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.Toxicolog

The use of transient lacZ expression in fish embryos for comparative analysis of activity of cloned regulatory elements

The introduction of foreign genes into fish embryos and the subsequent production of transgenic fish, have by now resulted in the accumulation of a substantial amount of data worldwide and there exists a large literature on the subject (for reviews see Maclean, Penman & Zhu, 1987; Fletcher & Davies, 1991; Hackett, 1993). The two major objectives of these studies are the production of valuable broodstock benefiting from the acquisition of desirable characteristics as well as the study of vertebrate gene regulation and the genetic basis of development. The production of transgenic fish with the desire to found genetically superior broodstock for food production has been an aim since Zhu and coworkers introduced a growth hormone gene into goldfish (1985). Attempts to increase the growth rate of farmfish species by introducing growth hormone genes have also been made by many research laboratories, some with success (Du et al., 1992; Chen et al., 1993; Devlin et al., 1994). Other goals include increased freeze tolerance (Fletcher, Davies & Hew, 1992) or improved disease resistance (Leong, 1994) using transgenic technology. Fish are also becoming a popular model for the study of development and gene regulation. One approach to the study of gene regulation is the isolation and characterization of regulatory sequences and subsequent testing of their functionality in vivo or in vitro. The number of genes isolated from fish grows steadily. In a survey carried out by Maclean and Rahman (1994), the isolation of over 70 cDNA and genomic DNA sequences from a wide range of fish species by the end of 1992 is listed

Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp β-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of β-galactosidase-expressing cells on an expression map. β-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the β-actin regulatory elements also showed a marked effect on the MyHC promoter activity

In vitro models of cancer stem cells and clinical applications

Cancer cells, stem cells and cancer stem cells have for a long time played a significant role in the biomedical sciences. Though cancer therapy is more effective than it was a few years ago, the truth is that still none of the current non-surgical treatments can cure cancer effectively. The reason could be due to the subpopulation called “cancer stem cells” (CSCs), being defined as those cells within a tumour that have properties of stem cells: self-renewal and the ability for differentiation into multiple cell types that occur in tumours. The phenomenon of CSCs is based on their resistance to many of the current cancer therapies, which results in tumour relapse. Although further investigation regarding CSCs is still needed, there is already evidence that these cells may play an important role in the prognosis of cancer, progression and therapeutic strategy. Therefore, long-term patient survival may depend on the elimination of CSCs. Consequently, isolation of pure CSC populations or reprogramming of cancer cells into CSCs, from cancer cell lines or primary tumours, would be a useful tool to gain an in-depth knowledge about heterogeneity and plasticity of CSC phenotypes and therefore carcinogenesis. Herein, we will discuss current CSC models, methods used to characterize CSCs, candidate markers, characteristic signalling pathways and clinical applications of CSCs. Some examples of CSC-specific treatments that are currently in early clinical phases will also be presented in this review