【作者】 王娟；

【导师】 刘忠华；

【作者基本信息】 东北农业大学 ， 发育生物学， 2016， 博士

【摘要】 在希腊神话中,泰坦巨人普罗米修斯因为受到宙斯的惩罚,肝脏不断地被神鹰啄食。神奇的是他被啄食的肝脏可以不断地再生,使得生命得以维持。现代社会,由于慢性病引起的组织器官衰竭或者意外事故引起的组织器官损伤病人越来越多。面对这类疾病,传统的医疗手段往往显得无能为力,人类急需把神话变为现实。再生医学是研究用正常的细胞、组织或者器官替换受损的细胞、组织或器官行使功能或者用一定的手段诱导受损的细胞、组织或器官再生的学科,是未来医学的一个重要研究方向。细胞、组织或者器官的供体不足是限制再生医学发展的重要因素之一。人多潜能干细胞(human Pluripotent Stem Cells,hPSCs)包括人胚胎干细胞(human Embryonic Stem cells,hESCs)和人诱导的多潜能干细胞(human induced Pluripotent Stem Cells,hiPSCs),具有无限的增殖能力和分化形成机体内所有细胞类型的潜能,理论上来说可以源源不断的为再生医学提供各种类型的细胞、组织和器官,应用前景巨大。自从Thomson实验室建立了第一株hESCs以来,科学家就一直在hPSCs的临床应用这条道路上不断地探索,并取得了一系列的成果。但是hESCs是从发育早期的人类胚胎中建系获取的,涉及到了伦理道德问题,其科学研究和临床应用一直备受争议。hiPSCs是通过一定的手段(导入外源基因或者使用化学小分子)将体细胞重编程获得的,和hESCs具有相同的特性和临床应用价值,并且能规避hESCs所涉及到的伦理问题。重编程效率低下,外源基因随机整合和自发激活,动物源性物质的使用,基因组和表观遗传学的稳定性等是影响hiPSCs临床应用的重要问题。随着科学研究的深入,这些问题也正在被逐步解决,hiPSCs的临床应用时机已经趋于成熟。日本科学家正在用hiPSCs分化形成的视网膜色素上皮细胞(Retinal Pigmented Epithelial cells,RPE)进行治疗年龄相关性黄斑变性的临床试验。到目前为止,接受细胞移植的患者没有出现不良反应,视力有所改善。为了保证临床应用的合法性、安全性、有效性和可追溯性,临床应用的hiPSCs应该满足以下几点要求:(1)供体细胞捐献过程符合《人体细胞治疗研究和制剂质量控制技术指导原则》和《组织捐献条例》中的相关规定及伦理道德规范;(2)供体细胞取材方便;(3)在临床应用过程中,难免会涉及到一些年纪较大个体细胞的重编程,细胞供体的年龄越大,细胞越不容易被重编程,为了保证成功率,应该选择高效的重编程方法;(4)为了避免外源基因随机整合到基因组中引起的插入突变和外源基因自发激活导致的致瘤性,以临床应用为目的的hiPSCs应该不含有外源重编程因子;(5)国际上的干细胞协会和大多数国家都要求或者鼓励临床应用细胞的操作规程符合优良制造标准(Good Manufacturing Practice,GMP),所以从供体细胞的采集,hiPSCs的诱导到hiPSCs的定向分化等操作流程和制造环境都应该符合GMP标准;(6)为了避免动物源性病原体在人畜之间交叉传播,在以临床应用为目的的细胞培养过程中最好要避免动物源性成分的添加:(7)对于以临床应用为目的的细胞,我们要做细菌、真菌、支原体、一些特殊种类病原体的检测,确保其具有生物安全性。虽然目前世界上有一些临床级别hiPSCs的报道,但是在这些研究中,科学家基本上都是将研究级别的hiPSCs通过在无动物源性的培养液中培养转化成所谓的临床级别的hiPSCs,或者是将研究级别的供体细胞通过在无动物源性的培养液中重编程,获得所谓的临床级别的hiPSCs。所获得的hiPSCs都与含有动物源性的培养液接触过,给其临床应用增加了潜在动物源性病原体感染的风险。在仅有的一项完全从无动物源性培养液中建系的hiPSCs的报道中,研究者没有对其生物安全性进行检测,也没有描述供体细胞的捐献过程是否符合《组织捐献条例》。所以以符合《组织捐献条例》细胞为供体细胞,在无动物源性的培养液中和符合GMP操作规范和环境下从头建立临床级别的hiPSCs并对其进行全面的生物安全性检测对于hiPSCs更加安全有效和规范的应用于临床是十分必要的。基于临床用hiPSCs的基本要求和目前临床级别hiPSCs的研究现状,我们进行了以下研究:(1)初步用慢病毒体系优化取材方便的人脐带血单个核细胞(human Umbilical Cord Blood Mononuclear Cells,hUCBMCs)重编程为hiPSCs的流程,提高其重编程效率和成功率;(2)在不使用原癌基因C-Myc的情况下,优化用携带Oct4、Sox2、Klf4、Nanog四因子的非整合型质粒诱导hiPSCs的方法,高效稳定获得不携带外源基因的hiPSCs;(3)在前期优化重编程方法的研究中,我们发现不同个体来源的人体细胞的重编程效率不同,这种效率的差异和供体的年龄无关。对这些细胞进行RNA-Seq测序,并对其基因表达谱进行分析,找出了不同个体来源的人体细胞重编程效率不同的机制:(4)基于以上探索,最后,以符合《组织捐献条例》的人包皮成纤维细胞为供体细胞,在符合GMP环境规范和操作规范的条件下和Xeno-free培养液中从头建立非整合型临床级别hiPSCs,并对其细胞特性,分化能力和生物安全性做全面检测。上述研究结果表明:(1)在重编程之前,将悬浮生长的hUCBMCs接种到包被MesenCult-XF基质的培养皿中,使其转化成贴壁生长状态;在重编程的过程中,用向贴壁不牢固的供体细胞中加饲养层细胞的方式取代将供体细胞消化下来接种到饲养层细胞上的方式,提高供体细胞贴壁和存活效率;能大幅度提高hUCBMCs的重编程效率和成功率;(2)在采用效率比较高的电转染方法将质粒转染到供体细胞中的情况下,在重编程过程中使用重编程效率比较高的X培养液的前提下,携带Oct4、Sox2、Klf4、Nanog四因子的非整合型质粒(PCEP4-OSKN)能够高效重编程HFF-21WF细胞,诱导效率显著高于已经报道的不含有C-Myc等原癌基因的非整合型质粒的重编程效率,并且可以在不使用任何和表观遗传学调控相关小分子的情况下诱导获得hiPSCs;(3)携带Oct4、Sox2、Klf4、Nanog四因子的非整合型质粒(PCEP4-OSKN)不能成功重编程脐带间充质干细胞(human Umbilical Cord Mesenchymal Stem Cells,hUCMSCs)和其它个体来源的HFF细胞;(4)对四个不同个体来源的 HFF 细胞(HFF-21WF,HFF-19WM,HFF-16WM 和 HFF-6Y)和一个个体来源的hUCMSCs(UCMSC-21WF)进行RNA-Seq测序,数据分析结果表明,能被PCEP4-OSKN重编程的HFF细胞和不能够被PCEP4-OSKN重编程的细胞基因表达差异主要集中在细胞连接分子和TGFβ信号通路上;(5)以符合《组织捐献条例》的在Xeno-free培养液中和符合GMP操作规范和环境规范的条件下原代和传代培养的人包皮成纤维细胞为供体细胞,在Xeno-free培养液中和符合GMP操作规范和环境规范的条件下能够成功建立非整合型临床级别hiPSCs;随机挑选的一株临床级别hiPSCs能够在Xeno-free培养液中和符合GMP操作规范和环境的条件下被定向诱导分化形成多巴胺能神经前体细胞和心肌细胞。生物安全性检测结果表明我们建立的临床级别hiPSCs是安全的。该研究可以为未来hiPSCs的临床应用提供较好的种子资源细胞和重编程方法。更多还原

