Cell Stem Cell, E-pub Dec. 19, 2013. In Vivo Direct Reprogramming of Reactive Glial Cells into Functional Neurons after Brain Injury and in an Alzheimer’s Disease Model Ziyuan Guo, Lei Zhang, Zheng Wu, Yuchen Chen, Fan Wang, and Gong Chen. http://dx.doi.org/10.1016/j.stem.2013.12.001 Nature Research highlight: http://www.nature.com/nature/journal/v505/n7482/full/505134b.html News press: http://medicalxpress.com/news/2013-12-brain-injury-alzheimer-disease.html Science China (Chinese 中文）： http://www.sciencemagchina.cn/highlights131220.aspx Highlights • Conversion of reactive glial cells into functional neurons in vivo using NeuroD1 • NeuroD1 induces glia-neuron conversion in an Alzheimer’s disease mouse model • NeuroD1 also reprograms NG2 cells into glutamatergic and GABAergic neurons • Human astrocytes can also be converted into glutamatergic neurons with NeuroD1 Summary Loss of neurons after brain injury and in neurodegenerative disease is often accompanied by reactive gliosis and scarring, which are difficult to reverse with existing treatment approaches. Here, we show that reactive glial cells in the cortex of stab-injured or Alzheimer’s disease (AD) model mice can be directly reprogrammed into functional neurons in vivo using retroviral expression of a single neural transcription factor, NeuroD1. Following expression of NeuroD1, astrocytes were reprogrammed into glutamatergic neurons, while NG2 cells were reprogrammed into glutamatergic and GABAergic neurons. Cortical slice recordings revealed both spontaneous and evoked synaptic responses in NeuroD1-converted neurons, suggesting that they integrated into local neural circuits. NeuroD1 expression was also able to reprogram cultured human cortical astrocytes into functional neurons. Our studies therefore suggest that direct reprogramming of reactive glial cells into functional neurons in vivo could provide an alternative approach for repair of injured or diseased brain.
【点评】 2012年诺贝尔生理学或医学奖已于昨日公布，名花有主归英国人戈登（“咯噔”）和日本人山中伸弥（“山中生霉”），其获奖理由是“发现成熟细胞可被重编程（reprogrammed）变成多能性（pluripotent）”。 由于我们今年中了一项有关“肿瘤干细胞”起源的国家自然科学基金面上项目，因而我对“重编程”和“多能性”的内涵也很感兴趣，于是想跟同行们分享其生物学意义及医学应用前景。恰好遇ScienceDaily发表了一篇相关文章，我就此点评一番，配以自己的理解和体会。 首先申明，我不是这方面的专家，一切知识皆来自网络，融合了一些过去做植物细胞全能性研究的经验，本人现买现卖，一方面让自己更新知识，另一方面希望对同行有所启发。当然，谬误、不当之处在所难免，敬请各位专家不吝批评指正！ 就在山中伸弥出生的1962年，戈登（1933年出生）就发现细胞的分化（专门化）是可以被逆转的。他将爪蟾成熟的小肠细胞核取出，注射到卵细胞去核的未成熟细胞内，结果使这些修饰过的卵细胞全部发育成蝌蚪。 这个经典实验表明，成熟细胞的DNA仍然保留了发育成各种细胞的信息，并非单向性，也并非不可逆转。 在40年后的2006年，山中伸弥创造性地发明了如何将小鼠完整的成熟细胞实现重编程而转变成未成熟干细胞的技术。令人惊讶的是，他仅仅在成熟细胞中导入4个转录因子基因（Oct4、Sox2、cMyc、Klf4），就能将它们重编程而成为多能干细胞（pluripotent stem cell，PSC）。所谓的PSC就是能发育成各种细胞类型乃至组织、器官和个体的未成熟细胞。 用计算机程序做比喻，重编程就是把已经被修改了的程序恢复到默认（缺省）状态，多能性就是从默认状态出发衍生出无数不同的新程序。更形象地说，重编程可使成年人变回婴儿状态，让“一张白纸可以画最新最美的图画”；多能性就是赋予这种“返老还童”以可能性，正如在这张白纸上有人喜欢画画，有人喜欢写字！ 至于重编程的机理就十分复杂，可能涉及表观遗传控制。记得看过一份文献上曾说，随着年龄的增长，人的基因组甲基化程度是逐步升高的。对双胞胎的研究表明，甲基化修饰还受到环境的影响，导致双胞胎的甲基化程度存在差异。这似乎能解释双胞胎中的一个患上某种表观遗传疾病，而另一个则不患这种病。 利用为数不多的转录因子基因导入成熟细胞而获得的诱导性多能干细胞（iPSC）可以很好地解决干细胞研究中细胞来源的伦理问题，也为有效地进行个性化干细胞治疗奠定了基础。1999年12月干细胞研究在Science公布的年度十大科学突破中名列榜首，2007年再度被Science杂志评为世界十大科学突破之一。 为什么细胞重编程及多能性的发现被定义为重大突破呢？因为一直以来人们虽然认为动物细胞像植物细胞一样具有“全能性”（totipotency），但只能在体外将分化的植物成熟细胞（如根茎叶等）培养成苗，而无法将分化的动物成熟细胞（如皮肤）培育成完整的个体。也就是说，以往动物细胞仅仅具有理论上的全能性。 戈登发明的核移植技术可以说是现代克隆动物培育的基础。