Genomic Instability Associated with p53 Knockdown in the Generation of Huntington’s Disease Human Induced Pluripotent Stem Cells

Abstract

Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.

Efficacy of remote limb ischemic conditioning on poststroke cognitive impairment

“…RLIC treatment alters the expression of multiple genes and proteins, including hypoxia-inducible factor-1α (HIF-1α), HIF-1β , the anti-apoptotic gene Bcl-2, and superoxide dismutase (SOD), all of which are observed to be elevated. RLIC has also been observed to inhibit the expression of the apoptotic protein, p53 (Jin et al, 2016;Salido et al, 2013;Tidball et al, 2016). These changes can affect a range of processes, such as improving the metabolism of nerve cells, inhibiting oxidative damage, and reducing neuronal apoptosis.…”

Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules

“…Among the non-integrative methods for iPSC production, episomal plasmids have been a popular choice due to their relatively lower costs and simplicity of use. One of the few caveats of this expression system is that the episomes currently used by most groups contain immortalization factors, such as large T antigen (Yu et al, 2009) or shRNA for p53 knockdown (Okita et al, 2011), which in some cases have been shown to cause genomic instability (Schlaeger et al, 2015;Tidball et al, 2016). In addition, episomal plasmid expression may persist in up to 33% of the reprogrammed cells even after P11 (Okita et al, 2011;Schlaeger et al, 2015).…”

Variant-specific changes in persistent or resurgent Na+current inSCN8A-EIEE13 iPSC-derived neurons

“…Skin punch biopsies were obtained from 3 EIEE13 patients (P1-3) and two healthy controls (C1 and C4) without known genetic disorders with consent under a protocol approved by the Institutional Review Board of Michigan Medicine and reprogrammed by previously published methods (Tidball et al, 2017). Two additional control lines used in this study were previously reported as CC1 (now C2) and CHD2 WT/WT2 (now C3) from (Tidball et al, 2016;Tidball et al, 2017), respectively, and were reprogrammed using identical methods. See Supplemental materials for further details of reprogramming and cell line validation.…”

“…1A). We used two additional control lines, C2 and C3, ( Table 1) that have been previously published (Tidball et al, 2016;Tidball et al, 2017). Each line used for subsequent experiments was negative for episomal reprogramming vector integration, had no observable genomic abnormalities as tested by either g-band karyotype or SNP chip microarray analyses, and expressed markers of pluripotency ( Supplemental Fig.…”