In Chapter 7, we saw that fibroblasts and some other cell types can be driven to switch their character and differentiate as muscle cells if the master muscle-specific transcription regulator MyoD is artificially expressed in them. Could the same technique be used to convert adult cell types into ES cells, through forced expression of factors such as Oct4? This question was tackled by transfecting fibroblasts with retroviral vectors carrying genes that one might hope to have such an effect. A total of 24 candidate ES-critical genes were tested in this way. None of them was able by itself to cause the conversion; but in certain combinations they could do so. In 2006, the first breakthrough experiments whittled down the requirement to a core set of four factors, all of them transcription regulators: Oct4, Sox2, Klf4, and Myc, known as the OSKM factors for short. When coexpressed, these could reprogram mouse fibroblasts, permanently converting them into cells closely similar to ES cells (Figure 22–41). ES-like cells created in this way are called induced pluripotent stem cells, or iPS cells. Like ES cells, iPS cells can continue dividing indefinitely in culture, and when incorporated into a mouse blastocyst they can participate in creation of a perfectly formed chimeric animal. In this animal, they can contribute to the development of any tissue and can turn into any differentiated cell type, including functional germ cells from which a new generation of mice can be raised (see Figure 22–40).
iPS cells can now be derived from adult human cells and from various other differentiated cell types besides fibroblasts. Numerous methods can be used to drive up regulation of up regulation ofembryonic stem cell proliferationcell genes down regulation of loosening of differentiation genes chromatin structure
Figure 22–41 Reprogramming fibroblasts to IPS cells with the OSKM factors. as indicated, the master gene regulator proteins oct4, Sox2, and klf4 (oSk) induce both their own and each other's synthesis (gray shading). this generates a self-sustaining feedback loop that helps to maintain cells in an embryonic stem cell-like state, even after all of the experimentally added oSkm initiators have been removed. myc overexpression speeds up early stages of the reprogramming process through the mechanisms shown (see Figure 17–61). Stable reprogramming also involves the permanently induced expression of the Nanog gene, which produces an additional master transcription regulator. (adapted from
J. kim et al., Cell 132:1049–1061, 2008.) expression of the transforming OSKM factors, including methods that leave no trace of foreign DNA in the reprogrammed cell. Variations of the original cocktail of transcription regulators can drive the conversion, with different specialized cell types having somewhat different requirements. Myc overexpression, for example, turns out not to be absolutely necessary, although it enhances the efficiency of the process. And differentiated cell types may express some of the required factors as part of their normal phenotype. For example, cells of the dermal papilla of hair follicles already express Sox2, Klf4, and Myc; to convert them into iPS cells, it is enough to force them artificially to express Oct4. Oct4, indeed, seems to have a central role and to be generally indispensable for the creation of iPS cells.
Reprogramming involves a massive upheaval of the Gene Control System
Converting a differentiated cell into an iPS cell is not like flicking a switch on some predictable, precisely engineered piece of machinery. Only a few of the cells that receive the OSKM factors will actually become iPS cells—one in several thousand in the original experiments, and still only a small minority with more recent, improved techniques. In fact, the success of the original experiments depended on clever selection to pick out those few cells where the conversion had occurred (Figure 22–42).