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本帖最后由 tpwang 于 2011-2-27 19:41 编辑 9 }! L! `( [; z' d- {. c" ~9 d
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% k# B! n- s P9 b5 Q干细胞科学家喜欢对公众科普说，干细胞能够再造各种各样的人体细胞（甚至器官）。大多数听众听而信之，感觉很神奇。遇到过少数怀疑心比较重的人会问：干细胞造出来的跟“原来的”细胞一模一样吗？这就难说了，起码是还无法完全证伪，即使放到身体里跟原来的细胞一样有用，也难肯定。其实同样性质的问题还可以问克隆动物，大多数公众对克隆的想法似乎比再造细胞更“激进”，认为克隆出来的一定是一模一样的。7 L. a. O6 n# e0 ]) O
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后来有了iPS，这个问题换成了iPS是否与胚胎干细胞一模一样，这回问问题的变成了科学家里面有“想法”的人。过去一两年有几篇文章从表观遗传层面质疑iPS与胚胎干的相似性，研究表明iPS与胚胎干之间在表观遗传特点上有很大差异。有人认为这是不完全重编程所致，有人认为这里面有来源细胞“无法抹去”的记忆，有研究说iPS带有更多的致癌性基因和更少抑癌基因。老鼠的不完全重编程现象可以似乎通过培养而消除，人的iPS似乎无法做到这一点。有人认为这个差异是个非特异性差异，有人认为iPS与胚胎干是完全不同的”物种”（creature)。有人从更广的角度来看这个问题，即胚胎干与iPS某种意义上都是“人造”的重编程现象，相比较成体干细胞，都带有这样那样的表观遗传的某种“变异”。
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( h: o) {$ Y" V, W# s一些年前有过特定胚层细胞类型之间转分化现象（transdifferentiation），去年出现几例更激进的所谓跨胚层细胞系之间的直接转分化（direct conversion）。目前还没有见有人比较这些直接转分化的来的细胞与胚胎干分化来的、iPS分化来的以及成体细胞之间的差异。再说得进一步，这些个差异是否能“抹平”，是否对后续分化的细胞有不可逆的（负面）影响，这些影响是否会严重干扰大家所预期的细胞移植临床价值，套用一位干细胞科学家的话说：The problem is that we don’t know if any of these differences are going to be consequential.
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iPS出世也第五个年头了，对iPS重编程完全程度也就是iPS是否与胚胎干一样的争议去年直至今年初达到了高峰。去年底接受今年初发表的三篇文章很有代表性，估计还有一些在制造过程中。一篇Salk研究所研究人员发表在一月份Nature上的文章Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells号称第一篇比较了多株iPS、胚胎干和成体细胞表观遗传单碱基分辨率的全基因谱差异，除发现iPS表现出多种更显著的表观遗传水平重编程变异外，更证明这些iPS重编程的表观遗传特征可以在随后分化为胚胎滋养层细胞中系统地存在。这样一来，iPS技术不仅存在体细胞重编程“不理想”的问题，而且再分化为成体细胞也似乎存在问题。这篇文章引起了不小的震动，Nature杂志甚至写了语气颇为负面的编者按。有趣的是，这篇文章的作者里包括了James Thomson。
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R' O$ S9 N( Z; }然而，紧随其后，Nature Biotechnology和Cell有各自分别发表了一篇不同作者但相互关联的文章。这两篇文章都来自波士顿的研究者，说有关联是因为其中一篇文章刻意采用了另一篇文章所用的干细胞系，这样一来两篇不同角度的文章就有了相互旁证的意义。这也预示了很久以来有人一直在推动的模式，即设法避免不同实验室所生产的干细胞系之间的差异对实验结果比较的不利影响。
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Nature Biotechnology的文章题目是A functionally characterized test set of human induced pluripotent stem cells，它通过两个独立的实验室对同一组16株iPS和几株胚胎干细胞分化为功能性运动神经细胞的潜能进行了严格的比较，结果发现其中的13株与胚胎干没有差别，3株效果不好的分化结果也能通过特殊的“拯救”措施而达到满意的效果。其结论是尽管这两种干细胞之间在核型、早期多潜能标记物以及外源基因等方面存在广泛差异，但其分化效率与效果可能没有显著差异。这似乎是对上面提到的iPS与胚胎干之间的差异是否具有必要的“后果”问题的尝试回答。也就是说，iPS与胚胎干之间的差异已是共识，尽管这种差异是否可以用技术手段消除仍有不同意见，甚至这两种干细胞是否等同也有严重分歧，但一些研究者着眼于“终极产品”是否有分别。换句话说，尽管存在差异，如果分化来的细胞产品本身没有明显差别的话，这些“原材料”的差别是否可以相对忽略不计了。当然，这只是一个初步的尝试，与前一篇研究分化为滋养层细胞的目标细胞也不同，尚无法比较。但本研究的作者认为，本项研究所采用的材料即iPS细胞系具有前所未有的内在品质，而且数量足够，同时结果是由两个独立的实验室分别作出的。
