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本帖最后由 tpwang 于 2011-3-3 14:09 编辑
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% _) S! H. g% u: n回复 zhulihuazzz 的帖子+ `) I c) d% @3 z
, Q/ Y8 m/ F& U7 [, m& u这下好，毛病更多了。圣地亚哥、波士顿、威斯康星，这几家出了大部分的iPS表观遗传和基因screen研究。而日本人是在那里闷头提高诱导iPS效率，忙着分化为各种细胞……2 \( c5 u1 X$ n2 m4 V9 g* g0 h! g
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. t* I; Z9 I7 y6 D9 _# ]Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.5 d4 }, L' L0 m3 c0 }- J( u7 H. w" U
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Discussion
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Taken together, our results demonstrate that pre-existing and new mutations that occur during and after reprogramming all contribute to the high mutational load we discovered in hiPS cell lines. Although we cannot completely rule out the possibility that reprogramming itself is ‘mutagenic’, our data argue that selection during hiPS cell reprogramming, colony picking and subsequent culturing may be contributing factors. A corollary is that if reprogramming efficiency is improved to a level such that no colony picking and clonal expansion is necessary, the resulting hiPS cells could potentially be free of mutations.
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( @* z2 w; D5 {) [Despite the power of our experimental approach to identify and characterize reprogramming-associated mutations accurately, their functional significance remains to be shown. This issue parallels a general problem facing the genomics community: high-throughput sequencing technologies have allowed data generation rates to greatly outpace functional interpretation. Additionally, when considering the biological significance of reprogramming-associated mutations, there are two separate functional aspects to consider:whether some of thesemutations contributed functionally to the reprogramming of cell fate, and whether some of these mutations could increase disease risk when hiPS-cellderived cells/tissues are used in the clinic. These two aspects are not necessarily connected. Although the functional effects of the 124 mutations remain to be characterized experimentally, it is nonetheless striking that the observed reprogramming-associated mutational load shares many similarities with that observed in cancer. Furthermore, the observation of mutated genes involved in human Mendelian disorders suggests that the risk of diseases other than cancer needs to be evaluated for hiPS-cell-based therapeutic methods. Future long-term studies must focus on functional characterization of reprogramming-associated mutations to aid further the creation of clinical safety standards.
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+ }+ b, w7 E' N1 _Safe hiPS cells are critical for clinical application. Therefore, just as previous findings of large-scale genome rearrangements in hiPS cell lines led to the introduction of karyotyping as a standard postreprogramming protocol, routine genetic screening of hiPS cell lines to ensure that no obviously deleterious point mutations are present must become a standard procedure. Complete exome or genome sequencing of hiPS cell lines might be an efficient way to screen out hiPS cell lines that have a high mutational load or have mutations in genes implicated in development, disease or tumorigenesis. Further rigorous work on mutation rates and distributions during in vitro culturing and reprogramming of hiPS cells, and perhaps human embryonic stem cells, will be essential to help establish clinical safety standards for genomic integrity. |
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发表于 2011-3-3 13:18
发表于 2011-3-3 14:31
