Human iPSC Genome Stabilization During the Gene Correction Process
This proposal seeks ways to measure and minimize damage to the chromosomes and DNA of cells to be used for stem cell therapy. These cells are called iPSCs, which stands for induced pluripotent stem cells, and they serve as stem cells for replacing cells that have been lost due to disease.
In order to make iPSCs, we and others use normal cells from the body (called somatic cells) and use genetic methods to push them into being stem-cell like. This is a promising approach because we can use the patient's own cells for this. However, we do not know how much genetic damage that we cause when we manipulate and then push the cells to become stem cells. We must have ways to measure and decide whether any genetic damage is excessive. Excessively damaged iPSCs could be quite dangerous because they might not behave normally, and they could even become tumors.
The goals here are:
1. Measure the amount of genetic damage in the iPSCs compared to the cells from which they are created (Aim 1A). It turns out that it is not trivial to measure the amount of genetic damage across the entire human genome. Our research team has a remarkably strong background in genetic and biochemical measurement of chromosomal and DNA damage.
2. Measure the amount of genetic damage in iPSCs after gene correction procedures. Gene correction will be a central step in using human stem cells to cure disease. For example, if a person has a disease, then, often, one of their genes predisposes to this disease. In these cases, it is helpful if we can correct that one gene before making the iPSCs or after making them. Our team is the most experienced in the world in human gene correction. In Aim 1B, C and Aim 2, we assess the amount of genetic damage that arises during the gene correction process.
3. Develop gentler ways of handling somatic cells and the iPSCs derived from them, so as to keep genetic damage to a minimum (Aim 3). Cells of our body are normally accustomed to seeing only about 3% oxygen in the dissolved gas around them when in our tissues. This is much lower than the 20% oxygen in room air and in the liquids (including laboratory solutions) that we use to wash and nourish such cells in the laboratory. Too much oxygen results in oxidation of the molecules that compose our cells, including the DNA that makes up our genome. We will develop methods that help the iPSC cells (or their somatic cell precursors) to make proteins that help protect them from high oxygen and oxidative conditions (Aim 3A). We will also develop reagents to add to the liquid in which we grow the cells in the laboratory to protect them from the oxidative environment (Aim 3B). We will also examine how the highly oxidative conditions affect the gene correction process (Aim 3C).
Genetic quality is a critical aspect of stem cells, including iPSCs. This project will lay the foundation for minimizing any unwanted genetic damage.
The research proposed here will benefit the state of California in at least three major ways.
First, the research here will move the application of stem cells to human disease forward. The progress that we will make will provide a firm foundation for assessing the genetic quality and genetic problems with various preparations of iPS cells. It will also generate better methods for preserving iPS cells in a genetically undamaged state.
Second, the work here will advance biotechnology on stem cells within California.
Third, the most immediate impact of this work will be to fund employment within the health research sector within California. The companies to which we planning to send samples or with who we are collaborating are all located in California (see Letters of Collaboration).