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inactivation of the p53/p21 pathway and the
Ink4/Arf
locus is an early step
in reprogramming (
Li et al., 2009
), this inactivation may explain the genome
instability reported in some iPSCs (
Gore et al., 2011; Mayshar et al., 2010;
Mikkelsen et al., 2008
).
4. CRITICAL STAGES AND EVENTS IN EPIGENETIC
REPROGRAMMING
Regardless of the strategy used, reprogramming is widely acknowl-
edged to be a multifactorial process that relies to some level on stochastic
events. When reprogramming is attempted using a reduced number of
iPS factors, conversion takes weeks to months. This suggests that successful
reprogramming may rely on other factors that are either preexisting in a sub-
set of somatic cells or that occur stochastically during the reprogramming
process. To examine this question, Hanna et al. characterized the repro-
gramming efficiency and kinetics of over 1000 somatic-cell-derived mono-
clonal populations expressing reprogramming factors (
Hanna et al., 2009
).
They found that most pro-B lymphocytes and most monocytes had the
potential to generate iPSCs when cultured for extended period of time. This
favors the idea that reprogramming depends on stochastic events rather than
the preselection of a subset of target cells. Importantly, all iPSCs derived
from these cells at different times of culture had normal karyotypes and each
was capable of generating teratomas and chimeras.
Once triggered, reprogramming appears to proceed in an ordered man-
ner starting with a downregulation of somatic markers followed by
activation of less-stringent pluripotency markers (such as alkaline phos-
phatase, SSEA-1, and Fbxo1) and then more stringent markers (such as
Nanog and Oct4) (
Brambrink et al., 2008; Stadtfeld, Maherali, Breault, &
Hochedlinger, 2008
). Downregulation of somatic markers can occur with-
out successful induction of pluripotency as ESCs lacking PRC2 (that cannot
induce full reprogramming) can still extinguish the expression of somatic
markers upon fusion with B lymphocytes (
Pereira et al., 2010
).
A comparison of genome-wide profiles of gene expression and chroma-
tin status between ESCs, iPSCs, somatic cells, and partially reprogrammed
cells (pre-iPSCs;
Mikkelsen et al., 2008; Sridharan et al., 2009
) has revealed
that independently isolated iPSCs are similar at molecular level, even when
different protocols were applied and cells of different origins (mouse fibro-
blasts and B lymphocytes) were used. Interestingly, pre-iPSCs also appeared
similar, suggesting a common intermediate stage may exist where transiting