根据克隆动物培育的细胞来源不同，该技术包括以下4种不同情况： 1）胚胎细胞和胚胎干细胞克隆，如1999年获得的克隆小鼠胡博； 2）胎儿体细胞克隆，如法国的克隆牛玛格丽特（1998）、美国的克隆牛乔治和查尔斯（1998）、新西兰的克隆牛吉尔、中国的克隆山羊（1998）、日本的克隆猪（2000）； 3）成年体细胞克隆，如来自乳腺细胞的克隆羊多莉（1997）、来自输卵管上皮细胞和卵丘细胞的克隆牛（1998）、来自乳腺上皮细胞、肌细胞、皮肤及耳成纤维细胞的克隆牛（1999）、来自成纤维细胞和颗粒细胞的克隆山羊（中国，2000）、来自粒细胞的克隆猪（2000，英国）、体细胞克隆小鼠（澳大利亚，2001）、矮山羊（加拿大，2001）和牛（中国，2001）。 4）转基因成体或胎儿细胞克隆，如1997年6月英国PPL制药公司的科学家与英国罗斯林研究所的威尔穆特研究小组合作，用绵羊转基因胚胎细胞克隆出表达人凝血因子9的转基因克隆绵羊波莉。1997年12月，他们又报道用绵羊转基因胎儿成纤维细胞克隆出表达人抗胰蛋白酶的转基因克隆绵羊、表达半乳糖苷酶的转基因克隆牛、表达抗凝血酶3的转基因克隆山羊。2000年，他们以原胶原蛋白基因为靶基因，通过人抗胰蛋白酶基因对胎儿成纤维细胞的基因打靶，已培育出能在乳腺表达人抗胰蛋白酶的基因打靶克隆绵羊，羊奶中人抗胰蛋白酶含量高达650微克/毫升，比上述转基因克隆绵羊要高出35倍！ 山中伸弥利用基因导入逆转分化细胞的创意是独创的，但相关技术得益于1982年帕米特尔的转基因“超级鼠”培育技术和1985年哈默的转基因兔、羊、猪培育技术，也可能受威尔穆特转基因克隆绵羊波莉培育技术的启发。 尽管多莉羊和波莉绵羊都能正常分娩和成长，但衰老体细胞的年龄决定了克隆动物的年龄。例如，多莉羊出生后不久就因早衰而很快死去。 山中伸弥的重编程技术就能将“老年”体细胞拉回到“童年”，可以说是真正的“起死回生”和“返老还童”！ 尽管iPSC研究实现了前所未有的突破，但还有一些问题有待进一步解决，至少应该涉及以下两个方面： 1）iPSC与内源性胚胎干细胞仍然有很大的不同，包括基因组的甲基化分布及略有延长的细胞周期，这些差异对发育的影响尚不十分清楚。 2）这些导入的转录因子在体细胞重编程过程中的作用机理有待进一步研究，阐明其对所调控的下游基因及其作用机制可能对于进一步理解体细胞重编程有所帮助，并将为提高iPSC的体外诱导效率提供依据。 最后，为当时因培育多莉羊而轰动天下的威尔穆特鸣不平，他为何不能与戈登和山中伸弥一起获得今年的诺贝尔生理学或医学奖呢？ 附： http://www.sciencedaily.com/releases/2012/10/121008082955.htm Nobel Prize in Physiology or Medicine 2012 Awarded for Discovery That Mature Cells Can Be Reprogrammed to Become Pluripotent ScienceDaily (Oct. 8, 2012) — The Nobel Assembly at Karolinska Institutet has decided to award The Nobel Prize in Physiology or Medicine 2012 jointly to John B. Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent. The Nobel Prize recognizes two scientists who discovered that mature, specialised cells can be reprogrammed to become immature cells capable of developing into all tissues of the body. Their findings have revolutionised our understanding of how cells and organisms develop. John B. Gurdon discovered in 1962 that the specialisation of cells is reversible. In a classic experiment, he replaced the immature cell nucleus in an egg cell of a frog with the nucleus from a mature intestinal cell. This modified egg cell developed into a normal tadpole. The DNA of the mature cell still had all the information needed to develop all cells in the frog. Shinya Yamanaka discovered more than 40 years later, in 2006, how intact mature cells in mice could be reprogrammed to become immature stem cells. Surprisingly, by introducing only a few genes, he could reprogram mature cells to become pluripotent stem cells, i.e. immature cells that are able to develop into all types of cells in the body. These groundbreaking discoveries have completely changed our view of the development and cellular specialisation. We now understand that the mature cell does not have to be confined forever to its specialised state. Textbooks have been rewritten and new research fields have been established. By reprogramming human cells, scientists have created new opportunities to study diseases and develop methods for diagnosis and therapy. Life -- a journey towards increasing specialisation All of us developed from fertilized egg cells. During the first days after conception, the embryo consists of immature cells, each of which is capable of developing into all the cell types that form the adult organism. Such cells are called pluripotent stem cells. With further development of the embryo, these cells give rise to nerve cells, muscle cells, liver cells and all other cell types -- each of them specialised to carry out a specific task in the adult body. This journey from immature to specialised cell was previously considered to be unidirectional. It