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3.6.3 Morphology of hESCs
The morphology of the hESCs were checked after 13 passages and the phase
contrast images shown (Figure 6B). hESCs cultured on DxSDOCDOC matrix
still retained a pluripotent morphology with round, tight colonies with clearly
defined margins. These colonies consist of cells with high nuclear to
cytoplasm ratio. In contrast, hESC cultured on Matrigel grew in larger
colonies, albeit still with defined colony edges. These control cells still
retained their high nuclear to cytoplasm ratio, but there were more pockets and
areas consisting of differentiated cells with flatter and larger morphologies
(arrows).

3.6.4 Pluripotency markers levels by flow cytometry
At the 5
th
passage, hESCs cultured on DxSDOCDOC and on Matrigel were

harvested and analyzed by flow cytometry in a similar fashion to that of
section 3.5.4. The same 5 pluripotency markers were used to characterize the
hESC samples (Figure 6C).

It was found that 60.5% of the hESCs cultured on Matrigel were positive for
the marker Oct-4, while 76.4% of hESCs on DxSDOCDOC were positive for
Oct-4, which showed a significant increase compared to the control.
Conversely, SSEA-3 levels were lower in hESCs on DxSDOCDOC at 44%
compared to 57.2% in hESCs on Matrigel. SSEA-4 levels were similarly
strong in both samples of hESCs on Matrigel and DxSDOCDOC, at 93.6%.
TRA-1-60 levels were both high in both samples on Matrigel and

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DxSDOCDOC, at 73.3% and 77.1% respectively. 84.8% of hESCs on
DxSDOCDOC were positive for the marker TRA-1-81, which is higher than
that of hESCs on Matrigel at 75.2%.

Hence, at passage 5, hESCs on DxSDOCDOC expressed comparable
pluripotency marker levels compared to the control and markers Oct-4 and
TRA-1-81 were markedly increased, whereas SSEA-3 expression was
decreased.

At the 13
th
passage, again, the hESCs cultured on either Matrigel or
DxSDOCDOC were harvested for immunolabeling using the same 5
pluritpotent markers and analyzed by flow cytometry.

Oct-4 levels were very much reduced at 39.9% for hESCs on Matrigel and
39.3% for hESCs on DxSDOCDOC, but the two samples were comparable to

each other. SSEA-3 levels were also not significantly different between the
two samples at 41.8% and 38.3% for hESCs on Matrigel and DxSDOCDOC
respectively. SSEA-4 levels were much higher in hESCs on DxSDOCDOC at
82.7% compared to 73.7% in hESCs on Matrigel. TRA-1-60 levels were
similar in both hESCs on Matrigel and DxSDOCDOC at 65% and 68.8%
respectively. TRA-1-81 levels were also comparable in hESCs on Matrigel at
71.2% versus 74.7% in hESCs on DxSDOCDOC.


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Hence, at passage 13, hESCs on DxSDOCDOC produced comparable
pluripotency marker levels compared to the control, except for SSEA-4, where
significantly more hESCs on DxSDOCDOC had the expression of SSEA-4.

3.6.5 Pluripotency markers levels by immunofluorescence
The colonies of hESCs were harvested after 13 passages for adherent
immunofluorescence and stained for same panel of pluripotency markers as
the previous propagation trial - Oct-4, SSEA-4, TRA-1-81, SSEA-3, and
TRA-1-60. hESCs cultured on DxSDOCDOC grew mostly in tight colonies
that had cells positive for the pluripotency markers (Figure 6E-G). Control
hESCs also mostly grew in colonies positive for the pluripotency markers, but
there were some cells that were not positive for the markers (arrows). The
above observations confirmed the pluripotent morphology seen under phase
contrast microscopy.

3.6.6 In vitro induced differentiation
hESCs cultured on DxSDOCDOC for 13 passages were induced into neural
differentiation using a protocol modified from WiCell Research Institute,
National Stem Cell Bank. In this protocol, the hESCs formed embryoid bodies
using non-adherent cell culture plates and were transferred onto laminin-

coated plates and incubated in neural induction medium. Under these
conditions, hESCs were able to form extensions between embryoid bodies that
were stained positive for β III tubulin, a marker for neural differentiation.
These results indicate that after 13 passages on DxSDOCDOC, the hESCs

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were still receptive to neural induction and differentiation, showing their
retention of differentiation capabilities.

3.6.7 Karyotype
Both hESCs cultured on Matrigel and on DxSDOCDOC were found to have
normal female karyotype. All 20 tested metaphases of hESCs on Matrigel had
normal karyotypes, while 19 out of 20 tested metaphases of hESCs on
Matrigel had normal karyotypes; 1 metaphase showed non-clonal random loss.
Again, as mentioned in section 3.5.6, these cells can be considered as
karyotypically normal due to only 1 metaphase out of 20 having non-clonal
random loss, which could have been a result of technical artifacts or random
mitotic error.

