indicating that these cells were not visceral endoderm cells. Morphologic, phenotypic, and functional analysis proved that hES cells can differentiate into authentic hepatocytes in vitro.
3. Establishment of hES cell strain stably transfected with pAlb-EGFP.
Although we have developed improvements on strategies to differentiate hES cells into liver fates, the induced cells still a complex, which might be a danger in future application. Albumin (Alb) is expressed both in embryonic and adult liver, which make it as a specifically hepatic marker. In this study, we aimed to generate a hES cell strain expressing enhance green fluorescent protein (EGFP) under the control of the Alb promoter. Because the pAlb-EGFP contained an SV40-driven neomycin selectable marker, which confers resistance to G418 antibiotic, we can select positive clone by G418 conditional culture. The results showed that 200μg/ml is the optimum G418 concentration for selection. Next, we transfected hES cells with pAlb-EGFP by EXGen 500 and selected positive clone with 200μg/ml G418. RT-PCR proved that there is an Alb-EGFP sequence in the gene of positive clone previously selected, which demonstrated the transfection is stable and may be applied in future.
Differentiation of hES cells into functional hepatocytes offer a novel method for cell therapies in liver failure. It is of great importance to China, a country with a high morbidity in liver disorders. hES cells, which have permanent self-renewal capability and developmental pluripotency, might be an ideal source of seed cells for BAL, cells transplantation and gene therapy based on liver (stem/progenitor) cells. More importantly, its application in liver dysfunction treatment will be enlarged by nuclear transfer and banking on hES cells. Furthermore, the induction of modern biotechnology will also make a contribution to its clinical application.
引文
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