Analysis of Signal Transduction Pathways in Induced Pluripotent Stem Cell Reprogramming

Embryonic stem cells have immense biomedical potential, since they are pluripotent and thus have the capability to form any cell type in the adult body. Human embryonic stem cell derivation, however, involves destroying "leftover" embryos - a non-ideal scenario both ethically and as the embryonic stem cells may not be representative of, or "tissue compatible" with, the general patient population. In 2006, researchers discovered a way to "reprogram" an adult cell into an embryonic-like cell by expressing only four genes, the "Yamanaka factors" (c-Myc, Klf4, Oct4, Sox2), work that was awarded the 2012 Nobel Prize in Medicine. The resultant embryonic-like cells were termed induced pluripotent stem (iPS) cells. With this technology, cell-replacement therapies, where a diseased cell type in an individual is replaced with stem cell-derived cells, will be able to utilize a patient's own cells. Additionally, biopsies from patients will allow for researchers to study and culture diseased cells, a benefit especially if the cause of the disease is unknown and the diseased cells are not easily isolated./nHowever, two limitations hinder the applications of iPS cell technology. First, the formation of iPS cells is very inefficient, with only 0.01-0.1% of cells converting back to an embryonic-like state. Second, there is an increased risk of cell abnormalities and cancer. The delivery of the factors to cells is mediated by viral delivery, which can modify the genome and activate cancer-causing genes. Additionally, the proteins KLF4 and C-MYC are oncogenes, or have the potential to cause cancer. Typically, replacing a factor or changing the gene delivery method from viral gene delivery results in an even lower efficiency and has remained a challenge for iPS cell technology./n/nSignal transduction, the relay of an input from outside the cell to the DNA inside the nucleus, is mediated through protein-protein interactions, and has been shown to be a central regulator of cell behavior. Signal transduction proteins directly affect the activation of transcription factors (such as C-MYC, KLF4, OCT4, and SOX2), which then bind to DNA to regulate gene expression. Because signal transduction proteins are more easily manipulated through small molecules (drugs) and external proteins, I have developed a way to study signal transduction during iPS cell reprogramming./nI have created a library of 38 genes encoding signal transduction proteins that can upregulate and downregulate major signaling pathways within the cell. The library can be used in a modular approach to study reprogramming using mouse cells. First, the library can be used by itself to see if there are any combinations of signaling proteins that can replace the Yamanaka factors. Second, the library can be used in conjunction with the Yamanaka factors to see how signaling proteins affect reprogramming. Third, the library can be used to see if any signaling proteins can replace OCT4, SOX2, and KLF4./nThe second aim of the library, to see how each signaling protein affects reprogramming, resulted in a group of genes that aided in reprogramming, a group that had no effect on reprogramming, and a group that was deleterious to reprogramming. Importantly, the results from the library showed that previously published results aligned with our data, verifying our methods to study reprogramming. Additionally, I identified several GTPases that improve or hinder iPS cell reprogramming, factors that to date have not been largely studied in reprogramming./nTo learn more about the individual signaling proteins and their role in reprogramming, I performed studies looking if each signaling protein could replace OCT4, SOX2, and KLF4. The most promising results were in replacing OCT4, which to date has been the most difficult to replace as SOX2, KLF4, and c-MYC have published small molecule replacements. I discovered an activated cell surface receptor, GNAS, that could replace OCT4 with approximately 8% of the efficiency of OCT4, SOX2, KLF4, and C-MYC. Forskolin, a small molecule that mimicked the activity of the receptor could also replace OCT4 with a 2.1-fold increase in reprogramming efficiency. To illustrate these cells are pluripotent, I did immunostaining to probe for the presence of embryonic stem cell markers, SSEA1, OCT4, and NANOG. The cells were able to differentiate into the three germ layers, resulting in cells from the brain, muscle, liver, and heart. Additionally, I have determined that forskolin acts via the EPAC signaling pathway and is necessary and sufficient to induce colony formation. Forskolin also increases epithelial gene expression, decreases mesenchymal gene expression, and increases cell proliferation to replace OCT4 during reprogramming/nTo date, this work shows a small molecule can replace OCT4 to result in pluripotent cells. Not only does this aid the understanding of reprogramming biology, but it is an initial step in finding a method to reprogram without viruses or oncogenes, which is ideal for downstream applications such as cell-replacement therapies and disease studies.

Advisor: Schaffer, David