Methods of Reprogramming to Induced Pluripotent Stem Cell Associated with Chromosomal Integrity and Delineation of a Chromosome 5q Candidate Region for Growth Advantage

Abstract

Induced pluripotent stem cells (iPSCs) have brought great promises for disease modeling and cell-based therapies. One concern related to the use of reprogrammed somatic cells is the loss of genomic integrity and chromosome stability, a hallmark for cancer and many other human disorders. We investigated 16 human iPSC lines reprogrammed by nonintegrative Sendai virus (SeV) and another 16 iPSC lines generated by integrative lentivirus for genetic changes. At early passages we detected cytogenetic rearrangements in 44% (7/16) of iPSC lines generated by lentiviral integration whereas the corresponding figure was 6% (1/16) using SeV-based delivery. The rearrangements were numerical and/or structural with chromosomes 5 and 12 as the most frequently involved chromosomes. Three iPSC lines with chromosome 5 aberrations were derived from one and the same donor. We present in this study the aberrant karyotypes including a duplication of chromosome 5q13q33 that restricts a candidate region for growth advantage. Our results suggest that the use of integrative lentivirus confers a higher risk for cytogenetic abnormalities at early passages when compared to SeV-based reprogramming. In combination, our findings expand the knowledge on acquired cytogenetic aberrations in iPSC after reprogramming and during culture.

Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells

“…In addition this instability was not observed when using a non-integrative strategy even in the presence of this constitutive translocation. Even if controversial results exist in human iPSCs 22 27 and do not clarify whether integrative or non-integrative strategies are causal to genome and chromosome instabilities it seems that it is not the continuous expression of exogenous factors that may explain this instability. Such, in one of these studies, exogenous factors are silenced but the use of integrative reprogramming technologies strongly increases the number of chromosome abnormalities 27 supporting the fact that viral integrations can cause chromosomal aberrations as shown during papillomaviruses infection 28 .…”

“…Even if controversial results exist in human iPSCs 22 27 and do not clarify whether integrative or non-integrative strategies are causal to genome and chromosome instabilities it seems that it is not the continuous expression of exogenous factors that may explain this instability. Such, in one of these studies, exogenous factors are silenced but the use of integrative reprogramming technologies strongly increases the number of chromosome abnormalities 27 supporting the fact that viral integrations can cause chromosomal aberrations as shown during papillomaviruses infection 28 . Indeed, the reactivation of endogenous retrovirus and retrotransposable elements presents in the host genome after retro- or lentiviral infection and their genomic integrations 29 may increase genomic instability and favor the emergence of new rearrangements 30 31 .…”

Genomic Instability of Pluripotent Stem Cells: Origin and Consequences

“…One major hurdle is the efficiency of differentiation that still remains very low. In addition, PSCs show several signs of genetic instability, not only in culture but also in vivo [71,106], yet embryos manage to keep this instability under control by generating viable and healthy organisms. Hence, the question arises of how this control is achieved.…”

“…First, cells with unstable genomes can be eliminated by apoptosis during differentiation, which is actually what it is observed during in vitro differentiation. However, γH2AX detection in blastocysts shows that most of the cells stain positive for this marker [71,106], which makes unlikely that most of them bear indeed highly unstable genomes. Another possibility is that the γH2AX observed in these cells is not only a mark of genetic instability but perhaps also a marker of other DNA transactions, including chromatin remodeling.…”

“…Indeed, a very recent report that analyzed the "metabolome" of naïve ESCs compared to that of primed ESCs show significant differences between these two cell types, which in turn may impinge on the level of ROS [104]. Further, recent work suggests that the use of non-integrative vectors to induce reprogramming significantly reduces the number of CNVs in the resulting iPSCs [105,106]. Furthermore, a recent report that analyzed the mutational load of three distinct pluripotency induction methods shows that a non-integrative approach results in lower mutation load than either retrovirus or Sendai virus-based reprogramming methods [104].…”