Conversion to an iPS character by the OSKM factors is not only inefficient but also slow: fibroblasts take ten days or more from introduction of the conversion factors before they begin to express markers of the iPS state. This suggests that the transformation involves a long cascade of changes. These changes are being extensively studied, and they affect both the expression of individual genes and the state of the chromatin. The results of one such study are outlined in Figure 22–43. The process begins with a Myc-induced cell proliferation and loosening of chromatin structure that promotes the binding of the other three master regulators to many hundreds of different sites in the genome. At a large proportion of these sites, Oct4, Sox2, and Klf4 all bind in concert. The binding sites include the endogenous Oct4, Sox2, and Klf4 genes themselves, which eventually creates the types of positive feedback loops just described that makes expression of these genes self-sustaining (see Figure 22–41). But self-induction of Oct4, Sox2, and Klf4 is only a small part of the transformation that occurs. The three core factors activate some target genes and repress others, producing a cascade of effects that reorganize the gene control system globally and at every level, changing the patterns of histone modification, DNA methylation, and chromatin compaction, as well as the expression of innumerable proteins and noncoding RNAs. By the end of this complex process, the resulting iPS cell is no longer dependent on the artificially generated factors that triggered the change: it has settled into a stable, self-sustaining state of coordinated gene expression, making its own Oct4, Sox2, Klf4, and Myc (and all the other essential ingredients of a pluripotent stem cell) from its own endogenous copies of the genes.
factors O, S, K and M cell state unchanged, resistance gene not expressed Fbx15 G418-resistance gene iPS cell survives promoter rare change of state to iPS character, resistance gene expressed
Figure 22–42 A strategy used to select cells that have converted to an iPS character. the experiment makes use  of a gene (Fbx15) that is present in all  cells but is normally expressed only in  eS and early embryonic cells (although not required for their survival). a fibroblast cell line is genetically engineered to contain a gene that produces an enzyme that degrades G418 under the control of the Fbx15 regulatory sequence. G418 is an aminoglycoside antibiotic that blocks protein synthesis in both bacteria and eukaryotic cells. when the oSkm factors are artificially expressed in this cell line, a small proportion of the cells undergo a change of state and activate the Fbx15 regulatory sequence, driving expression  of the G418-resistance gene. when G418 is added to the culture medium, these are the only cells that survive and proliferate. when tested, they turn out to have an  ipS character.
~99% of cells transcription wave 2 ~<1% of cells become IPS cells an experimental manipulation of Factors that modify Chromatin Can increase Reprogramming efficiencies
The low efficiency and slow rate of conversion suggest that there is some barrier blocking the switch from the differentiated state to the iPS state in these experiments, and that overcoming this barrier is a difficult process that involves a large element of chance. Likewise, the outcome is variable, with significant differences between the individual lines of transformed cells that are generated, even when the initial differentiated cells are genetically and phenotypically identical. Only some of the candidate iPS lines pass all the tests of pluripotency. At a molecular level, there are differences even among the fully validated iPS cells: although they share many features, they vary in details of their gene expression patterns and, for example, in their patterns of DNA methylation.
Overcoming these difficulties will be critical for improving our understanding of how cell specialization is controlled and organized in multicellular organisms; it should also facilitate many medical advances. Thus, intensive research is being carried out on the reprogramming process. One approach aims at obtaining a much clearer picture of the role that chromatin structures play in gene regulation in eukaryotes.