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Molecular characterization of a toxicological tipping point during human stem cell differentiation.

ABSTRACT: Endoderm gives rise to the gut tube in the early embryo. We differentiated human induced pluripotent stem cells (hiPSCs) to embryonic endoderm to identify a "tipping point" at which the developing system did not recover from perturbations caused by exposure to all-trans retinoic acid (ATRA). Differentiating hiPSC-derived endoderm exposed to five concentrations of ATRA between 0.001 and 10 μM at 6 h, 96 h, or 192 h was assessed for forkhead box A2 (FOXA2) protein expression and global gene transcript expression. A tipping point of 17 ± 11 nM was identified where patterns of differentially expressed genes supported a developmental trajectory shift indicating a potential teratogenic outcome. This concentration is between women's endogenous ATRA blood plasma levels and teratogenic levels of circulating isotretinoin, an ATRA isomer used to treat acne. Taken together, these data suggest that this approach is a sensitive method to identify a point of departure for chemicals that impact early embryonic development.

SUBMITTER: Saili KS

PROVIDER: S-EPMC9728154 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Molecular characterization of a toxicological tipping point during human stem cell differentiation.

Saili Katerine S KS Antonijevic Todor T Zurlinden Todd J TJ Shah Imran I Deisenroth Chad C Knudsen Thomas B TB

Reproductive toxicology (Elmsford, N.Y.) 20191007

Endoderm gives rise to the gut tube in the early embryo. We differentiated human induced pluripotent stem cells (hiPSCs) to embryonic endoderm to identify a "tipping point" at which the developing system did not recover from perturbations caused by exposure to all-trans retinoic acid (ATRA). Differentiating hiPSC-derived endoderm exposed to five concentrations of ATRA between 0.001 and 10 μM at 6 h, 96 h, or 192 h was assessed for forkhead box A2 (FOXA2) protein expression and global gene transc ...[more]

PMID: 31600526

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Project description:Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk assessment based on in vitro and in silico testing requires utilizing toxicity pathway information to distinguish adverse outcomes from recoverable adaptive events. Little work has focused on oxidative stresses in human airway for the purposes of predicting adverse responses. We hypothesize that early gene expression-mediated molecular changes could be used to delineate adaptive and adverse responses to environmentally-based perturbations. Here, we examined cellular responses of the tracheobronchial airway to zinc (Zn) exposure, a model oxidant. Airway derived BEAS-2B cells exposed to 2–10 µM Zn2+ elicited concentration- and time-dependent cytotoxicity. Normal, adaptive, and cytotoxic Zn2+ exposure conditions were determined with traditional apical endpoints, and differences in global gene expression around the tipping point of the responses were used to delineate underlying molecular mechanisms. Bioinformatic analyses of differentially expressed genes indicate early enrichment of stress signaling pathways, including those mediated by the transcription factors p53 and NRF2. After 4 h, 154 genes were differentially expressed (p <0.01) between the adaptive and cytotoxic Zn2+ concentrations. Nearly 40% of the biomarker genes were related to the p53 signaling pathway with 30 genes identified as likely direct targets using a database of p53 ChIP-seq studies. Despite similar p53 activation profiles, these data revealed widespread dampening of p53 and NRF2-related genes as early as 4 h after exposure at higher, unrecoverable Zn2+ exposures. Thus, in our model early increased activation of stress response pathways indicated a recoverable adaptive event. Overall, this study highlights the importance of characterizing molecular mechanisms around the tipping point of adverse responses to better inform HTS paradigms.

Project description:Brain-state transitions are readily apparent from changes in brain rhythms,1 but are difficult to predict, suggestive that the underlying cause is latent to passive recording methods. Among the most important transitions, clinically, are the starts of seizures. We here show that an 'active probing' approach may have several important benefits for epileptic management, including by helping predict these transitions. We used mice expressing the optogenetic actuator, channelrhodopsin, in pyramidal cells, allowing this population to be stimulated in isolation. Intermittent stimulation at frequencies as low as 0.033 Hz (period = 30 s) delayed the onset of seizure-like events in an acute brain slice model of ictogenesis, but the effect was lost if stimulation was delivered at even lower frequencies (1/min). Notably, active probing additionally provides advance indication of when seizure-like activity is imminent, revealed by monitoring the postsynaptic response to stimulation. The postsynaptic response, recorded extracellularly, showed an all-or-nothing change in both amplitude and duration, a few hundred seconds before seizure-like activity began-a sufficient length of time to provide a helpful warning of an impending seizure. The change in the postsynaptic response then persisted for the remainder of the recording, indicative of a state change from a pre-epileptic to a pro-epileptic network. This occurred in parallel with a large increase in the stimulation-triggered Ca2+ entry into pyramidal dendrites, and a step increase in the number of evoked postsynaptic action potentials, both consistent with a reduction in the threshold for dendritic action potentials. In 0 Mg2+ bathing media, the reduced threshold was not associated with changes in glutamatergic synaptic function, nor of GABAergic release from either parvalbumin or somatostatin interneurons, but simulations indicate that the step change in the optogenetic response can instead arise from incremental increases in intracellular [Cl-]. The change in the response to stimulation was replicated by artificially raising intracellular [Cl-], using the optogenetic chloride pump, halorhodopsin. By contrast, increases in extracellular [K+] cannot account for the firing patterns in the response to stimulation, although this, and other cellular changes, may contribute to ictal initiation in other circumstances. We describe how these various cellular changes form a synergistic network of positive feedback mechanisms, which may explain the precipitous nature of seizure onset. This model of seizure initiation draws together several major lines of epilepsy research as well as providing an important proof-of-principle regarding the utility of open-loop brain stimulation for clinical management of the condition.

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Molecular characterization of a toxicological tipping point during human stem cell differentiation.

Publications

Molecular characterization of a toxicological tipping point during human stem cell differentiation.

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