Regulation of Apoptosis and Differentiation by p53 in Human ...

CHAPTER 1: Introduction
2007). Recently, iPs cells were generated from human adult dermal fibroblasts with the same 4
factors mentioned above and from human fetal fibroblasts with Oct4, Sox2, Nanog, and LIN28 ,
opening the possibility of generating patient and disease-specific pluripotent stem cells
(Takahashi et al., 2007; Yu et al., 2007).
1.1.3- Culture methods for hESC propagation: advantages and limitations
Two culture methods are widely used to propagate hESC (Figure 1.2): the original technique
used to culture the cells, referred to herein as the standard method and the serum-free method.
The standard method of culturing hESC involves weekly mechanical passaging of
morphologically undifferentiated parts of a hESC colony onto full density (6x10 4 cell/cm 2 ) mouse
or human fibroblast feeder (MEF and HEF, respectively) layers mitotically inactivated in Dulbeco’s
modified Eagle’s medium (DMEM) containing 20 % fetal calf serum (FCS) or human serum
(Reubinoff et al., 2000; Richards et al., 2002; Thomson et al., 1998). This protocol is demanding
in terms of time, money and effort, but has proven to be a safe and reliable method for the long
term propagation of hESC. This is highlighted by the fact that hESC thus cultured have rarely
been reported to develop genetic abnormalities during culture. However, the major drawback of
this method is the low number of hESC available for experimentation. Some laboratories
therefore opt to maintain hESC stock cultures using this standard method and regularly establish
fresh bulk cultures from these stocks for experimentation (Amit et al., 2000). The serum-free
method of culturing hESC is performed according to a protocol developed by Amit and colleagues
which involves passaging of enzymatically (for example collagenase and trypsin) or nonenzymatically
(cell dissociation solution, EDTA, etc) dissociated clumps of hESC on 1/3 density
feeders (2x10 4 cell/cm 2 ) in DMEM/F12 supplemented with 20% Knockout serum replacement
(KSR) from Invitrogen, and supplemented with fibroblast growth factor 2 (FGF-2). Despite the fact
that KSR contains fewer components than FCS, its composition is still undefined and it contains a
large amount of Albumax, a lipid-rich preparation of bovine serum albumin (BSA). This method
allows for dramatic expansion of hESC. However, a number of reports have indicated that this is
accompanied by an increase in genetic instability, methylation changes and mitochondrial
mutations, abnormalities also seen in cancer and hECC (Draper et al., 2004; Mitalipova et al.,
2005; Brimble et al., 2004; Allegrucci et al., 2007). Allegrucci and colleagues examined the DNA
methylation profiles of more than 2000 genomic loci by restriction landmark genome scanning
and showed that the culture conditions can induce DNA methylation instability in the hESC
epigenome (Allegrucci et al., 2007). The unstable loci have been previously associated with a
tumour phenotype. They also showed that the adaptation of the cell lines to serum-free conditions
result in additional epigenetic instability (Allegrucci et al., 2007). This study highlights the
importance of using appropriate techniques to analyze epigenetic changes and evaluate their
consequences on hESC experimental utility and biosafety, as well the need for optimization of
derivation and culture protocols of hESC in order to minimize culture-induced instability.
In addition to optimized culture methods that prevent the occurrence of genetic instability, novel
technologies to purge abnormal hESC from large scale hESC cultures will have to be developed.
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