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标题: 【Nature】iPS细胞的未来渺茫啊！Stem cells: The dark side of induced pluripotency [打印本页]

作者: linlicau 时间: 2011-3-3 13:28 标题: 【Nature】iPS细胞的未来渺茫啊！Stem cells: The dark side of induced pluripotency

News & Views

http://www.nature.com/nature/journal/v471/n7336/full/471046a.html
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Induced pluripotent stem cells have great therapeutic potential. But genomic and epigenomic analyses of these cells generated using current technology reveal abnormalities that may affect their safe use.
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See also Article by Hussein et al.
See also Article by Gore et al. 7 l5 H9 }" Z+ x7 z. U
See also Article by Lister et al.
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Induced pluripotent stem cells (iPSCs) are generated through the reprogramming of differentiated adult cells and can be coaxed to develop into a wide range of cell types. They therefore have far-reaching potential for use in research and in regenerative medicine. But the ultimate value of these cells as disease models or as sources for transplantation therapy will depend on the fidelity of their reprogramming to the pluripotent state, and on their maintenance of a normal genetic and epigenetic (involving aspects other than DNA sequence) status. Five recent surveys1, 2, 3, 4, 5, including three in this issue1, 2, 3, show that the reprogramming process and subsequent culture of iPSCs in vitro can induce genetic and epigenetic abnormalities in these cells. The studies raise concerns over the implications of such aberrations for future applications of iPSCs.
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It has long been known6 that, during cultivation in vitro, human embryonic stem cells (ESCs) can become aneuploid; that is, they acquire an abnormal number of chromosomes. The new papers have applied various state-of-the-art genomic technologies to assess in detail the occurrence and frequency of genetic and epigenetic defects in both human iPSCs and ESCs.% t! |, f8 N# ~. _

Hussein et al.1 ( page 58) studied copy number variation (CNV) across the genome during iPSC generation, whereas Gore and colleagues2 ( page 63) looked for point mutations in iPSCs using genome-wide sequencing of protein-coding regions. Lister et al.3 ( page 68) examined DNA methylation — an epigenetic mark — across the genomes of ESCs and iPSCs at the single-base level. These studies, along with other investigations into changes in chromosome numbers4 and CNV5 in the two kinds of stem cell, lead to the conclusion that reprogramming and subsequent expansion of iPSCs in culture can lead to the accumulation of diverse abnormalities at the chromosomal, subchromosomal and single-base levels. Specifically, three common themes, regarding the genetic and epigenetic stability of ESCs and iPSCs, emerge.4 J$ w, w% t# A$ ^8 D0 j
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First, by several measures, iPSCs display more genetic and epigenetic abnormalities than do ESCs or fibroblasts — the cells from which they originated. Chromosomal abnormalities appear early during the culturing of iPSCs5, a phenomenon not generally observed in ESCs. Also, the frequency of mutations in iPSCs is estimated to be ten times higher than in fibroblasts2. And there are greater numbers of novel CNVs (CNVs not found in the cell of origin or in human genomes of comparable background) in iPSCs than in ESCs1, 5. Similarly, the epigenome of iPSCs features incomplete reprogramming (with cells retaining epigenetic marks of the cell of origin), aberrant methylation of CG dinucleotides, and abnormalities in non-CG methylation — an epigenetic feature seen only in pluripotent cells3./ b- t. p! t: S$ \& W) G( c

Second, the studies show that genetic abnormalities can arise at different stages of iPSC generation. Some lesions are inherited from the cell used for reprogramming. Gore et al.2 employ a particularly sensitive approach to demonstrate that a subset of point mutations found in iPSC lines pre-existed in a small minority of fibroblasts used for reprogramming. Other lesions seem to arise early on in reprogramming, as mentioned previously. For example, Hussein et al.1 found large numbers of new CNVs during early passages (subcultures) following reprogramming, but noted that subsequent growth in vitro seemingly selected against most of the changes, which implies that they are deleterious for the cells that bear them. The studies also report changes that apparently relate to long-term adaptation to cell culture. These include over-representation either of the short arm of chromosome 12 (12p) or of this entire chromosome4, 5, and of a subregion in the long arm of chromosome 20 (ref. 5). Both of these changes have been observed6 in ESC lines, with an increased number of 12p being a hallmark of testicular germ-cell tumours — the malignant prototype of human pluripotent stem cells.) |1 Q6 M  d" N* v

