From our discussion of nuclear transplantation, one might expect that any reprogramming of a differentiated cell would require a radical and widespread change in the chromatin structure of selected genes. Not only are such changes observed, but a large number of different experiments reveal that the efficiency of the reprogramming process can be substantially increased by altering the activity of proteins that affect chromatin structure. Figure 22–44 categorizes some of the factors that have been shown to enhance the transformation of fibroblasts to iPS cells; those in the top three rows—chromatin remodelers, histone modifying enzymes, and histone variants—are especially well known to have profound effects on the organization of nucleosomes in chromatin (discussed in Chapter 4).
We can only touch briefly here on the massive amounts of data that have been accumulating in this exciting research area. The major challenge that remains is to obtain a systems-level model for the complex set of biochemical changes that are involved in reprogramming. For example, which chromatin changes come first, and which then follow? How can these be triggered by the master transcription regulators through their binding to specific DNA sequences, and why do many cells in a population appear resistant to these effects?
eS and ipS Cells Can be Guided to Generate Specific adult Cell types and even whole organs
We can think of embryonic development in terms of a series of choices presented to cells as they follow a road that leads from the fertilized egg to terminal
Figure 22–43 A summary of some of the major events that accompany the reprogramming of mouse fibroblasts to iPS cells. by sorting cells at various times after the oSkm induction shown, one can carry out detailed biochemical analyses on the different cell populations shown. this led to the discovery that two major waves of new gene transcription are induced, but that the second wave occurs only in the subset of cells expressing an embryonic marker protein. Some 1500 genes are found to be differentially expressed in these cells, compared to the large majority of cells that fail to progress toward ipS cells. as indicated, major dna methylation changes are observed only after the alteration of chromatin structures.
in the first transcription wave, among the genes prominently induced are those for cell proliferation, metabolism, and cytoskeletal organization; in contrast, genes associated with fibroblast development are repressed. in the second transcription wave, genes required for embryonic development and for stem cell maintenance are induced. (adapted from J.m. polo et al., Cell 151:1617–1632, 2012.) specifc miRNAs specifc IncRNAs histone deacetylases specifc histone methyl transferases specifc histone demethylases histone acetyl transferases histone variant H2AZ histone variant macroH2A histone variant H3.3 DNA demethylases differentiation. After their long sojourn in culture, the ES cells or iPS cells and their progeny can still read the signs at each branch in the highway and respond as normal embryonic cells would. If ES or iPS cells are implanted directly into an embryo at a later stage or into an adult tissue, however, they fail to receive the appropriate sequence of cues; their differentiation then is not properly controlled, and they will often give rise to a tumor of the type known as a teratoma, containing a mixture of cell types inappropriate to the site in the body.
In culture, by exposing the ES or iPS cell to an appropriate sequence of signal proteins and growth factors, delivered with the right timing, it is possible to guide the cell along a pathway that approximates a normal developmental pathway, so as to convert it into one of the standard specialized adult cell types (Figure 22–45 and Movie 22.5). Success requires trial and error, but has now been achieved for many different final specialized states, including neuronal, muscular, and intestinal cell types. In a few cases, it has even been possible, by careful manipulation of the culture conditions, to get ES or iPS cells to interact with one another so as to construct an entire organ, albeit on a small scale (Figure 22–46).
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