Erratum: Genome editing reveals a role for OCT4 in human embryogenesis.

This corrects the article DOI: 10.1038/nature24033.

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

Fluorescent tagging of endogenous Heme oxygenase-1 in human induced pluripotent stem cells for high content imaging of oxidative stress in various differentiated lineages.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

Protectiveness of NAM-based hazard assessment - which testing scope is required?

Hazard assessment (HA) requires toxicity tests to allow deriving protective points of departure (PoDs) for risk assessment irrespective of a compound's mode of action (MoA). The scope of in vitro test batteries (ivTB) thereby necessitated for systemic toxicity is still unclear. We explored the protectiveness regarding systemic toxicity of an ivTB with a scope, which was guided by previous findings from rodent studies, where examining six main targets, including liver and kidney, was sufficient to predict the guideline scope-based PoD with high probability. The ivTB comprises human in vitro models representing liver, kidney, lung and the neuronal system covering transcriptome, mitochondrial dysfunction and neuronal outgrowth. Additionally, 32 CALUX®- and 10 HepG2 BAC-GFP reporters cover a broad range of disturbance mechanisms. Eight compounds were chosen for causing adverse effects such as immunotoxicity or anemia in vivo, i.e., effects not directly covered by assays in the ivTB. PoDs derived from the ivTB and from oral repeated dose studies in rodents were extrapolated to maximum unbound plasma concentrations for comparison. The ivTB-based PoDs were one to five orders of magnitude lower than in vivo PoDs for six of eight compounds, implying that they were protective. The extent of in vitro response varied across test compounds. Especially for hematotoxic substances, the ivTB showed either no response or only cytotoxicity. Assays better capturing this type of hazard would be needed to complement the ivTB. This study highlights the potentially broad applicability of ivTBs for deriving protective PoDs of compounds with unknown MoA.

Animal tests are used to determine which amount of a chemical is toxic ('threshold of toxicity') and which organs are affected. In principle, the threshold can also be derived solely from tests with cultured cells. However, only a limited number of cell types can practically be tested, so one challenge is to determine how many and which types shall be tested. In animal studies, only few organs including liver and kidney are regularly among those most sensitively affected. We explored whether a cell-based test battery representing these sensitive organs and covering important mechanisms of toxicity can be used to derive protective human thresholds. To challenge this approach, eight chemicals were tested that primarily cause effects in organs not directly represented in our test battery. Results provided protective thresholds for most of the investigated compounds and gave indications how to further improve the approach towards a full-fledged replacement for animal tests.

Integration of temporal single cell cellular stress response activity with logic-ODE modeling reveals activation of ATF4-CHOP axis as a critical predictor of drug-induced liver injury.

Drug-induced liver injury (DILI) is the most prevalent adversity encountered in drug development and clinical settings leading to urgent needs to understand the underlying mechanisms. In this study, we have systematically investigated the dynamics of the activation of cellular stress response pathways and cell death outcomes upon exposure of a panel of liver toxicants using live cell imaging of fluorescent reporter cell lines. We established a comprehensive temporal dynamic response profile of a large set of BAC-GFP HepG2 cell lines representing the following components of stress signaling: i) unfolded protein response (UPR) [ATF4, XBP1, BIP and CHOP]; ii) oxidative stress [NRF2, SRXN1, HMOX1]; iii) DNA damage [P53, P21, BTG2, MDM2]; and iv) NF-κB pathway [A20, ICAM1]. We quantified the single cell GFP expression as a surrogate for endogenous protein expression using live cell imaging over > 60 h upon exposure to 14 DILI compounds at multiple concentrations. Using logic-based ordinary differential equation (Logic-ODE), we modelled the dynamic profiles of the different stress responses and extracted specific descriptors potentially predicting the progressive outcomes. We identified the activation of ATF4-CHOP axis of the UPR as the key pathway showing the highest correlation with cell death upon DILI compound perturbation. Knocking down main components of the UPR provided partial protection from compound-induced cytotoxicity, indicating a complex interplay among UPR components as well as other stress pathways. Our results suggest that a systematic analysis of the temporal dynamics of ATF4-CHOP axis activation can support the identification of DILI risk for new candidate drugs.

Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9.

The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease.

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.