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Unformatted text preview: or human feeder cells) (Xu et al. 2001, 2004).
Matrigel is a complex mixture of mouse sarcoma origin
which contains extracellular molecules, such as laminin,
collagen IV, and also growth factors and other substances.
Also other research groups have successfully used Matrigel
as substrate for hESC (Sato et al. 2004, Pebay et al. 2005,
Wang et al. 2005a, Xu et al. 2005). Matrigel is not an
optimal matrix for cells because it contains many unknown
components and is of non-human origin. To replace
Matrigel with alternatives, several research groups have
used laminin (Xu et al. 2001, Beattie et al. 2005, Genbacev
et al. 2005), ﬁbronectin (Amit et al. 2004, Noaksson et al.
2005) and human serum (Stojkovic et al. 2005a) -coated
plates for hESC. Table 3 summarizes different coating
materials which have been used as replacement of feeder
cells in hESC cultures. A synthetic human matrix, such as
recombinant human laminin (Nikolova et al. 2006), would
be optimal for hESC cultures.
Reproduction (2006) 132 691–698 Culture medium supplements
Unlike in the mouse (Smith et al. 1988), leukemia
inhibitory factor (LIF) is not able to maintain the nondifferentiated growth of hESC without feeder cells
(Thomson et al. 1998) and so far no such component
having an important effect on hESC as LIF has for mouse
ESC has been identiﬁed. To replace feeder cells and
conditioned medium in hESC cultures, identiﬁcation of
conditioned medium components facilitating non-differentiated growth would be crucial. Conditioned medium
most likely contains variable amounts of growth factors
with batch-to-batch variation. There are also differences
between conditioned media from different feeder cells in
supporting the growth of undifferentiated hESC (Xu et al.
2001). In our laboratory, we have not succeeded in
culturing non-differentiated hESC in feeder-free cultures
for longer than a few passages using conditioned
medium from human foreskin ﬁbroblasts (unpublished).
Several research groups have reported successful use of
supplements in hESC culture medium, such as bFGF and
hyaluronic acid (Heins et al. 2004), transforming growth
factor-b (TGF-b), bFGF and LIF (Amit et al. 2004), bFGF
and GSK3 inhibitor (Sato et al. 2004), activin A and bFGF
(Xiao et al. 2006), and spingosine-1-phosphate with
platelet-derived growth factor (Pebay et al. 2005).
Recently, two groups found that a combination of the
BMP signaling antagonist Noggin and high concentration
of bFGF was sufﬁcient to maintain undifferentiated hESC
in feeder-free culture conditions (Wang et al. 2005a, Xu
et al. 2005). In our laboratory, these factors in similar high
concentrations proved unsuitable to our hESC (unpublished). Beattie et al. (2005) showed that hESC cultured in
the presence of activin A, nicotinamide (NIC), and
keratinocyte growth factor (KGF) remained undifferentiated. They suggested that activin A may block the
differentiation and NIC and KGF may have function in
hESC proliferation. In addition to this, Vallier...
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This document was uploaded on 09/21/2013.
- Spring '13