[brijnbhatt]

The steady experience of cells to endogenous and exogenous agents that inflict DNA damage requires active repair processes to get rid of potentially mutagenic events in stem cells leading to cancer. The similar agents menace early human embryos with DNA damage that can eventually lead to mutations, cancer, and birth imperfections. In vitro, human embryonic stem cells (HESCs) impulsively undergo events leading to genetic instability and mutations. All these three types of genetic complications can have similar links to malfunctions in DNA repair systems, but little information now exists for HESCs. Consequently, the first step in accepting the causes of HESC genetic instability is to understand which DNA repair systems are faulty. We shall investigate the basis for this phenomenon in HESCs by evaluating their capacity to either repair DNA or form mutations.

We can culture two HESC lines and contrast HESC repair and mutation formation to that of control cells. We can use a new technique which simplifies the production and use of the feeder cells that support the growth of the HESCs. Genetic stability can be tested for HESCs grown on conventional feeder cells, as well as those grown in feeder free culture. Three types of DNA repair assays can be used to monitor the genetic stability of the two HESC lines grown in these different ways. As a first assays, DNA molecules with dissimilar randomly-induced damage are transferred into HESCs, and DNA repair is followed by the re-establishment of the activity of a reporter protein that is coded for in the damaged DNA. As a second assay we shall introduce specific DNA damage at a unique site in DNA that is transferred to HESCs and repair is determined using a polymerase chain reaction-based technique.

Since aneuploidy is also recognized to be sourced by double-strand DNA breaks, one can use two other assays to evaluate capacity of HESCs to repair that type of damage. These experiments will point toward DNA repair pathways that eradicate DNA damage and cause genetic instability. The final endpoint for these first round experiments is the formation of mutations. To study this, one can modified an assay system so that it will function in normal human cells to monitor mutations which arise instinctively or those which are induced by various agents.

As a conclusion, these investigations can provide the basis for understanding genetic instability in HESCs that can direct cells to tumor. The employment of HESCs clinically will necessitate such knowledge. Additionally, these results will also yield information on susceptibility to mutations of cells early in embryo development from stem cells.