Transcript Slide 1

Regulation of sonic hedgehog
expression and activity during
differentiation of human pluripotent
stem cells
Ishmam A. Ahmed
DOB: 02.18.1994
Wayzata High School
McGuire Translational Research Facility
Minneapolis, MN
Background
Following conception, the zygote develops into the body’s three germ
layers. The focus of this study was placed on the endoderm, the germ layer
from which the pancreas develops.
Image 1. Schematic diagram of germ layer and organ development
Background
continued
Most organs that develop from the endoderm, such as those in the central neural
system, the liver, and the intestine, require a high expression of the sonic hedgehog
(Shh) gene in order to do so.
The pancreas, however, and it’s cellular components, i.e. duct cells, acinar cells, and
endocrine cells (including insulin-producing), require low or no levels of sonic
hedgehog expression. Additionally, the pancreas requires the expression of a pancreasspecific gene, Pdx-1, in order to form.
Image 2. Characteristic gene expression during endoderm development
Background
continued
Pancreas formation is dependent on the
inhibition of the Shh expression and the
expression of its downstream pathway
genes.
The expression for the genes Gli-1, Ptc-1,
and Pdx-1 were used to verify the
expression of Shh, determine the affect
of Shh on the behavior of surrounding
cells, and to understand the affect that
certain differentiation conditions,
namely those that inhibit the Shh
pathway, on gene expression levels in
vitro.
Image 3. The sonic hedgehog (Shh) gene expression pathway
Questions and Hypothesis
Is Shh expression during in vitro differentiation of human stem cells
consistent with patterns observed during vertebrate development?
To what extent does Shh expression in developing gut cells affect gene
expression of surrounding cells?
Altering cellular growth conditions during human stem cell differentiation
will induce sonic hedgehog (Shh) expression in the gut endoderm and
subsequently increase the expression of Shh target genes. The
development of insulin producing cells can be favored by utilizing growth
conditions in which Shh expression is limited and in which Pdx1
expression is augmented.
Materials and Methods
The degrees of expression for the genes Shh, Gli-1, Ptc-1, and Pdx1 were measured
in each of four different sets of human embryonic “H9” cells, Groups 1-4. In order
to determine the effects that each medium’s components had on Shh expression
and downstream gene behavior, each set of cells was grown and differentiated in a
specific growth medium. Expression data was harvested through quantitative
polymerase chain reaction (qPCR) and further analyzed for trends.
The table below outlines the four different sets of tested growing mediums and
their components.
Table 1. Differentiation scheme
Data Collection
Complimentary DNA (cDNA) synthesis
Messenger RNA (mRNA) was reverse-transcribed by standard laboratory
procedure, i.e., by a series of enzymatic chemical reactions and heating/cooling
cycles.
Quantitative polymerase chain reaction (qPCR)
To analyze levels of gene expression for the cell in each group, the
corresponding cDNA was used in a standard qPCR machine. qPCR is a process
in which a DNA polymerase enzyme allows the multiplication of specific
strands of DNA by annealing primers, making a copy, melting the strands apart,
and then repeating the process continuously, doubling the number of copies
with each cycle.
Controls
Positive control: Gapdh (universal gene); guaranteed gene expression
Negative control: purified water; guaranteed zero gene expression
qPCR analysis
The standard qPCR machine indirectly measures the cycle threshold, the number
cycles it takes to obtain substantial expression, for each set of cells. In order to
quantify this raw data, a very specific algorithm is used.
