WARNER GREENE, MD, PhD

		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 The ability to convert human skin cells to induced pluripotent stem cells (IPSCs) represents a seminal break-through in stem cell biology. This advance effectively circumvents the problem of immune rejection because the patient's own skin cells can be used to produce iPSCs. This exciting technology could accelerate treatments for a number of presently incurable diseases. However, a paramount unanswered question is whether these cells or their derivatives are truly safe for administration. Specifically, it is unknown whether the integrity of the iPSC genome is maintained during the tissue culture steps required to generate, maintain, expand and differentiate iPSCs.
Every cell contains roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of the human genome. This contrasts with the fact that the roughly 21,000 human genes occupy only 1.5% of genome. While many of these retroelements have been permanently silenced during evolution, many others remain active and capable of replicating and moving to new chromosomal locations potentially producing disease-causing mutations or cancer. Somatic cells limit the jumping of these mobile genetic elements (retrotransposition) chiefly by methylating the DNA in and around these elements. Strikingly, the process of converting a skin cell to an iPSC results in a profound loss of DNA methylation potentially opening the door for high level retroelement activity that could corrupt genomic integrity. These insertions can disrupt key genes, create double strand DNA breaks or lead later to loss of large sections of DNA. Whether retroelement activity contributes to the fact that only 0.01% of skin cells are successfully reprogrammed to iPSCs is unknown. Thus, key questions regarding the safety of these cells remains. We now propose to determine the level of retroelement retrotransposition occurring in iPSCs and hESCs and to develop potentially safer ways to generate and maintain iPSCs in culture by blocking a key retroelement enzyme. Further, we will assess whether differentiation of these cells triggers retroelement activity. Finally, we will explore potential additional cellular defenses brought into action to oppose these retroelements with the goal of further enhancing these defenses. The APOBEC3 Gene Family as Guardians of Genome Stability in Human Embryonic Stem Cells The successful use of human embryonic stem cells (hESCs) as novel regenerative therapies for a spectrum of currently incurable diseases critically depends upon the safety of such cell transfers. hESCs contain roughly 3 million “jumping genes” or mobile genetic retroelements that comprise up to 45% of their genetic material. While many of these retroelements have been permanently silenced during evolution by crippling mutations, many 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) by methylating the DNA comprising these elements. Strikingly, such DNA methylation is largely absent in hESCs because these cells must be able to develop into a wide spectrum of different tissues and organs. Thus, in order to protect the integrity of their genomes, hESCs must deploy an additional defense to limit retroelement retrotransposition. Recent studies of HIV and other exogenous retroviruses have identified the APOBEC3 family of genes (A3A-A3H) as powerful anti-retroviral factors. These APOBEC3s interrupt the conversion of viral RNA into DNA (reverse transcription), a key step also used by retroelements for their successful retrotransposition. We hypothesize that one or more of the APOBECs function as guardians of genome integrity in hESCs. We propose to compare and contrast which APOBEC3s are expressed in one federally approved and nine nonapproved hESC lines and to assess the natural level of retroelement RNA expression occurring in each of these lines. Next we will test whether the knockdown of expression of these APOBEC3s in the hESCS lines by RNA interference leads to a higher frequency of retrolement retrotransposition. Finally, if higher levels of retrotransposition are detected, we will examine whether these cells display an impaired ability to differentiate into specific tissue types corresponding to the three germ cell layers (ectoderm, mesoderm, and endoderm) and whether increased retrotransposition is associated with a higher frequency of malignant transformation within the hESC cultures. These studies promise to provide important new insights into how genomic stability in is maintained in hESCs and could lead to the identification of specific GMP culture conditions that minimize the chances of such unwanted retrotransposition events in cells destined for infusion into patients. These studies are directly responsive to the CIRM request for application. If funded, these studies would allow the entry of my laboratory with extensive APOBEC experience, into the exciting field of stem cell biology.

 
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