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Abstract
Fluorescent ubiquitination-based cell cycle indicator (FUCCI) embryonic stem cells (ESCs), which fluoresce green during the S-G2-M phases, generate an S-shaped curve for the accumulation of cells during normal stemness (NS) culture with leukemia-inhibitory factor (LIF). Since it was hypothesized that a culture of ESCs was heterogeneous in the cell cycle, it was expected that increased S-G2-M-phases of the cell cycle would make an S-shaped curve parallel to the accumulation curve. Unexpectedly, it was observed that the fraction of FUCCI ESCs in green decreases over time to a nadir at ∼24 h after previous feeding and then rapidly enters S-G2-M-phases after medium change. G1 delay by infrequent medium change is a mild stress, as it does not affect growth significantly when frequency is increased to 12 h. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) were used as examples of members of the per- and polyfluoroalkyl substances (PFAS) and phthalate families of chemicals, respectively. Two adverse outcomes were used to compare dose- and time-dependent effects of PFOA and DEP. The first was cell accumulation assay by time-lapse confluence measurements, largely at Tfinal/T74 h. The second was by quantifying dominant toxicant stress shown by the suppression of mild stress that creates a green fed/unfed peak. In terms of speed, PFOA is 26 times faster than DEP for producing a time-dependent LOAEL dose at 100 uM (that is, 2 h for PFOA and 52 h for DEP). PFOA has 1000-fold more sensitive LOAEL doses than DEP for suppressing ESC accumulation (confluence) at day 3 and day 2. There were two means to compare the magnitude of the growth suppression of PFOA and DEP. For the suppression of the accumulation of cells measured by confluence at Tfinal/T74h, there was a 13-fold suppression at the highest dose of PFOA > the highest dose of DEP. For the suppression of entry into the cell cycle after the G1 phase by stress on day 1 and 2, there is 10-fold more suppression by PFOA than DEP. The data presented here suggest that FUCCI ESCs can assay the suppression of accumulated growth or predict the suppression of future growth by the suppression of fed/unfed green fluorescence peaks and that PFOA’s adverse effects are faster and larger and can occur at more sensitive lower doses than DEP.
Highlights
Comprehensive in vitro developmental toxicity (DevTox) assays are controversial, and the current standard is in vivo rodent gestational testing for the Extended One-Generation Reproductive Toxicity Study (Beekhuijzen et al, 2016)
5 INTERESTINGLY, the instantaneous fed/unfed state analysis picks up something that the cumulative growth stimulation index and direness index do not pick up, an apparent extremely sensitive
We report the slowing of the cell cycle here with environmental toxic stressors, consistent with the slowing of the cell cycle using positive control hyperosmotic stress reported previously (Slater et al, 2014; Li et al, 2016a; Li et al, 2016b; Li et al, 2019)
Summary
Comprehensive in vitro developmental toxicity (DevTox) assays are controversial, and the current standard is in vivo rodent gestational testing for the Extended One-Generation Reproductive Toxicity Study (Beekhuijzen et al, 2016). An important concept for DevTox in vitro HTS is that the cells used retain the developmental, molecular, and cellular strategies to respond to toxicant stress in the same way that they used to in the milieu of the early in vivo post-fertilization embryo they were isolated from (Rappolee et al, 2012; Puscheck et al, 2015a; Yang et al, 2017a). Embryonic and placental trophoblast stem cells (ESCs and TSCs, respectively) are isolated from the early embryo just prior to implantation into the uterus. In vitro, these stem cell lineages do seem to emulate the strategies needed for the survival of the stressed embryo, in vivo
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