SummaryHuntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs.

Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response.

HIGHLIGHTS d Generation of isogenic HD (IsoHD) hESCs to enable the study of CAG length effects d IsoHD hESCs and neural cells exhibit CAG-dependent phenotypic abnormalities d CNS and peripheral cell types of interest can be differentiated from IsoHD hESCs d Large-scale, unbiased-omics analyses reveal cell-typespecific, CAG-dependent effects

SummaryThe modular DNA recognition code of the transcription-activator-like effectors (TALEs) from plant pathogenic bacterial genus Xanthomonas provides a powerful genetic tool to create designer transcription factors (dTFs) targeting specific DNA sequences for manipulating gene expression. Previous studies have suggested critical roles of enhancers in gene regulation and reprogramming. Here, we report dTF activator targeting the distal enhancer of the Pou5f1 (Oct4) locus induces epigenetic changes, reactivates its expression, and substitutes exogenous OCT4 in reprogramming mouse embryonic fibroblast cells (MEFs) to induced pluripotent stem cells (iPSCs). Similarly, dTF activator targeting a Nanog enhancer activates Nanog expression and reprograms epiblast stem cells (EpiSCs) to iPSCs. Conversely, dTF repressors targeting the same genetic elements inhibit expression of these loci, and effectively block reprogramming. This study indicates that dTFs targeting specific enhancers can be used to study other biological processes such as transdifferentiation or directed differentiation of stem cells.