187feeders and a serum-free medium, defi ning a totally animal-free culture system
(Amit et al. 2003 ). Further, Lee et al. ( 2004 ) showed that human adult uterine endo-
metrial cells, adult breast parenchymal cells and embryonic fi broblasts can be used
as feeder cells for the growth of human ES cells (Lee et al. 2004 ). By comparing
different types of feeders, more effective human feeder cells should be selected
based on the expression of various factors such as extracellular matrices, growth
factors and cytokines that induce proliferation and inhibit differentiation of human
ES cells. These selective human feeder cells will aid the progress of cell-based
therapies. Several chemically defi ned media systems have been reported since then
for the maintenance and proliferation of pluripotent stem cells to try and overcome
culture-induced variability.
Subsequently, feeder-independent human ES cell cultures were developed com-
prising of protein components derived exclusively from recombinant sources or
purifi ed from human material (Ludwig et al. 2006 ). Braam et al. ( 2008 ) demon-
strated that human ES cells express integrin receptors for laminin, fi bronectin, col-
lagen and vitronectin which are all functional in facilitating adhesion. They found
recombinant vitronectin as a suitable and functional alternative to Matrigel as it
supported human ES cell growth just as well (Braam et al. 2008 ). More recently,
Baxter et al. ( 2009 ) derived a feeder-/serum-free culture system containing fi bro-
blast growth factor 2 (FGF2), activin A, neurotrophin 4 (NT4) and the N2 and B27
supplements along with a human fi bronectin substrate. This culture system was
reported to support the long-term proliferation of several euploid human ES cells
without the need for manual propagation and showing little or no spontaneous dif-
ferentiation. These components were explained to have discrete functions: both
FGF2 and activin A were necessary to maintain ES cells in an undifferentiated state
while NT4 to promote cell survival and the fi bronectin substrate to sustain a rapid
rate of ES cell culture expansion (Baxter et al. 2009 ). The cell-substrate interface
provided by the adsorbed fi bronectin that promotes human ES cell interactions is
quite specifi c. This can be used to develop the therapeutic potential of human ES
cells if the fi bronectin layer could be characterised such that it could be reproduced
synthetically (Kalaskar et al. 2013 ). Figure 9.4 shows the development of culture
since the fi rst human ES cells were cultured.
9.2.4 Differentiation
When removed from feeder layers and transferred to suspension culture, ES cells
begin to differentiate and form embryoid bodies (EB) which are multicellular
groups of differentiated and undifferentiated cells. In vitro differentiation is consis-
tently disorganised and often variable between different EBs even within the same
culture (Odorico et al. 2001 ). Initially, as human ES cell lines were not clonally
derived, pluripotency was only demonstrated by populations of cells and not indi-
vidual cells, so within a colony there were subpopulations of cells, and no individ-
ual cell showed the ability of differentiating into cells of all three germ layers
9 Human Embryonic Stem Cells and Associated Clinical Concerns