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几乎同时，另一篇发表在Cell上的文章Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines采用了颇为复杂的设计与技术，通过20株胚胎干细胞建立了一个DNA甲基化和基因表达变异参考谱系（reference map of variations)，然后将20株胚胎干以及12株iPS干细胞分别与这个参考谱系进行了对比，再将这两大组细胞系与参考谱系的对比差异进行比较。这种策略的好处是显而易见的。结果表明，两者DNA甲基化和基因表达的“全局特征征”（global profiles）颇为相似。每株干细胞与参考谱系的平均差基本相同，尽管iPS的值稍大。而单个基因平均差的对比也呈现类似的结果。在这种普遍的相似性之下，这些繁杂的比较也发现了一些特定iPS干细胞系的个别基因较大变异。这种微小但明显的差异有两种可能的解释，一种是这些差异可能系统地影响所有的iPS细胞系，另一种情况是这些差异只存在于一些“偏离”常态的iPS系。作者采用了将所有的DNA甲基化和基因表达数据分类，然后用来“预测”即辨别iPS与胚胎干。研究者又尝试用上述数据库来区分iPS和胚胎干，结果表明把iPS辨认为胚胎干的几率比反过来的方法要大。随后，研究者将几种genomic assays数据集中起来，建立了一个所谓“deviation scorecard”和一个“lineage scorecard"，用于评估各种多潜能干细胞的质量和使用价值。这里可以提及的一个实验数据，即采用这个“记分卡”，研究者成功地预测了前述研究采用的iPS和胚胎干分化为运动神经细胞的潜能。通过这个繁杂的研究，作者认为不同来源的iPS和胚胎干细胞系不应当被看作两种互不相干或者完全一样的东西，而毋宁是两团有交集的星云。作者的意图是提供一个可用于方便地评估所有不同来源和种类多潜能细胞的重编程质量和潜在应用价值的工具。其价值有待观察。# E( p) ]3 s& {5 L- U" O I* c
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需要指出的是，这两篇文章的研究者们普遍认为iPS与胚胎干的所谓差异未必是严重的问题，后面这篇的作者之一甚至认为还不能说iPS无法最终取代胚胎干（采访新闻语）。以上三篇研究可以看作是关于iPS争论的进一步深入。个人认为建立一个可以用来方便地比较独立研究结果的参考系确实是很必要的，除了显而易见的价值外，也给广大的实验室提供了一个方便的手段，否则很多人玩不起了。
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说到较新的iPS相关研究，不能不提丁盛刚发表在Nature Cell Biology的一篇文章Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy。去年对iPS冲击比较大的除了前述证明iPS与胚胎干的差异以外，就是所谓的“直接转分化”，即把一种成熟细胞直接转为另一种细胞，从而省去了倒回“全能状态”的必要。直接转分化采用的是lineage特异性因子，而丁盛这个conversion，采用的是iPS方法。可以看作是诱导重编程与直接转分化相结合的一个“变种”。这个研究采用所谓经典重编程因子（“四人帮”后来减为三人帮）的“一过性”高表达，来提高细胞表观遗传的“弹性”或“不稳定性”，然后在促心肌细胞分化的小分子和培养条件下，诱导小鼠fibroblasts变为功能性心肌细胞。据说分化效率成倍数地提升。这个思路是建立在iPS诱导的阶段性和随机性现象之上的，即重编程的过程可以分为一些明显的步骤。在特定的阶段，通过重编程转录因子使得细胞的表观遗传弹性显著升高（抹去了成体细胞的表观遗传特征，增高了多潜能的表观遗传弹性特征）。而且，很可能他们在以往的实验中看到过这种弹性升高的状态是需要不断维持的，否则就会退回到基础状态。大概从此想到，如果操纵这个“退回”过程的培养条件，是否就可以引导一过性多潜能状态的细胞走向指定的细胞类型。丁盛文章的做法是采用重编程因子短时间提高细胞的表观遗传弹性，在其随后“relex back” 到稳定状态过程中，通过指向靶细胞（心肌）的培养条件，加抑制多潜能机制的手段（JAK/STAT inhibition），使得一过性重编程状态的fibroblast细胞变成了功能性心肌细胞。看起来还是一个通过修改经典iPS重编程模式和条件为指定靶细胞转分化的一个技术平台。有点结合了这两者并提升效率的意思。其中采用了丁盛专长的小分子技术。三合一，达到省略诱导为iPS的中间阶段，并提升直接转分化的效率。
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这个技术平台的有趣之处是它突出显示了所谓多潜能的本质就是一个flexibility。连带的问题可以问，采用lineage特异因子的直接转分化是否连这个flexible的一过性过程也省略了，还是有这个过程而没有观察到（叠加、并行或是线性），拟或是完全不同的一种生物细胞内在flexibility？当然，这些问题的最终回答还是要等到所有这些“现象”能够广泛复制，系统建立之后才有可能。就好比iPS和胚胎干的争论，也许问题的本质不是哪个像哪个，而是哪个更有可控的可塑性，直接转分化可能也只是在这个双人博弈中插了一杠子，成了三足鼎立。说到这里，胚胎干、iPS和直接转分化可能开启了一个三国时代，现在还是乱战时期，后面的“大一统”何时到来，以何种方式实现，值得期待。这又是戏说了：）
