Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

1256 Chapter 22: Stem Cells and Tissue Renewal
ACTIVATE OSKM
EXPRESSION
differentiated
fibroblast
LOSS OF FIBROBLAST-
SPECIFIC MARKER PROTEIN
GAIN OF EMBRYONIC
MARKER PROTEIN
transcription
wave 1
ENDOGENOUS OCT4 GENE SWITCHED ON
0 3 6 9 12 15 days
histone
modifications,
chromatin
remodeling
transcription
wave 2
~99%
of cells
new DNA methylation
patterns established
~<1% of cells
become IPS cells
An Experimental Manipulation of Factors that Modify Chromatin
MBoC6 n22.142/22.43
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.
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?
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.)
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