was thought that the cell changes in such a way during maturation that it would no longer be possible for it to return to an immature, pluripotent stage. Frogs jump backwards in development John B. Gurdon challenged the dogma that the specialised cell is irreversibly committed to its fate. He hypothesised that its genome might still contain all the information needed to drive its development into all the different cell types of an organism. In 1962, he tested this hypothesis by replacing the cell nucleus of a frog's egg cell with a nucleus from a mature, specialised cell derived from the intestine of a tadpole. The egg developed into a fully functional, cloned tadpole and subsequent repeats of the experiment yielded adult frogs. The nucleus of the mature cell had not lost its capacity to drive development to a fully functional organism. Gurdon's landmark discovery was initially met with scepticism but became accepted when it had been confirmed by other scientists. It initiated intense research and the technique was further developed, leading eventually to the cloning of mammals. Gurdon's research taught us that the nucleus of a mature, specialized cell can be returned to an immature, pluripotent state. But his experiment involved the removal of cell nuclei with pipettes followed by their introduction into other cells. Would it ever be possible to turn an intact cell back into a pluripotent stem cell? A roundtrip journey -- mature cells return to a stem cell state Shinya Yamanaka was able to answer this question in a scientific breakthrough more than 40 years after Gurdons discovery. His research concerned embryonal stem cells, i.e. pluripotent stem cells that are isolated from the embryo and cultured in the laboratory. Such stem cells were initially isolated from mice by Martin Evans (Nobel Prize 2007) and Yamanaka tried to find the genes that kept them immature. When several of these genes had been identified, he tested whether any of them could reprogram mature cells to become pluripotent stem cells. Yamanaka and his co-workers introduced these genes, in different combinations, into mature cells from connective tissue, fibroblasts, and examined the results under the microscope. They finally found a combination that worked, and the recipe was surprisingly simple. By introducing four genes together, they could reprogram their fibroblasts into immature stem cells! The resulting induced pluripotent stem cells (iPS cells) could develop into mature cell types such as fibroblasts, nerve cells and gut cells. The discovery that intact, mature cells could be reprogrammed into pluripotent stem cells was published in 2006 and was immediately considered a major breakthrough. From surprising discovery to medical use The discoveries of Gurdon and Yamanaka have shown that specialised cells can turn back the developmental clock under certain circumstances. Although their genome undergoes modifications during development, these modifications are not irreversible. We have obtained a new view of the development of cells and organisms. Research during recent years has shown that iPS cells can give rise to all the different cell types of the body. These discoveries have also provided new tools for scientists around the world and led to remarkable progress in many areas of medicine. iPS cells can also be prepared from human cells. For