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Volume 357:1469-1472 October 11, 2007 Number 15
Pluripotency Redux — Advances in Stem-Cell Research
John Gearhart, Ph.D., Evanthia E. Pashos, B.Sc., and Megana K. Prasad, B.Sc.
A cell's ability to give to rise to all the cell types of the embryo and the adult organism is called pluripotency. Pluripotent cells are found within mammalian blastocysts and persist briefly in embryos after implantation. Embryonic stem cells, derived from the inner cell mass of blastocysts, are a renewable source of pluripotent stem cells that are proving valuable in basic science studies and may eventually become a source of cells for safe, effective cell-based therapies. Much embryonic stem-cell research has focused on determining the molecular signature of pluripotency, and a picture is emerging of a complex interaction among transcription factor networks, signaling pathways, and epigenetic processes involving modifications in the structure of DNA, histones, and chromatin.
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.)
