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Low dose irradiation enhances gene targeting in human pluripotent stem cells

ABSTRACT: Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy. However, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. Here we show that limited low dose irradiation (LDI) using either γ-ray or X-ray exposure (0.4 Gy) significantly enhances HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, Histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by over 30- fold when combined with the targeting tools zinc finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPRs). Whole exome sequencing confirmed that the LDI to hPSCs did not induce gross genomic alterations, or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new targeted hPSC lines that are crucial for the research community as this growing field develops.

ORGANISM(S): Homo sapiens

PROVIDER: GSE68328 | GEO | 2016/03/22

SECONDARY ACCESSION(S): PRJNA282497

REPOSITORIES: GEO

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Project description:Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many of the potential applications are still limited by the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. These challenges could be overcome by the use of adult tissue stem cells derived from hPSCs, as their restricted potential could limit the differentiation towards other undesired linages, and allow in vitro expansion and long- term propagation of fully differentiated tissue. To isolate adult stem cells from hPSCs, we applied genome-editing to generate an LGR5-GFP reporter system and subsequently developed a differentiation protocol for human intestinal tissue comprising an adult stem cell niche and all major cell types of the adult intestine. This novel derivation protocol is highly robust and even permits the isolation of intestinal organoids without the LGR5 reporter. Transcriptional profiling, electron microscopy and functional analysis revealed that such human organoid cultures could be derived with high purity, and a composition and morphology similar to that of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. With our ability to genetically engineer hPSCs using site-specific nucleases, this adult stem cell system provides a novel platform by which to study human intestinal disease in vitro. RNA from primary organoid samples was isolated from organoid lines that were both cultured for 1-6 months and derived from duodenum, ileum, or rectum biopsies of human subjects as described previously (Sato et al., Gastroenterology 2011) grown in media called WENR+inhibitors. RNA was also isolated from various steps in the culturing and differentiation protocol.

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Low dose irradiation enhances gene targeting in human pluripotent stem cells

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