Third, several of the groups2, 4, 5 report clues to the potential function of the genetic lesions that arise in ESCs and iPSCs. For example, regions prone to amplification, deletion or point mutation seem to be enriched in genes involved in cell-cycle regulation and cancer. Although the changes observed do not strongly implicate any particular gene functionally as a target for change during the amplification of iPSCs or during their adaptation to culture conditions, the frequent association of the affected genes with cancer gives cause for concern.% T) ?& _4 ?7 O% }$ L5 ?  L

This highly significant body of data1, 2, 3, 4, 5 provides a revealing, in-depth portrait of the status of the genome and the epigenome during cellular reprogramming. But it also leaves open some fairly challenging questions.
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The studies provide little insight into the crucial question of what aspects of the reprogramming methods might predispose the cells to the accumulation of recurrent genetic or epigenetic lesions. Although recurrence of change in specific genomic regions across a number of cell lines strongly implies a selective process, in several studies the researchers noted that there was no obvious correlation between the extent of genetic damage in a given population of reprogrammed cells and the methods used for their reprogramming or propagation. Hussein et al.1 provide some evidence that CNV occurred more frequently at sites prone to replication stress. It is not clear, however, whether this stress is unique to the reprogramming process, or whether it would be common to any experimental situation in which a cultured cell is subjected to strong selection and replication pressures in vitro.

Despite extensive evaluation of recurrent genetic change in a vast number of cell lines, we are only slightly closer to identifying which particular genes within the larger chromosomal regions that are commonly subject to duplication in iPSCs and ESCs might be under selection. Years of cytogenetic studies of germ-cell tumours have also identified large genomic regions that are commonly over-represented in these cancers, but the identification of the specific genes involved in the transformation of these pluripotent cells has remained elusive6. A possible interpretation of the data on the genetics of germ-cell tumours is that multiple genetic regions, or large regulatory regions, are crucial to the process of oncogenesis in vivo. Perhaps a similar mechanism is in play during in vitro adaptation of ESCs or iPSCs.) f5 @( k- ~& e% [! }: _9 U
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With regard to evaluating the safety of ESCs and iPSCs, a key issue is the biological significance of the changes that these studies1, 2, 3, 4, 5 report. Clearly, aneuploid cell lines would not be used in therapy (although they might be useful for research into the basis of genetic disorders associated with anomalies in chromosome number or other genetic abnormalities). Cell lines bearing mutations of established functional consequence in oncogenes or tumour suppressors, or in genes associated with Mendelian disorders (those usually due to a single gene), could equally not be used therapeutically. However, the many subchromosomal changes, CNVs or point mutations that are not obviously associated with known disease-related genetic abnormalities pose challenges to interpretation. This is because it is unclear how best to assess the effects of new genetic lesions on the growth, differentiation, tumorigenicity and functionality of pluripotent stem cells or their differentiated progeny. High-throughput functional genomics will probably be required to answer these questions. Pluripotent cells themselves will provide the most promising platform for such studies.

补充内容 (2011-3-3 15:16):- o) i: u# u4 g" ~5 f/ a0 d
不过对于不同方法的iPS可能还会有不同的结果。

作者: tpwang 时间: 2011-3-3 14:02

回复 linlicau 的帖子- R9 l/ t$ ~# U! ^1 j; V
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上一次的评论还是Flaw in inducedstem-cell model，这次升级成The dark side of induced pluripotency，再发现些什么，是否就成了Deadly reprogramming，最后就干脆Doomed iPS了：）

发现一大堆问题，后面的研究倒是有很多事情可做了。把基因和表观遗传变异搞清楚后的最大的问题其实是it is unclear how best to assess the effects of new genetic lesions on the growth, differentiation, tumorigenicity and functionality of pluripotent stem cells or their differentiated progeny.即怎么去验证这些变异的后果，还不知道。 X$ x$ ~; a( f

作者: have-a-rest 时间: 2011-3-3 14:13

当一个评定体系下认为女生都是美女时，这个评价体系有点问题。" F, B( E4 p" w- D; n' L
ips自身没错，也许是借鉴的ES的评价体系误诊了许多特殊群体细胞。

