Cell：哈佛iPS取得重大突破

　　

将成熟体细胞经过遗传程序重排可使体细胞转化成为类胚胎干细胞称为，诱导多能性干细胞（iPS）。在iPS的制备过程中还存在多种技术障碍，现在Cell杂志出版的两篇独立的研究性文章为iPS的研究扫清障碍。相关的文章发表在9月11日的Cell子刊《Cell Stem Cell》上。研究者开发了新的研究平台，可从分子、遗传和生化机制上改造细胞程序重排技术。这些新的发现不仅为细胞程序重排技术带来新的契机，也为改造细胞的临床应用打下基础。

 

尽管科学家们已经通过导入转录子成功地将成熟的人体皮肤细胞改造成诱导多能样干细胞（iPS），但是基因重排的效率不高一直是iPS技术的主要障碍。该研究的高级作者Konrad Hochedlinger博士说，效率不高的原因之一在于人们对个中复杂的机制仍不了解，导致无法改善重排技术，效率不高。Konrad Hochedlinger博士，哈佛大学干细胞研究所研究员。此外，iPS诱导的过程需要有逆转录病毒的参与，这也是限制iPS技术临床应用的原因之一。

 

Hochedlinger的研究小组开发了一种新的iPS诱导技术，只需要用药物-病毒系统就能产生iPS细胞，制备的iPS从结构和功能上都与人类胚胎干细胞很相似。这种方法的独特之处是，用doxycycline来控制转录因子的表达。

 

研究者发现，只要清除掉doxycycline，原代的iPS细胞就会分化成为成熟的细胞，当再次接触doxycycline时，又促进细胞第二次程序重排，产生第二代iPS细胞。上述的idea首先是麻省理工学院的Rudolf Jaenisch博士想到的。

 

Hochedlinger博士认为，iPS双重诱导系统有助筛选影响iPS程序重排技术的关键分子，这些影响iPS程序重排的关键分子就是制约iPS技术效率的关键障碍。而我们新的技术平台可最大效率地优化iPS技术。

 

在第二篇文章中，Jaenisch博士的研究小组报道，他们使用doxycycline诱导转基因成功地制备iPS细胞。Jaenisch博士说，新的药物诱导系统是一种新的，可预测，重复性好的研究平台。更有甚者，我们可以通过筛选影响iPS的化学和遗传因子以提高iPS细胞的转化效率，或是改善iPS制备技术，改变原有的重排因子用高效的重排因子取代原来的。

 

两个研究小组都发现两次重排产生iPS细胞的效率比一次重排产生iPS的效率要高。有趣的是，不同的皮肤细胞类型用于诱导产生iPS的时间也不同。比如说，人类成纤维细胞转化成iPS需要几周的时间，而角质化细胞只需要10天左右的时间。Hochedlinger博士说，通过观察角质化细胞转化的动力过程，我们可以改变程序优化iPS技术。

 

将两组研究结果综合起来看，可以发现通过这一平台可以对不同的细胞制备iPS的技术进行优化。新的技术平台不仅为基因重排技术带来改进，同时可以缩短iPS细胞从理论研究到临床应用的发展周期，因为通过该平台可以取代对人体不利的病毒和不利的基因修饰。

 

两原文摘要：

A High-Efficiency System for the Generation and Study of Human Induced Pluripotent Stem Cells

 

【Summary】

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.

 

A Drug-Inducible System for Direct Reprogramming of Human Somatic Cells to Pluripotency

 

【Summary】

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.

 

（生物通 张欢）