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Audio Interview
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- 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) 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. 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.) The proto-oncogene c-Myc is believed to regulate the expression of 15% of all genes, including genes involved in cell division, cell growth, and apoptosis. It exerts its effects on transcriptional targets through various mechanisms — there are positive effects from recruitment of histone-modifying enzymes, general transcriptional machinery, and chromatin-remodelin Klf4 promotes self-renewal of mouse embryonic stem cells, probably through a leukemia-inhibitory The identification and characterization of the responsive cells in the target population of primary fibroblasts may also help us understand these results. The low efficiency with which induced pluripotent stem cells were generated (<0.01%, despite a 50% transduction rate) may indicate that only a subgroup of cells can be induced to pluripotency. The skin, a complex tissue with robust regenerative capabilities, contains a variety of stem cells, including epidermal, mesenchymal, neural crest–derived, and stem cells or progenitors from the circulation. Fibroblasts derived from the skin represent a heterogeneous population of cells. Skin fibroblasts from various anatomical sites have distinct gene-expression patterns, varying in terms of the genes involved in pattern formation, cell–cell signaling, extracellular matrix synthesis, and fate determination. Furthermore, fibroblasts from a single skin region have widely varying morphologic and physiological characteristics. Perhaps the induced stem cells are derived from a rare fibroblast subpopulation that is already multipotential and more easily induced to pluripotency. Determining the identity of such a subpopulation may aid in increasing the efficiency of reprogramming. Repeating these experiments with a variety of differentiated cell types and subpopulations of fibroblasts from various tissue sources will be informative. Whether these four factors (or others) will be capable of inducing pluripotency in human cells that will prove safe for use in cell therapies remains unknown. Differences between human and mouse embryonic stem cells in the mechanisms of pluripotency suggest that other factors may be required to achieve similar results with human cells. Further investigation of the factors is needed to elucidate their roles in reprogramming and to ensure that we can avoid any detrimental effects they may have on cells. Transient expression of factors (using vectors that do not integrate into the genome) in fibroblasts or the identification and use of small molecules that mimic the effects of the factors would enable researchers to avoid the possibility of generating mutations in the genome through random insertions and reactivation of transgenes in the retroviral vectors. Reprogramming of adult cells to generate patient-specific therapies represents the future for stem-cell biologists. Inducing pluripotent stem cells is the first successful way of instructing somatic cells to become pluripotent by introducing defined factors. A recent report on identifying induced pluripotent stem cells on the basis of morphologic criteria alone brings us a step closer to translating this work safely into human cells. Such identification would obviate the need for transgenic reporter genes in the donor fibroblasts.5 Despite these encouraging results, research on human embryonic stem cells should not be impeded; such cells remain the gold standard for determining the molecular basis of human tissue development and for developing cell-based therapies for human diseases. Dr. Gearhart is a professor of gynecology and obstetrics, physiology, comparative medicine, and biochemistry and molecular biology and the director of the Stem Cell Biology Program at the Institute for Cell Engineering, Johns Hopkins Medical Institutions, Baltimore, where Ms. Pashos and Ms. Prasad are Ph.D. candidates in the Human Genetics and Molecular Biology Program. References
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Induction of Pluripotent Stem Cells through Retroviral Transduction.
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-modificat
Source Information
An interview with Dr. Douglas Melton, a scientific director of the Harvard Stem Cell Institute and a professor in the Department of Molecular and Cellular Biology at Harvard University, can be heard at www.nejm.org.
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