Using Live Imaging and FUCCI Embryonic Stem Cells to Rank DevTox Risks: Adverse Growth Effects of PFOA Compared With DEP Are 26 Times Faster, 1,000 Times More Sensitive, and 13 Times Greater in Magnitude

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.

Using high throughput screens to predict miscarriages with placental stem cells and long‐term stress effects with embryonic stem cells

“…Hypoxic stress with FGF4 removal (d) causes imbalanced differentiation to TGC like normal balanced differentiation (c). In ESC (a), the key comparison is between NS after 3 days of culture and 300 mM sorbitol override of NS (LIF+/stemness‐maintaining growth factor (modified from Abdulhasan et al (2021) and Li et al (2019)). In TSC (b), the same type of comparison as ESC is between baseline NS after 6 day of culture and 0.5% oxygen—sharply stressful compared with optimal 2% oxygen—and its override of NS (fibroblast growth factor [FGF]4/stemness maintaining growth factor).…”

“…Increase in the first lineage reported Pdgfra‐GFP viable reporter culture, have been reported in an HTS using about 20 toxicants also used during the ECVAM EST validation (Li et al, 2016; Li et al, 2019; Li et al, 2016). A further trial was done with fluorescence ubiquitinated cell cycle indicator which can show time lapse kinetics of diminished growth due to general stresses and endocrine disruptors (Abdulhasan et al, 2021) and mutagenic stress (Abdulhasan et al, 2022). Scaling up of these assays and translation to a huESC model needs to be done.…”

“…From normal stemness culture (NS) to a high, but viable, stress‐forced loss of stemness and gain of first lineage, bright first lineage sorted platelet growth factor receptor (Pdgfra)‐promoter embryonic stem cells (ESC) increase from nothing to about 1%, whereas first lineage trophoblast giant cells induced from trophoblast stem cells (TSC) increase from about 5 to 50%. In ESC (a), the key comparison is between NS after 3 days of culture and 300 mM sorbitol override of NS (LIF+/stemness‐maintaining growth factor) (modified from Abdulhasan et al (2021) and Li et al (2019)). In TSC (b), the same type of comparison as ESC is between baseline NS after 6 days of culture and 0.5% oxygen—sharply stressful compared with optimal 2% oxygen—and its override of NS (fibroblast growth factor [FGF4]/stemness maintaining growth factor).…”