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该研究距离制造更安全的干细胞又近了一步 " n( E" S+ h) \! S y, e) {
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图片说明：科学家距离制造更安全的干细胞又近了一步。
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* q1 M6 ^# X" b, [- D& b（图片来源：K. Woltjen et al.）- J% o/ S( E; x% U
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英国及加拿大的研究人员研发出不需使用病毒就可由皮肤细胞培育出人类万能细胞的方法，这种新法不易使基因癌化，可以说是再生医疗的一大突破。! e3 V: Z% b( d5 B' ^9 G! s1 k/ X
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英国爱丁堡大学教授木尾圭介（Keisuke Kaji）等所组成的研究小组及加拿大的研究小组，首度研发出不使用病毒研制的人类万能细胞，与使用病毒研制的效率相较，前者效率高逾25倍。这项研发成果3月1日在线发布于《自然》杂志。
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人类首度研发出的万能细胞是使用“反转录病毒”(retrovirus)将数种基因导入体细胞，但有科学家认为，为了将基因植入体细胞的染色体，反转录病毒容易使基因引发异常，导致癌化。
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为了制造出安全度更高的完能细胞，木尾圭介等研究人员是以“转位子”(transposon)将制造万能细胞所需的四个基因同时导入人类胎儿的纤维母细胞，成功地制造出万能细胞。+ p: {8 Q9 d" A+ C8 D3 f) v
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木尾圭介等研究小组初期采用的方法虽可去除被植入的基因，但会留下一些痕迹，安全上有影响。但在配合加拿大研究小组独立开发的技术后，成功地去除四个基因。染色体中的基因去除后，也具有分化成各种细胞的能力。
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研制出万能细胞的日本京都大学教授山中伸弥，去年没使用病毒研制出老鼠万能细胞，但他并未成功地制造出人类万能细胞。
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Virus-free induction of pluripotency and subsequent excision of reprogramming factors
* G z! ?# y, g; mKeisuke Kaji1, Katherine Norrby1, Agnieszka Paca1, Maria Mileikovsky2, Paria Mohseni2,3 & Knut Woltjen2 T/ f: S4 Q1 G3 o/ _
( f6 [2 Q. `4 e. O( c' }MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK
3 i' P& Q. T( [! k5 u# x' C* OSamuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada ! L4 Q2 o3 w/ `# |, G( @
Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada ' W* m% J5 U4 U7 s0 h; b% w
Correspondence to: Keisuke Kaji1 Correspondence and requests for materials should be addressed to K.K. (Email: keisuke.kaji@ed.ac.uk).. l$ Z( Z v5 ^5 a7 ^: J9 Y! G/ N
, h5 q. Q7 W! q, Z* {( ETop of pageReprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors1, 2, 3, 4, 5, 6, 7. However, such iPS cells contain a large number of viral vector integrations1, 8, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming9, 10, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia11, 12. Two transient transfection-reprogramming methods have been published to address this issue13, 14. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon15, 16, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.% K9 I# N- j5 U7 O! Z- u/ f& |
1 X$ ~- l7 d5 Q; R% k* N& qTop of pageNature advance online publication 1 March 2009 | doi:10.1038/nature07863; Received 6 November 2008; Accepted 12 February 2009; Published online 1 March 2009) \- s* ^4 b. ^" P3 u
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piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells0 m$ H; N1 w2 a! h7 f3 E
Knut Woltjen1, Iacovos P. Michael1,2, Paria Mohseni1,2, Ridham Desai1,2, Maria Mileikovsky1, Riikka Hämäläinen1, Rebecca Cowling1, Wei Wang3, Pentao Liu3, Marina Gertsenstein1, Keisuke Kaji4, Hoon-Ki Sung1 & Andras Nagy1,2
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Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada - I' J8 T( _! A( J! K' V
Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada
" a* Q. }5 c9 s- | _8 wThe Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK " r) Q+ H( W! c
MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK
# @- o) K' b6 {: j0 j' T' hCorrespondence to: Andras Nagy1,2 Correspondence and requests for materials should be addressed to A.N. (Email: nagy@lunenfeld.ca).
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/ O( v! n* j( i& Y: q3 G9 uTop of pageTransgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state1, 2, 3, 4. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral1, lentiviral5, adenoviral6 and plasmid7 transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (P transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines8, 9, 10, 11. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events12. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition13. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision12, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences14 delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.
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无需病毒诱导出人类万能细胞.pdf
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发表于 2009-3-7 21:36
