regulation of sonic hedgehog expression and activity during differentiation of human pluripotent...
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Regulation of sonic hedgehog expression and activity during differentiation of human pluripotent stem cells
Ishmam A. AhmedDOB: 02.18.1994
Wayzata High School
McGuire Translational Research FacilityMinneapolis, MN
BackgroundFollowing 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 pancreas-specific 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 MethodsThe 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) synthesisMessenger 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.
ControlsPositive control: Gapdh (universal gene); guaranteed gene expression Negative control: purified water; guaranteed zero gene expression
qPCR analysisThe 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 NameCt
SYBRAmount SYBR
Target SYBR
GAPDH Ct
Delta Ct
Fold Avg Fold Stan Dev
A9 D3 16.42 - GAPDH
A10 16.7 - GAPDH 16.56
A11 26.64 - Shh 10.08 1.952064
A12 26.19 - Shh 9.63 2.666597 2.30933 0.505252
Table 2. Expression quantification algorithm
Algorithm DetailsCtSYBR: raw data provided by machinePos: coordinate position on a standard qPCR plateTarget SYBR: primer used GAPDH Ct: average value of the two corresponding Ct SYBR valuesFold: relative expression value obtained by formula 2^(d0 average Delta Ct – Delta Ct of target gene)Average Fold: value between pair of Fold valuesStan Dev: standard deviation of two corresponding Fold values (used to construct error bars; see next)
Results
d0 d3 d6 d9 d15 d210
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H9 Shh Expression
Group 1Group 2Group 3Group 4
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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 -Shh stimulates Shh αexpression, and that it was added at d3, a spike in Shh after its addition is probable.
Results
d0 d3 d6 d9 d15 d210
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H9 Gli-1 Expression
Group 1Group 2Group 3Group 4
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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
d0 d3 d6 d9 d15 d210
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H9 Ptc-1 Expression
Group 1Group 2Group 3Group 4
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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 increasinglevels Shh, and vice versa. The same is reasonably true for decreasing levels of each.
d0 d3 d6 d9 d15 d210
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2500H9 Pdx1 Expression
Group 3Group 4Group 1Group 2
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Results
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.
ConclusionA 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.
DiscussionShh 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 DirectionsBy 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.