Regulation of sonic hedgehog expression during differentiation of human pluripotent stem cells
Ishmam Ahmed 2010-2011
Realizing the potential of stem cells to treat and replace defective pancreatic cells in diabetes patients, this experiment was an effort to better understand the role of sonic hedgehog (Shh)—a keystone protein during vertebrate organ development—in the context of in vitro human pluripotent stem cell differentiation into insulin-producing pancreatic β (beta) precursor cells. The understanding gained here is significant not only with regard to the ability of sonic hedgehog, but also its effects on the gene expression of human embryonic stem cells (HESCs) and whether these cells can serve as a model for in vivo vertebrate pancreas development.
In using hESCs, quantitative polymerase chain reaction (qPCR) was employed to gather and quantify gene expression data. The cells were grown in four chemically different conditions in order to direct expression. To determine the magnitude of expression for Shh and genes in the Shh pathway, Gli-1, Ptc-1, and Pdx-1, complimentary DNA (cDNA) was synthesized from the cells’ messenger RNA using standard laboratory procedure. The cDNA was then processed by qPCR machine which directly evaluated expression levels. These data were used to verify the expression of Shh, determine the effect of Shh on the gene expression of HESCs, and understand the effect of differentiation conditions on gene expression levels in vitro.
Shh activity was systematically regulated by the presence of the anti-Shh antibody and endoderm-inducing growth factors. The pancreatic differentiation of the HESCs was characterized by a decrease in Shh expression and a corresponding increase in Pdx-1 expression, a behavior that mimics in vivo pancreas development. Thus, this manipulation of Shh activity can be replicated to yield insulin-producing cells that, upon transplantation, may be viable for treatment of diabetes.
Click here for Detailed Research Report (PowerPoint)