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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-remodeling complexes and negative effects from recruitment of DNA methyltransferases. In many contexts, c-Myc drives cell proliferation and inhibits differentiation, as in mouse embryonic stem cells. Such cells proliferate and remain in an undifferentiated state in culture when the cytokine leukemia inhibitory factor is provided, but forced expression of c-Myc eliminates the requirement for this factor. Surprisingly, c-Myc behaves quite differently in human embryonic stem cells, where it induces apoptosis and differentiation. Given that tumors developed in 20% of mice derived from induced pluripotent stem cells owing to the reactivation of the c-Myc transgene,2 it is clear that we must determine which role of c-Myc is essential to reprogramming to obviate the need for introducing the proto-oncogene into cells. The use of a series of c-Myc–deletion mutants may clarify its role in reprogramming.
Klf4 promotes self-renewal of mouse embryonic stem cells, probably through a leukemia-inhibitory-factor–dependent pathway. A recent report suggests that Klf4 behaves in a context-dependent manner, mediating a cytostatic function by repressing p53, a repressor of Nanog, or by promoting cell proliferation in collaboration with the H-RasV12 oncogene.
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.
Source Information
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.
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.