3.6.8 DNA Methylation
Epigenetic programming has been shown to play an important role in
regulating cellular differentiation, and because little is known about the
epigenetic factors that define the differentiation state or potency of stem cells
in comparison to differentiated cells, DNA methylation studies were done.
The goal was to identify methylation changes between hES cells grown on the
human matrices versus Matrigel. Samples from this propagation trial were
collected and analyzed by Illumina Methylation Assay – Infinium II (see
section 3.5.7.). Genes that were found to be overexpressed in iPSCs were
selected to act as a pluripotency signature and the methylation status of the
respective CpGs were presented in a heatmap (Figure 6J). Samples from the

previous propagation trial were combined into the same heatmap and labeled

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with a suffix (3.5.7), while samples from this propagation trial were labeled
with a suffix (3.6.8). In addition, neurally-differentiated hESCs that were
previously cultured on DxSDOCDOC were also included in the analysis and
labeled with a suffix (3.6.6). From the heatmap, it can be seen that there were
no major changes in the signature, which is in contradiction to observations
seen for the differences in cellular behaviour. Again, the epigenetic analysis of
the samples was performed in the same way as in section 3.5.7, which only
covers 2 CpGs out of all the CpGs found to be present in each gene of the
signature, as such, it only serves as an incomplete representation of the
epigenetic changes that can be found.

Interestingly, in the neurally differentiated hESCs, one of the two CpGs
representing the gene coding for Oct-4 (POU5F1) was found to be highly
methylated at 0.91 (arrow) compared to the other pluripotent samples ranging
from 0.45 to 0.57. It is possible that the neural differentiation of the hESCs has
led to DNA methylation changes in that particular CpG, which could lead to
changes in transcription levels. It is also interesting to note that the assay used
was able to pick epigenetic differences only in the situation when there are
major changes to the hESCs such as the changes brought about by neural
differentation. Conversely, when cellular changes are comparatively minor,
such as poor pluripotency maintenance on Matrigel, the epigenetic assay was
unable to detect any major epigenetic changes. This inadequacy of the assay
could be due to it only representing a small fraction of the CpGs found within
the genome, and unless major advances in technology allows for a far larger
representation of the genome, the assay may not be sufficient to detect

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epigenetic changes that truly reflects observations seen of cellular morphology
changes.

Deep (454) bisulfite sequencing was used to analyze the methylation status of
14 CpG sites found within the Oct4 promoter region. The statuses of hESCs
on Matrigel or human matrices are presented in a heatmap (Figure 6K). In all
conditions, the DNA methylation of 14 CpG sites remained low, hence the
Oct-4 promoter were unmethylated, indicating that the Oct-4 promoter region
was not yet silenced in all three conditions. Oct-4 is an important transcription
factor shown to be highly expressed in undifferentiated cells, and its
expression leads to the expression of other genes that maintain hES cells in an
undifferentiated state.

DNA methylation status of the 27,578 CpG sites tested for using the
Illuminium Infinium Chip revealed a high overall similarity between Matrigel
versus DxSDOC hESC cultures, and Matrigel versus DxSDOCDOC hESC
cultures (Figure 6L). However, there still remains methylation differences
between Matrigel-propagated hESCs and the DxSDOC-propagated hESCs, as
426 markers were found to be differentially methylated (Figure 6M). 194
markers were found to be differentially methylated between Matrigel-
propagated hESCs versus DxSDOCDOC-propagated hESCs. Between the two
groups of differential markers, there is an overlap of 49 markers there were
differentially methylated (Figure 6M). These 49 markers represent a diverse
group of genes with many different functions. While the functional
significance of this observation will have to be further analyzed, it is possible

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that the differential methylation of these 49 genes could have contributed to
the superior proliferation of pluripotent hESCs on the DxSDOC and
DxSDOCDOC matrices.

3.6.9 DxSDOCDOC matrices are able to maintain pluripotent hESCs
In this repeat propagation trial based on population doubling observations,
morphology, pluripotency marker expression, in vivo differentiation studies
and karyotype results, it was concluded once again that hESCs cultured on
DxSDOCDOC showed superior population doubling rates, while maintaining
pluripotent-like morphology compared to control hESCs on Matrigel.
Pluripotency marker expressions were comparable to the control hESCs by
flow cytometry and by adherent immunofluorescence studies, hESCs on
DxSDOCDOC had fewer areas of differentiation compared to the control. In
vitro differentiation studies showed that hESCs cultured on DxSDOCDOC
matrices retained their neural differentiation capacity after 13 passages and yet
maintained their karyotype despite a prolonged culture period of 18 passages.

3.7 Matrices that maintain hESC pluripotency using dispase
3.7.1 Population doublings
In section 3.4, 3.5 and 3.6, all hESCs were passaged by enzymatic passaging
using collagenase IV. There is another enzyme, dispase, which is commonly
used for enzymatic passaging of hESCs. In the propagation trial described in
this section, dispase is used for subculturing of the hESCs to illustrate that
irregardless of whether dispase or collagenase IV is used, DxSDOCDOC will
still prove superior at increasing the population doublings and also in

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maintaining the pluripotent characteristics of the hESCs during their
propagation.

Once again, hESCs were cultured onto Matrigel, DxSDOC and DxSDOCDOC
matrices for up to 20 passages. hESCs were maintained in defined culture
medium, mTeSR-1, and subcultured every 5-7 days depending on the

confluency of the cultures. At each passage, replicates of the cultures were
sacrificed for cell counting and the population doublings were calculated and
plotted (Figure 7A).

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