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3 i! W/ G) n% q: H/ `: Y题外话：丁盛的iPS转分化技术用的是心肌细胞模型，巧的是最近一些干细胞新进展也与心脏有关，包括用iPS分化模拟特定心脏病模型，这是另一个话题了。
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（下面是这几篇文章的abstract，供参考，相关文章都可以在本站找到。所涉及的研究比较复杂多样，必有理解不准之处，当不得严格的评述）
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; S: _; @1 K. V+ ]Nature
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/ w' f/ N! d( \Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells
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1 v' b: [, k7 E9 ^$ l( PInduced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNAmethylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.
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Flaw in inducedstem-cell model 1 M) b8 [& y; b
Adult cells do not fully convert to embryonic-like state.( i& Z+ h' q' w: m H
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Medical researchers’ hopes of replacing politically fraught embryonic stem (ES) cells with stem cells derived from adult tissues have suffered a setback. Induced pluripotent stem (iPS) cells, created by turning back the developmental clock on adult tissues, and ES cells display similar geneexpression patterns, and both can produce any of the various tissues in the human body. But patterns of epigenetic changes — alterations that affect gene expression without changing the DNA sequence — tell a different story about iPS cells, a team led by Joseph Ecker, a molecular geneticist at the Salk Institute in La Jolla, California, reports online in Nature this week1.. m# [! |2 z0 E
; x+ p2 c' `/ t [ `- s“They are slightly different creatures,” says Chad Cowan, a stem-cell biologist at Massachusetts General Hospital in Boston who was not involved in the work. The finding suggests that iPS cells may not be suitable substitutes for ES cells in modelling or treating disease.Ecker and his colleagues analysed patterns of DNA methylation, a type of epigenetic change, across the genomes of 15 cell lines. These included four human ES cell lines, five iPS cell lines and the tissues from which they came, as well as differentiated cells made from both kinds of stem cells.“If you look with blinders on, they look fairly similar,” says Ecker. “But if you zoom in you find different signatures of what an iPS cell is.”
