Biology Reference
In-Depth Information
cells accumulate before conversion is complete. On the other hand, several
studies have shown that iPSCs derived from different cell types (mouse
fibroblast, hematopoietic, and myogenic cells) have different gene expres-
sion patterns, especially at early passages after iPSCs derivation (
Polo
et al., 2010
). Although all early passage iPSCs were shown to have the
potential to differentiate into three different germ layers and produce chime-
ric mice, differences in their differentiation potential were noted that
became less evident with successive passage in culture (
Polo et al., 2010
).
A major roadblock to reprogramming is thought to be the need to
reverse DNA methylation at the promoter regions of pluripotency-
associated genes such as
OCT4
and
NANOG
(
Deb-Rinker, Ly, Jezierski,
Sikorska, & Walker, 2005; Gidekel & Bergman, 2002
). Demethylation is
presumably needed to allow the binding of transcriptional activators that
are required for gene reexpression. This step may require multiple rounds
of cell division to establish in the iPS system where reprogramming is
accomplished with exogenously supplied Oct4, but in the absence of Nanog
(
Theunissen et al., 2011
). Nanog is known to enhance the reprogramming
efficiency that can be achieved by Oct4, Sox2, Klf4, and c-myc alone
(OSKM) and has been shown to help overcome barriers blocking complete
conversion of pre-iPSCs (
Theunissen et al., 2011
). How DNA demethyla-
tion is accomplished during reprogramming remains a hotly debated issue
(
Hochedlinger & Plath, 2009
). One model proposes that DNA methylation
is gradually lost by a “passive” dilution where CpG methylation fails to
be copied onto newly synthesized DNA during S phase. An opposing
model invokes the active removal (by eviction or conversion) of
5-
m
ethyl
c
ytosine (5mC) from the genome. This can be achieved by one
of a number of candidate enzymes. A critical difference between the two
models is that while the first model depends on DNA replication and cell
division for DNA demethylation, the latter model does not. One of the
candidate proteins implicated in active DNA demethylation is the 5mC
deaminase AID. This enzyme is expressed in activated B cells and in mouse
primordial germ cells (
Popp et al., 2010
) although its expression in ESCs is
unclear (
Bhutani et al., 2010; Foshay et al., 2012
). It has been claimed that
AID is essential for the successful reprogramming of human fibroblasts in
heterokaryons formed with mouse ESCs (
Bhutani et al., 2010
). However,
others have found no evidence for contribution of AID to reprogramming
(
Foshay et al., 2012
). Recent studies have also shown that Tet (
t
en-
e
leven-
t
ranslocation) familymembers can oxidize 5mCto 5-hydroxymethylcytosine
and potentially to other oxidized forms (
Tahiliani et al., 2009
; reviewed in
