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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.

Reprogramming can occur by the introduction of key transcription factors (TFs) as well as by epigenetic changes. We demonstrated that histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) combined with a chromatin remodeling medium (CRM) induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. Furthermore, treatment with DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (5AZA) CRM did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in gene expression of endoderm, pancreatic endoderm, and endocrine markers was seen over levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore the role epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β -cell specific conditions.

This study’s aim is threefold: (I) Evaluate movement quality parameters of gait in people with hip or knee osteoarthritis (OA) compared to asymptomatic controls from a single trunk-worn 3D accelerometer. (II) Evaluate the sensitivity of these parameters to capture changes at 6-weeks, 3-, 6-, and 12-months following total knee arthroplasty (TKA). (III) Investigate whether observed changes in movement quality from 6-weeks and 12-months post-TKA relates to changes in patient-reported outcome measures (PROMs). We invited 20 asymptomatic controls, 20 people with hip OA, 18 people pre- and post-TKA to our movement lap. They wore a single trunk-worn accelerometer and walked at a self-selected speed. Movement quality parameters (symmetry, complexity, smoothness, and dynamic stability) were calculated from the 3D acceleration signal. Between groups and between timepoints comparisons were made, and changes in movement quality were correlated with PROMs. We found significant differences in symmetry and stability in both OA groups. Post-TKA, most parameters reflected an initial decrease in movement quality at 6-weeks post-TKA, which mostly normalised 6-months post-TKA. Finally, improved movement quality relates to improvements in PROMs. Thus, a single accelerometer can characterise movement quality in both OA groups and post-TKA. The correlation shows the potential to monitor movement quality in a clinical setting to inform objective, data-driven personalised rehabilitation.