Pos
Name
Ct
SYBR
Amount
SYBR
Target
SYBR
A9
D3
16.42
-
GAPDH
A10
16.7
-
GAPDH
A11
26.64
-
A12
26.19
-
GAPDH
Ct
Delta
Ct
Fold
Shh
10.08
1.952064
Shh
9.63
2.666597
Avg Fold
Stan Dev
2.30933
0.505252
16.56
Table 2. Expression quantification algorithm
Algorithm Details
CtSYBR: raw data provided by machine
Pos: coordinate position on a standard qPCR plate
Target SYBR: primer used
GAPDH Ct: average value of the two corresponding Ct SYBR values
Fold: relative expression value obtained by formula 2^(d0 average Delta Ct – Delta Ct of target gene)
Average Fold: value between pair of Fold values
Stan Dev: standard deviation of two corresponding Fold values (used to construct error bars; see next)
Results
H9 Shh Expression
300
Relative Expression to d0
250
200
150
Group 1
Group 2
Group 3
100
Group 4
50
0
d0
-50
d3
d6
d9
d15
d21
Harvest Day
Shh expression increased dramatically in groups 1, 3, and 4 at around d6, peaked at
d9, and dropped to relatively low levels at d15. Group 2 showed a sharp increase in
expression at d15 that continued onto d21. Considering that α-Shh stimulates Shh
expression, and that it was added at d3, a spike in Shh after its addition is probable.
Results
H9 Gli-1 Expression
8
7
Relative Expression to d0
6
5
4
Group 1
Group 2
3
Group 3
2
Group 4
1
0
d0
d3
d6
d9
d15
d21
-1
-2
Harvest Day
There is a general trend of increasing expression for all groups. Group 1 exhibits the
highest Gli-1 expression at d21. This may indicate that Gli-1 expression is continuous
and increases as differentiation occurs. Gli-1 expression is indicative of Shh
expression. This is reasonable when considering the Shh signaling pathway.
Results
H9 Ptc-1 Expression
25
Relative Expression to d0
20
15
Group 1
Group 2
10
Group 3
Group 4
5
0
d0
-5
d3
d6
d9
d15
d21
Harvest Day
The Ptc-1 expression for each group resembles the Shh and Gli-1 expression;
each has a peak expression around d9. This is reasonable, knowing that
transcription of Ptc-1 is induced by Shh. Increasing levels of Ptc-1 indicate increasing
levels Shh, and vice versa. The same is reasonably true for decreasing levels of each.
Results
H9 Pdx1 Expression
3000
Relative Expression to d0
2500
2000
1500
Group 3
Group 4
Group 1
1000
Group 2
500
0
d0
d3
d6
d9
d15
d21
Harvest Day
High, or increasing, Pdx-1 expression is indicative of pancreatic differentiation. The data are a
reflection of this. For group 1, Pdx-1 expression remains relatively low through d15, while Shh
expression is highest between d6 and 15. As Shh expression decreases, by d15 and d21, Pdx-1
expression begins to rise. The same general trend is seen in group 3. For group 2, Shh expression
rises sharply after d15 while Pdx-1 expression begins to decrease at d15. For group 4, Shh expression
noticeably increases around d15 and does so through d21 whereas Pdx-1 expression begins to
decrease from d15 and through d21.
Conclusion
A drop in Shh expression and simultaneous increase in Pdx-1 expression
during pancreas formation is expected. It is also expected that Gli-1 and Ptc-1
expression patterns mimic those of Shh; they are downstream genes.
Growth conditions yielded by Group 1 components allow embryonic stem
cells to most closely follow the differentiation of those that occur during in
vivo vertebrate development
Cells in Group 2 did not exhibit natural expression patterns. This was
expected; the differentiation in vivo relies on chemical and genomic stimuli.
Cells in Group 2 were left unstimulated.
Cells in Groups 3 and 4 were grown in a lesser concentration of growth serum
than those in Groups 1 and 2. Based on the observed trends, cells grown in 2%
serum do not grow or proliferate normally.
Certain human pluripotent cell lines can be used as an accurate in vitro model
of in vivo pancreas development when placed in specific differentiation
conditions, like those present in Group 1.
Discussion
Shh can be manipulated by cellular growth conditions in a way that will
favor pancreatic development, mimicking in vivo patterns. This was
especially apparent in Group1 cells, which were sequentially treated with
chemical factors; see Table 1.
This study has illuminated a potential method for streamlining the
production of pancreatic cells that can be used for diabetes treatment.
Future Directions
By further analysis of Shh’s role in pluripotent cells, the scientific
community may gain insight into their differentiation potential and even
uncover possibilities for clinical application.
Since this study was conducted on “H9” embryonic stem cells, it would be
reasonable to conduct the same study on other types of pluripotent cells,
namely, induced pluripotent stem (iPS) cells.