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News & Views9 n. ^( @5 k! j+ v
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http://www.nature.com/nature/journal/v471/n7336/full/471046a.html( S# d- ?1 N! s
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7 ]+ S* B; m' K5 lInduced 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. 0 ?" R& x3 W9 H2 V4 x
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See also Article by Hussein et al.
) N4 T; T( Z+ d% F' u3 f; B# |See also Article by Gore et al. . d- o: ?( [0 a0 V5 k
See also Article by Lister et al. + q" s$ t8 [1 j5 ?9 f
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, K8 j" f; t8 V8 g: k/ jInduced 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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8 r, m/ H( U9 o+ i: w/ VIt 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.: b: \3 w+ ]3 ]6 l1 I: ]9 g: t
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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.% N: V& R+ I$ {
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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 cells3.9 I# N, h! m2 P! r1 ?8 Q
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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.- S0 S" A$ x& h: x, X! }" Y2 C6 w
2 k8 G f* X( L6 m4 ^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.
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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.7 F$ l0 Q+ r' V9 l
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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.
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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.
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f2 W$ j/ X, i; r, ~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.
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补充内容 (2011-3-3 15:16):% Z2 W K1 Y/ x/ e) w \& l
不过对于不同方法的iPS可能还会有不同的结果。 |
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发表于 2011-3-3 13:28
发表于 2011-3-3 14:36
