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People may use serum starvation and abnormal cell cycle synchronization to aid in preprogramming Chen et al. Growth measured by confluence shows an S-shaped growth curve: In Figure 1A a live imager records confluence every 2 hours in five or six independent biological experiments for and h feeding frequency, respectively.
Standard ESC culture conditions require medium change every day to remove lactate from aerobic glycolytic. Warburg metabolism was used by stem cells to maintain good growth conditions and to feed the cells.
ESCs found heterogeneously in the cell cycle seem like they would have an exponential increase in green cells with a line similar in shape and lower than the confluence line as shown in Figure 1A. But in Figure 1B , the number of cells in the S-G2-M phase reaches a nadir in the last few hours prior to the h medium change and then increases in the hours following medium change. It should be noted that increase in green fluorescence in a single experiment varies from two—12 h after the nadir, although it is usually 6—8 h after the nadir.
FUCCI ESCs accumulate in an S-shaped curve, but medium change reveals that cells accumulate in the green-negative state G1 approaching a h interval feeding and then rapidly re-entering the green S-G2-M-phase after medium change.
The times for medium change may vary by 2 hours. Error bars are s. It was hypothesized that insufficient medium change effects would be ameliorated by more frequent medium changes. Two effects were expected; that the peak amplitude would decrease and the growth rate would increase with more frequent feedings. In Figure 1B , it is shown that h feeding does decrease the amplitude of the peak green number of cells entering the S-G2-M phase after medium change, but in Figure 1A there was no significant increase in cell growth mediated by more frequent medium changes.
As shown in Figure 1 , cells are not heterogeneous in the cell cycle, and we analyzed the nadirs in total green object integrated intensity before and after medium change by calculating the fold change between the peak divided by the nadir. Picking the nadir and peak for each replicate was performed to get the most accurate ratio as the timing of feeding and acquisition of green fluorescence may vary between experiments.
More detail of picking peaks is shown in the Statistics subsection of the Materials and Methods section. The nadirs from to h feeding frequencies were not significant, but the peaks for both frequencies were higher than the nadirs, and the h peak was significantly higher than the h peak.
The stimulation index of h feeding frequency peak is greater than the 12 h-feeding frequency peak. Same N as shown in Figure 1. As confluence is reached and contact inhibition increases, the velocity of the lagging indicator of growth measurements decreases.
Interestingly, the doubling rate is shortest, indicating fastest growth rate only on the first day, suggesting this would be a good period to assay for instantaneous changes in the growth rate caused by increase in toxicant stress doses.
Velocity of increase in confluence decreases with time, and doubling rates get slower with time as confluence and contact inhibition increases. A shows the fold change whereas B shows the doubling rate of confluence from the stated time to 2 h.
Five independent biological experiments from h feeding frequency and six independent biological experiments for h feeding frequency with paired replicate wells in each experiment for each dose and stimulus were used for statistical analysis.
Confluence at T68 was used instead of T74 for datasets without T After LIF removal, ESCs slowly lose stemness and pluripotency-maintaining transcription factors, but the culture conditions here support similar time-dependent increases in confluence Figure 4A. Similarly, instantaneous entry into the S-G2-M-phase showed a sharp increase from the unfed to fed states, though ND culture has a shallower increase than NS Figure 4B.
A Cells were measured in time-lapse every 2 hours for confluence in the live imager. Three highest PFOA doses suppress entry into the cell cycle, but only the two highest doses suppress the lagging phase accumulated growth measured by confluence. A Cells were measured in time-lapse every two hours for confluence in the live imager. As the cells grow for a longer period, the effect of the doses increases, causing the LOEL to be a lower dose. B Green cell cycle entry into the S-G2-M-phase was measured simultaneously with confluence measurements in A and after feeding at day 1 and 2.
In general, the green peak decreases as the dose of PFOA increases. NS 0 dose : N is described in Figure 1. PFOA: four biological experiments. Analyses of the stimulation indices from the line graphs, now graphed as histograms, also show that PFOA suppresses accumulated growth as a lagging indicator measured by confluence Figure 6A and suppresses entry into the S-G2-M phase after medium change as a leading indicator Figure 6B predicted during ESC culture with LIF.
In Figure 6A , Tfinal confluence means decrease with dose, but a second measure, the direness index, measures the first hour of departure of significantly decreased confluence compared with NS, 0 mM PFOA dose. In general, the green fold change decreases as the dose of PFOA increases. The day 1 peak, occurring after the T24 feeding, shows a clearer trend and is less noisy than the day 2 peak which occurs after the T48 feeding.
In Figure 6C , a benchmark analysis of the point of departure was used to assess the dose of significant decrease compared with 0 mM sorbitol and normal stemness and such as Tfinal in Figure 6A , is accompanied by the direness significant point of departure for diminished confluence.
As data of the confluences of the wells before the first feeding of the toxicant are not known, T0 was used 2 h after the first feeding for NS. The direness points of departure from Figure 5A are shown in the day 3 bars. B The amount of green fluorescence at the peak after feeding and the nadir before feeding is rationated.
Direness points of departure are shown in Figure 5A. All the models were deemed questionable. DEP suppresses accumulated growth as a lagging indicator measured by confluence and suppresses entry into the S-G2-M phase after medium change as a leading indicator predicted during ESC culture with LIF. Red asterisks show when confluence starts to significantly differ from NS.
