NEJM：引入基因诱导产生质量更高的多能干细胞
细胞所具有的向胚胎及成体器官所有类型细胞转化的能力称为全能性。全能干细胞仅存在于哺乳动物的胚泡和胚胎着床的早期阶段。一直以来人们进行了大量研究，试图使已丧失分化全能的成体细胞恢复全能性，用于临床疾病的治疗。
Takahashi和Yamanaka在去年发表的研究报告，让干细胞生物学家感到既惊奇又兴奋。他们用逆转录病毒将一些基因表达转录因子导入来自于成年鼠和胎鼠组织的成纤维细胞中，成功诱导其恢复了全能性。最初有24个基因候选，最后研究者发现只需要引入编码Oct3/4, Sox2, Klf4 和 c-Myc的四个基因就足以诱导出多能干细胞。这些诱导产生的多能干细胞具有胚胎干细胞的一些特性，但它们在基因表达和表观遗传方面与胚胎干细胞存在着实质性的区别，并且这些细胞被注入胚泡后不能形成胎生嵌合体幼体。
最近三个独立开展的研究报道他们诱导产生了质量更高的多能干细胞。这些新的细胞系不仅在转录和染色体修饰方面与胚胎干细胞更相似，而且在芽胚原基层细胞的参与下可以生成嵌合体鼠。这些细胞系生产的过程与Takahashi和Yamanaka的细胞系最主要的不同在于引入了一种以Nanog 或者Oct3/4为标记物的更有效的细胞筛选方式。
虽然Oct3/4, Sox2, Klf4和 c-Myc这四个因子的具体作用机制还未完全明了，现有的诱导效率也较低，但这些进展是鼓舞人心的，使我们离诱导生成的多能干细胞安全应用于临床的梦想更近了一步。
相关论文发表于《新英格兰医学杂志》（NEJM）2007年357卷15期。（科学新闻杂志）
原始出处:
The New England Journal of Medicine
| Volume 357:1469-1472 |  |
October 11, 2007 | |
Number 15 |
|---|
Deciphering the molecular basis of pluripotency will facilitate the development of procedures for efficiently deriving patient-specific stem cells. In somatic-cell nuclear transfer, which has held the greatest promise for generating such cell lines, the nucleus of a somatic cell is introduced into an enucleated oocyte or mitotic zygote and is "reprogrammed" to an embryonic state, resulting in the formation of a blastocyst from which embryonic stem cells can be derived. Although this procedure has been demonstrated in animals, it has yet to be accomplished with human oocytes or zygotes. An alternative approach to reprogramming a somatic cell is to fuse it with an embryonic stem cell, but the resulting hybrid pluripotent cell is tetraploid and of limited practical application.
Against this background, a study published last year by Takahashi and Yamanaka1 surprised and excited stem-cell biologists. Using a novel strategy, the investigators showed that fibroblasts derived from tissues of adult and fetal mice could be induced to become embryonic-stem-cell–like cells with the introduction of four genes expressing transcription factors. Twenty-four genes were initially chosen as candidates on the basis of their preferential expression in embryonic stem cells or their known roles in the maintenance of such cells or in cell-cycle regulation. These genes were introduced into fibroblasts isolated from mouse embryos and adult tail tips in a combinatorial manner through retroviral transduction.
Fibroblasts that are induced to become pluripotent stem cells were selected through the expression of Fbx15, a gene known to be expressed in pluripotent cells. The investigators discovered that only four factors — encoded by Oct3/4, Sox2, Klf4, and c-Myc — were sufficient to induce pluripotency (see diagram). The induced pluripotent stem cells had some properties of embryonic stem cells: they formed teratomas when grafted into immunocompromised mice, and they formed embryoid bodies (aggregates of embryonic stem cells that can spontaneously differentiate). However, they differed substantially from embryonic stem cells in their gene-expression and epigenetic profiles, and they failed to form live-born chimeric pups when injected into blastocysts.
Induction of Pluripotent Stem Cells through Retroviral Transduction.
Retrovirally encoded transcription factor genes were introduced into mouse embryonic and adult fibroblasts. After integration and expression of the transgenes, the fibroblasts were reprogrammed to pluripotency.
Recently, the generation of higher-quality induced pluripotent stem cells has been reported in three independent studies.2,3,4 The new lines not only resemble embryonic stem cells more closely in their transcriptional and chromatin-modification profiles but are capable of generating adult chimeric mice with contributions to the germ line — the most rigorous test for pluripotency. The main procedural difference between the production of these lines and that of Takahashi and Yamanaka's line was the selection scheme for identifying the reprogrammed cells. The initial strategy relied on the induced expression of Fbx15 in the transduced fibroblasts. This gene is expressed in embryonic stem cells but is not required for pluripotency. The recent studies were designed to select for expression of Nanog or Oct3/4, which are essential for pluripotency and embryonic stem-cell identity.
The molecular changes characteristic of pluripotency occurred gradually during weeks in culture. How these four factors induced reprogramming is unknown, but their known roles suggest hypotheses. Oct3/4 and Sox2, along with Nanog, form a core regulatory network for pluripotency in embryonic stem cells. Oct3/4–/– embryos die in utero because of defects in the inner cell mass; Oct3/4 repression in mouse embryonic stem cells results in a loss of pluripotency and differentiation into trophectoderm, and Oct3/4 overexpression leads to the loss of pluripotency and differentiation into primitive endoderm and mesoderm. Similarly, Sox2-null mice die during the peri-implantation period because of epiblast defects, and Sox2 knockdown in embryonic stem cells leads to trophectoderm differentiation. (Pluripotency is known to be maintained by a few transcription factors, including Oct3/4, Sox2, and Nanog. We hypothesize that the dispensibility of Nanog as an introduced factor in these experiments can be explained by the induced expression of the endogenous Nanog gene by cooperativity between Oct3/4 and Sox2.)
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