【Abstract】 In Greek mythology,the Titans Prometheus’ liver was constantly pecked by the Condor bucause of Zeus’ punishment.Amazingly,his liver could constantly regenerate after being pecked,so his life could be maintained.In modern society,patients with organ failure caused by chronics and organ injury caused by accidents are increasing year by year.Faced with these diseases,the existing traditional medical methods often seem powerless.So turning myth into reality is urgently needed.Regenerative medicine is the discipline that studying replacement of damaged cells,tissues or organs with normal ones to cure diseases,and it is an important medical research direction in the future.The shortage of cells,tissues or organs is one of the most important factors limiting the development of regenerative medicine.Human Pluripotent stem cells(hPSCs)can go indefinite self-renewal and hold the potential to give rise to all cell types in the human body,thus could continuously provide various types of cells,tissues and organs for regenerative medicine theoretically and have great prospects.Since the first human pluripotent stem cell line-human embryonic stem cells(hESCs)was established by Thomson laboratory in 1998,scientists have continued to explore the clinical application of hPSCs and have made a series of achievements.Since hESCs are derived from early human embryos,which is related to ethical issues,the basic research and clinical application of hESCs are controversial.Human induced pluripotent stem cells(hiPSCs),which share identical cell characteristics and clinical application values with hESCs,are derived by delivering exogenous genes into somatic cells or treating somatic cells with chemical molecules,so there are no ethical issues involved in hiPSCs.Low reprogramming efficiencies,exogenous genes randomly integration and spontaneously activation,usage of animal-derived reagents,genomic and epigenomic instability and others are major factors that affect clincial application of hiPSCs.With the development of technologies and theories in the basic research of hiPSCs,it is time to pave the way for their clinical applications.Now scientists in Japan are doing a hiPSCs based clinical trial,which uses Retinal Pigmented Epithelial cells(RPE)differentiated from hiPSCs for autologous transplantation to treat age-related macular degeneration[1]Until now,there are no side effects observed in patients who have received cell transplalntation and the patients’ eyesights have improved to some extent.To ensure the safety,effectiveness,traceability,reproducibility and legality of hiPSCs intended for clinical trials or therapies,the following principles should be followed:(1)Parental cell donation process must comply with the "Guidelines for Human Somatic Cell Therapies and Quality Control of Cell-based Products" and the "Tissue Donor Guidance".(2)Parental cells must be easy to obtain.(3)In clinical applications,cells from old individuals which are difficult to be reprogrammed will be inevitably involved,so high reprogramming efficiency are required to ensure success rate.(4)To avoid insertional mutations caused by exogenous genes random integration and tumorigenicity caused by exogenous genes spontaneous activation,hiPSCs intended for clinical researches or therapies should be exogenous factors free.(5)According to the current national and international regulation policies,most countries require a Good Manufacturing Practice(GMP)environment and operation procedure when handling the cells for clinical applications.So operation procedures and manufacturing environments of parental cell collection and cultivation,hiPSCs induction,hiPSCs differentiaions should be comply with the current GMP standards.(6)To avoid cross-transmission of animal pathogens between humans and animals,it is better to avoid using ingredients of animal origin in the cell culturing process.