作者: sunsong7 时间: 2011-3-3 14:36

天使还是魔鬼，认清iPS的真实面目有利于人们尽早丢掉侥幸的幻想，iPS是一场奢华的豪赌！

作者: 心醉夕阳1 时间: 2011-3-3 17:46

回复 sunsong7 的帖子: I2 p$ e! l& U' g% P# m
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一花一世界8 v4 [) l' J) [2 Q( {: I k
一叶一乾坤

作者: xiaoheilong 时间: 2011-3-3 18:12

iPS的未来会怎么样呢，值得期待

作者: tpwang 时间: 2011-3-3 22:11

回复心醉夕阳1 的帖子, a7 p+ _: e& K4 W

hehe,
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One cell one world+ ~. Z& s9 Y+ {0 g9 r
One gene one life

作者: inankai 时间: 2011-3-4 03:35

应该是“一花一世界，一叶一菩提”吧

作者: fananran2003 时间: 2011-3-4 08:32

个人认为iPS表观机制的阐明，将会影响iPS的后续研究及其作用的评价起到导向作用！

作者: songzxj 时间: 2011-3-4 12:29

have-a-rest 发表于 2011-3-3 14:13
0 O' k3 K. P1 |' S# H当一个评定体系下认为女生都是美女时，这个评价体系有点问题。8 G) L& p* Z/ z# z0 O
ips自身没错，也许是借鉴的ES的评价体系误诊 ...

赞同此观点，这个评价体系可能还是有些问题。- n7 M- N3 v' C& O/ G' m8 U
另外，既然IPS可以通过四倍体补偿，克隆的方法获得后代，说明重编程后获得的IPS也和体内的其它细胞一样，它的基因组并不是混乱的，它的问题出现在个别基因的开启闭合上，这些应该问题将来应该可以解决的。

作者: songzxj 时间: 2011-3-4 12:34

IPS的出现毕竟还是给我们提供了一个新的解决ES所不能解决的思路，虽然还有很多基因和表观遗传学的问题需要解决，但是我还是相信它的未来不会错，它不会昙花一现！

作者: songzxj 时间: 2011-3-4 12:35

IPS的出现毕竟还是给我们提供了一个新的解决ES所不能解决的思路，虽然还有很多基因和表观遗传学的问题需要解决，但是我还是相信它的未来不会错，它不会昙花一现！

作者: wujunyu 时间: 2011-3-4 17:39

应该说还有很多东西没弄明白，我们在这方面还有很多机遇

作者: dabai22222 时间: 2011-3-7 11:22

这里的评论也很谨慎吧，只是把这期发表的几篇paper的观点又复述了一遍而已，我觉得所谓的dark side可能只是说目前我们还不清楚的ips的一面

作者: 573639471 时间: 2011-3-7 12:32

认识越来越全面了

作者: 希望之光 时间: 2011-3-9 21:14

我觉得吧无论是IPS还是ESCs,大家都把他们得利用价值看的过高了，这就好比看到天上的星星就好像看到了整个银河系甚至是整个宇宙，就目前大家研究的领域来说，仅仅是冰山一角，所以很难就某些已经知道的东西来断定他就能有用或者没用，万事都是有联系得，只能说明这条路很艰辛。所以革命尚未成功，同志任需努力6 r4 f7 E# L6 N
:)

作者: haoyan222 时间: 2011-3-18 09:51

ips在再生医学方面的潜力还是巨大的，但是我们现在还是没有找到准确的利用ips细胞的最佳方法

作者: zsycgz 时间: 2011-3-18 10:35

很多iPSC株经过基因测定后发现产生的不仅仅是表观遗传学层面的变化，而是部分基因组也发生了变化，这种变化可能是病毒基因组的插入造成的，也可能是经过大量的小分子物质、转录因子等“暗箱操作”造成的。到底是什么还没有搞清楚。但是iPSC一方面是为了进行细胞替代治疗，现有的一些结果阻碍了这方面的作用；一方面也客观上对细胞到底是怎么进行分化的机制进行研究探讨，逐步的解开细胞基因-转录-翻译以及细胞信号转导系统的错综复杂的控制谜团或者说the mechanism controlled by god。科学研究成果不光是依据能不能很有临床应用价值，还要看它阐明的什么我们没有搞清楚的假说或机制，人们研究果蝇不也是一种工具么。我相信iPSC的研究还能够给我们带来更多的有用的东西，不要只把目光集中在细胞治疗上。个人不成熟的观点，欢迎拍砖。

