Maximizing the Safety of Induced Pluripotent Stem Cells as an Infusion Therapy: Limiting the Mutagenic Threat of Retroelement Retrotransposition during iPSC Generation, Expansion and Differentiation

Maximizing the Safety of Induced Pluripotent Stem Cells as an Infusion Therapy: Limiting the Mutagenic Threat of Retroelement Retrotransposition during iPSC Generation, Expansion and Differentiation

Funding Type: 
Early Translational I
Grant Number: 
TR1-01227
Award Value: 
$1,324,011
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
Status: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

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.

Year 2

Human embryonic stem (hES) cells and induced pluripotent (iPS) cells hold great promise as regenerative therapies for a number of currently incurable human diseases. However, before these cells or their progeny can be used as infusion therapies, the safety of these cells must be confirmed. One important safety concern is that during the generation, expansion, and manipulation of these pluripotent cells, mutations may be introduced into their genomes as a result of the activation and “jumping” of endogenous mobile genetic elements. Human cells contain roughly 3 million endogenous retroelements that comprise slightly less than half of the DNA present in the entire genome. Although many of these retroelements have been permanently silenced due to mutations, many others remain retrotransposition-competent and are capable of producing RNA, converting this RNA back into DNA (reverse transcription) and inserting this DNA at new sites in the cellular genome. These new insertions expand the cell’s pool of DNA and potentially produce disease-causing mutations or cancer. In somatic cells, the expression of these retroelements is strongly repressed through DNA methylation. However, because of the dynamic changes in DNA methylation that occur during the generation of iPS cells, high level retroelement retrotransposition may be unleashed. Our CIRM-sponsored work focuses on assessing endogenous retroelement activity as skin cells are reprogrammed into iPS cells. In the last 6 months, we have demonstrated that endogenous retroelements jump during iPS cell reprogramming and that the iPS cells generated contain new retroelement insertions in their genomes. We are now creating a new method to monitor the retroelement retrotransposition more accurately during reprogramming. In addition we are exploring both small molecules and cellular defenses that may counter retroelement retrotransposition in iPS cells (e.g., DNA methylation, APOBECs, TREX-1, and the RNAi machinery). Together, our goal is to provide a safer way to produce, culture and expand pluripotent cells.

Year 3

Human embryonic stem (hES) cells and induced pluripotent (iPS) cells holds great promise as regenerative therapies for a number of currently incurable human diseases. However, before these cells or their progeny can be used as infusion therapies, the safety of these cells must be confirmed. One important safety concern is that during the generation, expansion, and manipulation of these pluripotent cells, mutations may be introduced into their genomes as a result of the activation and “jumping” endogenous mobile genetic elements. Human cells contain roughly 3 million endogenous retroelements that comprise slightly less than half of the DNA present in the entire genome. Although many of these retroelements have been permanently silenced due to mutations, many others remain retrotransposition-competent and are capable of producing RNA, converting this RNA back into DNA (reverse transcription) and inserting this DNA at new sites in the cellular genome. These new insertions expand the cell’s pool of DNA and potentially produce disease-causing mutations or cancer. In somatic cells, the expression of these retroelements is strongly repressed through DNA methylation. However, because of the dynamic changes in DNA methylation that occur during the generation of iPS cells, high level retroelement retrotransposition may be unleashed. Our CIRM-sponsored work focuses on assessing endogenous retroelement activity as skin cells are reprogrammed into iPS cells. In the last 6 months, We made significant progress by applying whole-genome sequencing to investigate the LINE-1 and Alu retrotransposition in iPSCs. We also demonstrated that endogenous retroelements jump when iPSCs were differentiated to hematopoietic stem cell progenitors.

© 2013 California Institute for Regenerative Medicine