Regulation of sonic hedgehog expression and activity during differentiation of human pluripotent stem cells Ishmam A. Ahmed DOB: 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 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 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. PosName Ct SYBR Amount SYBR Target SYBR GAPDH Ct Delta Ct FoldAvg FoldStan Dev A9D GAPDH A GAPDH16.56 A Shh A Shh 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 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 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 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 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.