Analysis of Viral Integration Sites in Human Induced Pluripotent Stem Cells.

Abstract 1485

Poster Board I-508

The recently discovered approach for the direct reprogramming of somatic cells into induced pluripotent stem (IPS) cells by expression of defined transcription factors may provide new approaches for regenerative medicine, gene therapy and drug screening. Successful reprogramming currently requires at least temporary expression of one to four different transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog and Lin28) in the targeted cells. Non-viral based reprogramming technologies have been reported, but expression of the reprogramming factors after γ-retroviral or lentiviral gene transfer remains the most efficient and commonly used approach. Since the reprogramming frequency is consistently low in these studies, it has been speculated that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. Here we present for the first time an extensive analysis of the lentiviral integration profile in human IPS-cells. We analysed the IS of 8 IPS cell lines derived from either human fetal fibroblasts (IMR90) or newborn foreskin fibroblasts (FS) after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28, using linear amplification-mediated PCR (LAM-PCR). With 5 to15 IS per individual IPS clone we identified a total of 78 independent IS. Finally we assigned 75 IS to a unique chromosomal location. In addition to LAM-PCR, we confirmed the total number of IS via Southern blot. Interestingly, in 6 of 8 IPS clones some of these IS were found in pairs, integrated into the same chromosomal location within 4 base pairs of each other. This integration pattern has not been detected in our previous analysis of 702 IS in rhesus macaques transplanted with CD34+ cells transduced with retroviral vectors. Of the 75 valid IS 53 (70.7%) could be mapped to a gene-coding region, 52 located in introns and 1 in an exon, annotated in a human reference sequence in the UCSC Genome Browser RefSeq Genes track. The different IPS-clones had no integration site in common. To investigate the impact of integration on the regulation of vector targeted genes we analyzed the mRNA expression profiles using available microarray data from these clones. Out of 46 evaluable genes only two (WDR66 and MYST2 in clone IMR90-2, p<0.0001) were significantly over-expressed. The expression of two genes in clone FS-1 (ACVR2A p=0.01, RAF1 p=0.02) and one in FS-2 (KIAA0528, p=0.03) was decreased compared to the expression data of all other clones combined. In summary our data suggest that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes. Furthermore, identification of the insertion profile of the IPS cell clones IMR90-1 and -4 as well as FS-1 will be useful to other researchers using these cell lines distributed by the Wisconsin International Stem Cell (WISC) bank.