自從iPS發(fā)現(xiàn)以來,，科學(xué)家們已經(jīng)通過導(dǎo)入轉(zhuǎn)錄子成功地將成熟的人體皮膚細(xì)胞改造成誘導(dǎo)多能樣干細(xì)胞（iPS）,，但是基因重排的效率不高一直是iPS技術(shù)的主要障礙,。相關(guān)的文章發(fā)表在9月11日的Cell Stem Cell上。研究者開發(fā)了新的研究平臺,，可從分子,、遺傳和生化機(jī)制上改造細(xì)胞程序重排技術(shù)。這些新的發(fā)現(xiàn)不僅為細(xì)胞程序重排技術(shù)帶來新的契機(jī),，也為改造細(xì)胞的臨床應(yīng)用打下基礎(chǔ),。哈佛大學(xué)干細(xì)胞研究所研究員Konrad Hochedlinger博士表示，效率不高的原因之一在于人們對個(gè)中復(fù)雜的機(jī)制仍不了解,，導(dǎo)致無法改善重排技術(shù),。此外，iPS誘導(dǎo)的過程需要有逆轉(zhuǎn)錄病毒的參與,，這也是限制iPS技術(shù)臨床應(yīng)用的原因之一,。
Hochedlinger的研究小組開發(fā)了一種新的iPS誘導(dǎo)技術(shù)，只需要用藥物-病毒系統(tǒng)就能產(chǎn)生iPS細(xì)胞,，制備的iPS從結(jié)構(gòu)和功能上都與人類胚胎干細(xì)胞很相似,。這種方法的獨(dú)特之處是用doxycycline來控制轉(zhuǎn)錄因子的表達(dá)。
Hochedlinger博士認(rèn)為,，iPS雙重誘導(dǎo)系統(tǒng)有助篩選影響iPS程序重排技術(shù)的關(guān)鍵分子,，這些影響iPS程序重排的關(guān)鍵分子就是制約護(hù)s技術(shù)效率的關(guān)鍵障礙,。而我們新的技術(shù)平臺可最大效率地優(yōu)化iPS技術(shù)。
在第二篇文章中,，Jaenisch博士的研究小組報(bào)道,，他們使用誘導(dǎo)轉(zhuǎn)基因成功地制備iPS細(xì)胞。Jaenisch博士說,，新的藥物誘導(dǎo)系統(tǒng)是一種新的,，可預(yù)測，重復(fù)性好的研究平臺,。更有甚者,，我們可以通過篩選影響iPS的化學(xué)和遺傳因子以提高iPS細(xì)胞的轉(zhuǎn)化效率，或是改善iPS制備技術(shù),，改變原有的重排因子用高效的重排因子取代原來的,。
兩個(gè)研究小組都發(fā)現(xiàn)兩次重排產(chǎn)生iPS細(xì)胞的效率比一次重排產(chǎn)生iPS的效率要高。有趣的是,，不同的皮膚細(xì)胞類型用于誘導(dǎo)產(chǎn)生iPS的時(shí)間也不同,。比如說，人類成纖維細(xì)胞轉(zhuǎn)化成iPS需要幾周的時(shí)間,，而角質(zhì)化細(xì)胞只需要們天左右的時(shí)間,。Hochedlinger博士說，通過觀察角質(zhì)化細(xì)胞轉(zhuǎn)化的動力過程,，我們可以改變程序優(yōu)化iPS技術(shù),。
將兩組研究結(jié)果綜合起來看，可以發(fā)現(xiàn)通過這一平臺可以對不同的細(xì)胞制備iPS的技術(shù)進(jìn)行優(yōu)化,。新的技術(shù)平臺不僅為基因重排技術(shù)帶來改進(jìn)， 同時(shí)可以縮短iPS細(xì)胞從理論研究到臨床應(yīng)用的發(fā)展周期,，因?yàn)橥ㄟ^該平臺可以取代對人體不利的病毒和不利的基因修飾,。（生物谷Bioon.com）
生物谷推薦原始出處：
Cell Stem Cell, Vol 3, 340-345, 11 September 2008
A High-Efficiency System for the Generation and Study of Human Induced Pluripotent Stem Cells
Nimet Maherali,1,2,4,6 Tim Ahfeldt,1,5,6 Alessandra Rigamonti,1 Jochen Utikal,1,2 Chad Cowan,1,3, and Konrad Hochedlinger1,2
Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs), demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system, we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells, we obtained “secondary” hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.
Cell Stem Cell, Vol 3, 346-353, 11 September 2008
A Drug-Inducible System for Direct Reprogramming of Human Somatic Cells to Pluripotency
Dirk Hockemeyer,1,3 Frank Soldner,1,3 Elizabeth G. Cook,1 Qing Gao,1 Maisam Mitalipova,1 and Rudolf Jaenisch1,2
Current approaches to reprogram human somatic cells to pluripotent iPSCs utilize viral transduction of different combinations of transcription factors. These protocols are highly inefficient because only a small fraction of cells carry the appropriate number and stoichiometry of proviral insertions to initiate the reprogramming process. Here we have generated genetically homogeneous “secondary” somatic cells, which carry the reprogramming factors as defined doxycycline (DOX)-inducible transgenes. These cells were obtained by infecting fibroblasts with DOX-inducible lentiviruses, isolating “primary” iPSCs in the presence of the drug, and finally differentiating to “secondary” fibroblasts. When “secondary” fibroblast lines were cultured in the presence of DOX without further viral infection, up to 2% of the cells were reprogrammed to pluripotent “secondary” human iPSCs. This system will facilitate the characterization of the reprogramming process and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors.