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The researchers found that rather than being reset to an embryo-like state, methylation patterns near the tips and centres of chromosomes in the iPS cells resembled those in the adult tissues from which the iPS cells had been derived.This could constrain the types of tissues that the cells are capable of forming.“The reprogramming process, although fascinating, is a fundamentally different way of getting to pluripotency than deriving cells from [embryos],” says George Daley, a stem-cell expert at Children’s Hospital Boston in Massachusetts. “We’re still looking for reprogramming methods that return cells to the ES-cell-like state,” he adds.
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The finding that reprogrammed stem cells carry an epigenetic ‘memory’ dovetails with work published last year by Daley and others comparing mouse iPS and ES cells2,3. In mice, however, the methylation differences could be reset, either by continuing to culture the iPS cells or by differentiating the cells again to more specialized cell types. In the human cells,the epigenetic marks lingered even after the iPS cells had been coaxed to form new tissues.Regardless of their epigenetic differences, neither iPS cells nor ES cells may turn out to be perfect models of tissues in the body. Both cell types seem to harbour genomic abnormalities.In separate work published last month4, a team led by Jeanne Loring, a stem-cell researcher at the Scripps Research Institute in La Jolla, found that ES cells tended to contain duplicated chunks of DNA linked to genes associated with self-renewal, whereas iPS cells incorporated extra cancer-causing genes and fewer tumoursuppressor genes. These genomic differences between the two types of stem cells probably result from the culturing techniques used to derive and maintain them.2 M7 s. k* N W) R
3 s1 s& Y" v p6 z“When we culture cells outside a normal organism they can acquire features that may not be compatible with life once they go back into an organism,” says Richard Young, a stem cell biologist at the Whitehead Institute in Cambridge, Massachusetts. The impact of such discrepancies remain unclear, says William Lowry, a stem-cell biologist at the University of California, Los Angeles. “The problem is that we don’t know if any of these differences are going to be consequential.” ■$ o1 b9 ]9 W! ]- Z
0 v0 O7 N( N4 v. K1. Lister, R. et al. Nature doi:10.1038/nature09798 (2011).
& X) {: m" @7 U/ f$ m2. Kim, K. et al. Nature 467, 285–290 (2010).
' h) I4 z& K5 _. z K. f3. Polo, J. M. et al. Nature Biotechnol. 28, 848–855 (2010)." b& p" y$ z% F* t" d# m# e' t
4. Laurent, L. C. et al. Cell Stem Cell 8, 106–118 (2011).
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3 K0 s5 N1 o9 l; ^3 Z3 q1 T+ H4 M0 pNature Biotechnology7 M- ~. B: Z. p4 F( v5 u
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A functionally characterized test set of human induced pluripotent stem cells * W+ \) i2 Y% {
! L1 S; t+ U. S4 pHuman induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the
$ a5 D4 m5 i' L4 `( [5 t2 B4 R- riPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.( E7 i' u2 n Q; @
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) z( Q n4 ^0 _# X& j9 }Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines
^8 t& s: e/ F! E$ l/ bSUMMARY
) b6 N H6 ?; H) N9 JThe developmental potential of human pluripotent stem cells suggests that they can produce disease relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.
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8 q- j1 v+ W( z. t3 k: ENature Cell Biology2 U( X7 {& ~4 c q- l b
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Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy/ `! C7 a' i2 {3 j0 N0 b
' S5 k: G: v1 \+ c9 SHere we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11–12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our
( a/ ^* v$ ?; ^) v8 G2 J: estudy has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm. |
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发表于 2011-2-27 19:20
发表于 2011-2-28 13:10