作者: chips100 时间: 2011-3-18 12:10

很奇怪为什么一个dark side就引起了iPS如此多的负面评价。诚然iPS后突变率提高，拷贝变异，但引起的功能变化大吗？比如cell paper提到的reference，通过筛选把分化倾向性强的iPS系选出来就可以了。那篇nature biotechnology的功能评价也说明对于分化的影响有限。做细胞治疗毕竟不需要直接注射干细胞性质的细胞，而只需要分化细胞，那么，突变的分化细胞影响会有多大呢，能完成功能即可。在nature biotechnology的一片review曾经提到，比起风险来说，需要看带来的收益是否大。至少在诸如帕金森，糖尿病中，iPS是有潜力的。当然，长期风险仍然需要评估。

作者: linlicau 时间: 2011-3-21 13:15

回复 zsycgz 的帖子, H. E) I+ X4 m3 J1 i8 L: p
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iPSC很重要，只是可能并不像我们想象中的那么重要，就像基因组测序起初被渲染的那样，如果测完，就可以治疗人类癌症或者其他疾病了。其实，还任重而道远。

作者: zsycgz 时间: 2011-3-22 14:03

回复 linlicau 的帖子
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是的，iPSC只是认识细胞分化或者信号通路的重要工具，最终人类将通过这些工具，自然的高效的人工的对细胞进行调节，让细胞生长成我们需要的细胞，这是终极目标。但是在这之前，有些研究还是可以继续下去，积累经验，不管是正面的还是走了弯路的，科研就是这样，没有绝对的有意义的和没意义的。现在感觉诱导分化越来越热，但是很多诱导出来的细胞功能上和自然状态下的正常细胞存在很大的差距，比如iPSC诱导的肝样细胞ALB产生和P450等功能上都有几个数量级的差距，我觉得还是应该对自然状态下的分化再深入研究，现有的文献好多都是重复别人的工作，仅仅是进行少量的改进，很少有很深入的研究分化机制的文献，可能是我看的太少了吧，还要再努力。

作者: wubihappy 时间: 2011-3-22 14:13

一沙一世界，一叶一菩提

作者: qizhi502 时间: 2011-3-23 10:11

在定义ips渺茫时，大家用的都是人的ips的，很少用小鼠ips来说明其渺茫，因为有足够的手段可以找出和ES相同的小鼠ips，而当轮到对人的ips甚至ES进行评价时，科研工作者有些力不从心，没有一个很好的手段去证明。不像非灵长类动物，可以做生殖系嵌合，也可以做四倍体嵌合，但是对人却不可以，所以当大家在用一个目前认为已鉴定甚至根本没有鉴定完全（只是做了AP,pluripotent factors,teratoma,microarray）的hips去验证一些东西，这已经存在的选材的错误，所得结论也不能完全信之。

作者: FreeCell 时间: 2011-3-23 15:34

嘻嘻，对iPS的临床应用抱有任何不切实际的幻想都是浪费资源，这等于拿癌细胞去治病，好像还真有人这样做唉！

作者: zouwowo 时间: 2011-3-24 15:08

:'(用了几个包包下载的paper咋就打不开呢？

作者: snokey 时间: 2011-3-25 04:46

iPS和当年的转基因技术，knockout技术，siRNA技术一样，热闹一阵回归平淡之后才能真正体现它们的价值。现在转基因动/植物，knockout的老鼠，还有siRNA已经成为日常实验不可缺少的实验技术和平台。我觉得没有任何实验技术是完美的，iPS将来一定会成为一个有用的platform，问题在于目前过于强调regeneration medicine，泡泡吹得太大就容易破。

作者: 细胞海洋 时间: 2011-3-25 15:41

回复 zouwowo 的帖子
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不要使用迅雷使用默认下载

作者: whe022 时间: 2011-3-30 23:40

请问linlicau 有文章的supplementary informations吗？谢谢:)

作者: linlicau 时间: 2011-3-31 09:55

回复 whe022 的帖子
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具体哪篇？您可以在求助版块求助。

作者: whe022 时间: 2011-4-3 15:56

回复 linlicau 的帖子

ArticleHussein, S. M. et al. Copy number variation and selection during reprogramming to pluripotency. Nature 471, 58–62 (2011)的supplementary informations, 谢谢～～

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