instance, skin cells can be obtained from patients with various diseases, reprogrammed, and examined in the laboratory to determine how they differ from cells of healthy individuals. Such cells constitute invaluable tools for understanding disease mechanisms and so provide new opportunities to develop medical therapies. Sir John B. Gurdon was born in 1933 in Dippenhall, UK. He received his Doctorate from the University of Oxford in 1960 and was a postdoctoral fellow at California Institute of Technology. He joined Cambridge University, UK, in 1972 and has served as Professor of Cell Biology and Master of Magdalene College. Gurdon is currently at the Gurdon Institute in Cambridge. Shinya Yamanaka was born in Osaka, Japan in 1962. He obtained his MD in 1987 at Kobe University and trained as an orthopaedic surgeon before switching to basic research. Yamanaka received his PhD at Osaka City University in 1993, after which he worked at the Gladstone Institute in San Francisco and Nara Institute of Science and Technology in Japan. Yamanaka is currently Professor at Kyoto University and also affiliated with the Gladstone Institute. Journal References : Gurdon, J.B. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles . Journal of Embryology and Experimental Morphology , 1962; 10: 622-640 Kazutoshi Takahashi, Shinya Yamanaka. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors . Cell , 2006; 126 (4): 663 DOI: 10.1016/j.cell.2006.07.024 Nobel Foundation (2012, October 8). Nobel Prize in Physiology or Medicine 2012 awarded for discovery that mature cells can be reprogrammed to become pluripotent. ScienceDaily . Retrieved October 9, 2012, from http://www.sciencedaily.com /releases/2012/10/121008082955.htm Disclaimer : This article is not intended to provide medical advice, diagnosis or treatment. Views expressed here do not necessarily reflect those of ScienceDaily or its staff. The Nobel Prize in Physiology or Medicine 2012 has been awarded jointly to John B. Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent. 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1. yamanaka缺乏科学实验基本功，所用供试样品细胞未经过分离纯化，是混杂的细胞群，还包括什么muse之类的... 2. 欺世盗名，把人造畸形细胞强说成是类胚胎干细胞：iPS仅仅激活了部分全能性基因，并未对起始细胞的表观遗传特征进行抹除，有意忽略iPSC的表观遗传缺陷... 3. 所谓“返老还童”根本没实现，全能性验证试验具有欺骗性，硬是把胚性微环境中表观遗传的重构说成是iPS的全能性... 4. 哗众取宠，任何有常识的人都知道转分化是条捷径，而yamanaka偏偏多此一举搞所谓“拨回发育时钟”... 5. Yamanaka从未作出过真正的重编程多能干细胞（iPSC），只是硬着头皮把“转基因技术”说成是“细胞重编程”... 6. 和黄禹锡如出一辙，骗取经费； 7. Yamanaka很具有日本人的传统性格，宁死不认错 ！ 【小资料】诱导多能干细胞（induced pluripotent stem cells, iPS cells）最初是日本人 山中申弥 （Shinya Yamanaka）于2006年利用 病毒载体 将四个 转录因子 （Oct4, Sox2, Klf4 和c-Myc）的组合转入分化的体细胞中，使其 重编程 而得到的类似 胚胎干细胞 的一种细胞类型。2007年、2008年和2010年美国《科学》杂志都以iPS细胞倒转“生命时钟”为名将其评选为年度十大科学突破。 【名词解释】 细胞重编程（Reprogramming ） ：对已分化细胞的表观遗传特征抹除和重构（ refers to erasure and remodeling of epigenetic marks, such as DNA methylation , during mammalian development）。 转基因技术（Transgene）：通过自然或其它基因工程手段将基因及遗传物质从一种生物体导入到生另一个生物基因组中（a gene or genetic material that has been transferred naturallyor by any of a number of genetic engineering techniques from one organism to another）； 【寓言故事一】 “重编程”or“转基因”？ 日本男孩Yamanaka偷了邻家的四粒花种（基因：Oct4, Sox2,c-myc 和 Klf4），拌上肥料撒到自家园子里（转基因），花园里长出了原先没有的新花品种（所谓iPSC），邻家找上门来质问，Yamanaka辩称：我只是给花园施了些肥（重编程）； 【寓言故事二】中国男孩指着iPSC问yamanaka说：这家伙怎么不穿衣服？ 【魔鬼词典】重编程多能干细胞（iPS cell）是被转基因、转RNA、转蛋白、转小分子、吃Vc、乏氧等折腾得想让干什么就得干什么的“老顽童”细胞； 【疑问】教父级人物弄出如此低级的错误是主观故意还是客观无能？iPS的概念有无学术底线？
标签: 重编程