(7)Bacteria,fungi,mycoplasma and some types of pathogen must be detected to ensure the biological safety of clinical-grade cells.Previously,one group indicated that hiPSCs could be derived under fully defined conditions from the very beginning,however,whether the obtained iPSC lines were biologically safe or the"tissue donor guidance" was followed were not described.Several other groups have also successfully achieved the clinical-grade hiPSC lines by converting the existing non-clinical-grade hiPSC lines.However,in this process animal products were applied at the beginning,which could cause the risk of infectious agents transfer across-species.So it is urgent to derive clinical-grade hiPSC lines in GMP and Xeno-free conditions following the "tissue donor guidance" from the very beginning and evaluate the safety of the cell lines strictly.Based on the basic requirements and the current progresses in clinical-grade hiPSCs,we conducted the following researches:(1)Firstly,we optimized the reprogramming process from human umbilical cord blood mononuclear cells(hUCBMCs),which are easy to obtain,to hiPSCs,and achieved high reprogramming efficiency and success rate.(2)Secondly,we optimized the process of non-integrated plasmid mediated reprogramming.Without the proto-oncogene C-Myc,we sucessfully reprogrammed human fetal fibroblasts(HFF)into hiPSCs by a non-integrated plasmid carring Oct4、Sox2、Klf4 and Nanog with high efficiency.(3)In the process of optimizing non-integrated plasmid mediated reprogramming,we found that reprogramming efficiencies differs in HFF cells from different individual in an age independent manner.Then we conducted RNA-Seq among these cells and analyzed their gene expression profiles to identify the underlying mechanisms that leading to the different reprogramming efficiencies.(4)Finally,we sucessfully established several integration-free clinical-grade hiPSC lines in GMP and Xeno-free conditions following the "tissue donor guidance" from the very beginning and tested their cell characteristics,differentiation ability and biological safety comprehensively.Our results showed that:(5)Before reprogramming,converting the parental hUCBMCs from suspension culture state into adherent state by coating the Petri dish with MesenCult-XF attachment substrates,and during reprogaming,adding feeder cells to the lossely attached hUCBMCs instead of seeding the hUCBMCs onto feeder cells can greatly improve the reprogramming efficiency and success rate from hUCBMCs to hiPSCs.(6)HFF-21WF cells can be reproammed to hiPSCs by the non-integrated plasmid carrying Klf4 and VP 16 engineered Oct4,Sox2 and Nanog(PCEP4-OSKN)efficiently in the case of using the highly efficient electrotransfection gene transfection method for exogeneous genes delivery and using X medium during the reprogramming process.Our reprogramming efficiency was significantly higher than other episomal mediated oncogene-free reprogramming efficiencies,and hiPSCs lines can be established without the use of small molecules involved in epigenentic regulation.(7)The non-integrated plasmid,PCEP4-OSKN,could reprogram HFF-21WF efficiently.However,it could not fully reprogram UCMSC-21WF and and other HFF cells from different individuals.(8)RNA-Seq results of the four HFF ceels(HFF-21WF,HFF-19WM,HFF-16WM and HFF-6Y)and the human umbilical cord mesenchymal stem cells(UCMSC-21WF)indicate that differentially expressed genes between HFF-21 WF cell line(with high reprogramming efficiency)and other cell lines(with low reprogramming efficiency)are mainly cell junction related genes and TGFβ signaling related genes.(9)Integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture could be suceessfully established in line with the current guidance of international and national evaluation criteria.Neural cells and cardiomyocytes could differentiate from the established hiPSCs in Xeno-free culture medium.The established hiPSCs were biologically safe.This study may provide good seed cell source and reprogramming method for future hiPSCsbased clinical trials or therapies.更多还原