Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation

Abstract

Induced pluripotent stem (iPS) cells generated using Yamanaka factors have great potential for use in autologous cell therapy. However, genomic abnormalities exist in human iPS cells, and most mouse iPS cells are not fully pluripotent, as evaluated by the tetraploid complementation assay (TCA); this is most likely associated with the DNA damage response (DDR) occurred in early reprogramming induced by Yamanaka factors. In contrast, nuclear transfer can faithfully reprogram somatic cells into embryonic stem (ES) cells that satisfy the TCA. We thus hypothesized that factors involved in oocyte-induced reprogramming may stabilize the somatic genome during reprogramming, and improve the quality of the resultant iPS cells. To test this hypothesis, we screened for factors that could decrease DDR signals during iPS cell induction. We determined that Zscan4, in combination with the Yamanaka factors, not only remarkably reduced the DDR but also markedly promoted the efficiency of iPS cell generation. The inclusion of Zscan4 stabilized the genomic DNA, resulting in p53 downregulation. Furthermore, Zscan4 also enhanced telomere lengthening as early as 3 days post-infection through a telomere recombination-based mechanism. As a result, iPS cells generated with addition of Zscan4 exhibited longer telomeres than classical iPS cells. Strikingly, more than 50% of iPS cell lines (11/19) produced via this "Zscan4 protocol" gave rise to live-borne all-iPS cell mice as determined by TCA, compared to 1/12 for lines produced using the classical Yamanaka factors. Our findings provide the first demonstration that maintaining genomic stability during reprogramming promotes the generation of high quality iPS cells.

Role of Zscan4 in secondary murine iPSC derivation mediated by protein extracts of ESC or iPSC

“…It has been reported that the combination of Yamanaka 4 factors and Zscan4 attenuated DNA damage response and markedly promoted the efficiency of iPSC generation through stabilizing the genomes, resulting in p53 downregulation [23]. So, we compared p53 level in fibroblasts after treatment of extract from authentic mES and Zscan4-overexpressing mES cells.…”

“…Therefore, we analyzed our microarray data thoroughly with a focus on the genes related to reprogramming efficiency. It has recently been reported that the stabilization of genomic DNA and the rapid enhancement of telomere elongation improved the generation of iPS cells [23] and that telomeres were elongated by telomerase during reprogramming [24]. Accordingly, we selected telomere regulators that are involved in long-term genomic stability and telomere elongation and compared their expressions between mES and primary iPS cells (Fig.…”

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells

“…These studies indicate that many other factors contribute to the pluripotency state of iPSCs. Transcription factor-mediated reprogramming is a complex process, and the pluripotency of iPSCs can be influenced by the reprogramming strategies and culture conditions employed [9,10]. Therefore, systematic deciphering of genetic or epigenetic alternations in the iPSCs with consistent genetic background would help to identify some hotspots of iPSCs.…”

Tissue Engineering

“…Zscan4, a zinc finger protein that is expressed selectively in 2-cell embryos at the time when zygotic gene activation occurs, appears to have this property. The addition of Zscan4 to the 'Yamanaka' set of transcription factors decreased the DNA damage response during reprogramming of mouse somatic cells, and improved the yield of pluripotent cells [431]. Most tellingly, iPS cell lines produced using Zscan4 were of higher quality than those obtained with the standard factor set alone, as judged by significantly enhanced ability to give rise to live-borne mice derived entirely from the reprogrammed cells in a tetraploid complementation assay.…”