Year 1
The use of human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells holds great therapeutic promise for a number of currently incurable human diseases. However, successful deployment of these cells requires successful negotiation of several “bottlenecks” some of which involve the safety of the derived cells for infusion. One important safety concern is that during the generation, expansion, and manipulation of these pluripotent cells, mutations may be introduced into the genome due to increased “jumping” of mobile genetic elements.
Every cell contains roughly 3 million “jumping genes” or endogenous retroelements that comprise up to 45% of the DNA present in the human genome. Fortunately, many of these retroelements have been permanently silenced during evolution by crippling mutations. Nevertheless, some remain active and capable of moving to new chromosomal locations potentially producing disease-causing mutations or cancer. More mature differentiated cells control retroelement movement (retrotransposition) chiefly by methylating the DNA comprising these retroelements. Strikingly, such DNA methylation is highly dynamic in hES cells because these cells must be able to differentiate into a wide spectrum of different cell types leading to tissue and organ generation. During reprogramming of skin cells to iPS cells, DNA methylation patterns are erased, potentially making iPS cells vulnerable to heightened retroelement activity. Our CIRM-sponsored work focuses on assessing endogenous retroelement activity as skin cells are reprogrammed into iPS cells. During the first year of the funding, we have shown that endogenous retroelements are very active in iPS cells and thus potentially endanger the genomic integrity of these cells. We are now exploring whether diminishing retroelement mobilization by treatment with specific drugs may provide a safer way to produce, culture and expand pluripotent cells.
In addition we are exploring several cellular defenses beyond DNA methylation that may counter retroelement retrotransposition in iPS cells (e.g., APOBECs, TREX-1, and the RNAi machinery). One goal is to assess whether one or more of these pathways is particularly active in iPS and hES cells and specifically used to control retroelement activity. Together, these studies are systematically addressing endogenous retroelement activity in pluripotent stem cells with an eye to limiting their activity and thus making the generation of these cells and their progeny safer.