For higher doses, the divergence points are realized earlier T58for 10 uM and T52 for uM. The rest of the doses are not significantly different than NS at T B Simultaneously, confluence green fluorescence was measured in time-lapse every two h in the live imager. Green intensity increases cells enter the S-G2-M phase after cells are fed at approximately T24 and T Offsets in the timing of the zero dose and the rest of the curves are due to feeding differences between experiments.
NS 0 dose : N described in Figure 1. As data of the confluences of the wells before the first feeding of the toxicant are not known, T0 used was the first time-lapse recorded T2 for NS. B The amount of green intensity at the peak after feeding and the nadir before feeding are rationated.
In general, the green fold change decreases as the dose of DEP increases. DEP: N is described in Figure 7. A FUCCI, Pdgfra, and Oct4 ESC were cultured and assayed as shown in Figure 4 , except confluence increase and doubling rate were both measured in the first 24 h when rate of increase was highest and not as affected by contact inhibition.
Confluence fold change from timepoints T2 to end of day 1 is shown. B During the culture of the same three ESC lines from T2—T26, change in confluence was used to compute the doubling rate. First, the suppression of accumulated cells at Tfinal looks backward at stress effects through culture, and both PFOA and DEP suppress ESC accumulation albeit at different doses and levels of suppression.
Second, the prediction of future cell growth can be done early in culture before contact inhibition obscures the growth of fastest growing stress-free controls. This predictive assay is based on the suppression of the progression into the green S-G2-M phase of the cell cycle after medium change relieves G1 delay. Third, live imaging provides a time lapse report of when the first significant adverse suppression of ESC accumulation occurs compared to lesser stress doses.
Thus, DEP suppression of future growth on day 1 is reversible by day 2. Toxicity can be thought of as a product of the lowest dose that a LOAEL is detected at and the magnitude of the toxic effects. Thus, for PFOA the time-lapse point of departure is much earlier. Is it reasonable to see early time point effects? Diminished cell growth is not the cause of lower accumulation at the highest dose— uM PFOA; since fewer cells are present than at Tzero, death must account for this first point of departure.
This is consistent with the effects of stress enzymes that diminish Oct4, Sox2, Nanog, and Rex1 protein levels by 4 h and are activated highly by 1 h of hyperosmotic stress which slows growth rapidly Slater et al.
Although Oct4, Sox2, and Nanog protein levels return to normal by 24 h of hyperosmotic stress, Rex1 is maintained at lower levels by Mek1 at 1—4 h and SAPK at 24 h, respectively Slater et al. Yet, the effects of Rex1 suggest that some stemness features have decreased within 2—4 h, and these decreases persist.
Rex1 and Oct4 control the metabolic programs of stemness that control growth rates Frum et al. Time-lapse direness assayed here is also likely to be based on the large programmatic effects of these rate-limiting stemness factors.
So, it can be concluded that hyperosmotic stress at mM sorbitol or similar effects of 10— uM PFOA have early higher effects both on growth and stemness at higher doses and lesser effects at longer durations, as indicated by data here and in previous reports where the metabolism and stemness change over time. It will be important to determine using transcriptomic analysis if PFOA also induces changes in metabolism and stemness that are induced by hyperosmotic stress Abdulhasan et al.
This delay in G1 by h feeding was not anticipated, and the analysis of re-entry and its suppression by stress produced interesting outcomes. Unlike the assay of accumulation, re-entry after feeding into the S-G2-M-phase predicts future growth and its suppression by environmental toxicants. Thus, early suppression of re-entry by DEP is exceedingly small at the highest dose and is very sensitive, occurring at the lowest dose, but is reversible.
It will be interesting to test for long-term changes in anabolic and epigenetic transcriptomes in reversible DEP vs irreversible PFOA exposures to find whether brief reversible stress episodes have long-term effects. This may be because the green suppression is only on day 1 and it reverses so that overall, the cell growth is not suppressed by a large amount. In one report, PFOA exposure alone in the first trimester did not affect later pregnancy, but PFOA and cigarette smoke co-exposure negatively affected fetal growth at a later stage Mamsen et al.
A study of human embryonic and fetal tissues obtained in Denmark from —15 found that by the end of the first trimester, PFOA was the second highest PFAS family member in maternal blood, placenta, and many fetal organs with PFOS being the highest Mamsen et al.
Thus, PFOA crosses the placenta early and accumulates in the fetal organs. Taken together, the data suggest that first trimester PFOA exposures cross the placenta and enter fetal organs and are associated with increased pregnancy loss or decreased fetal growth. In the environment, point exposures such as ground water in Little Hocking OH were 1 uM Vestergren and Cousins, , and worldwide average blood exposures were 5—20 nM Vestergren and Cousins, The highest level was found in cord blood, and thus the fetus or embryo was also 20 nM Negri et al.
Thus, the BMDL doses reported to affect ESC accumulation of cell cycle progression are within the limit of maternal blood and cord blood exposures. DEP harms animal and human reproduction Radke et al. DEP increases the body weight of F1 after in utero exposure in rodents, decreased the growth rate of cord blood—derived hematopoietic stem cells, and decreased BMI in humans.
Phthalates affect the first trimester placenta by changing its transcriptome and methylome Grindler et al. Although DEP was not studied, many phthalates elevated in first trimester maternal blood are also associated with DNA methylation changes in the offspring LaRocca et al.
Thus, phthalates, in general, and DEP, specifically, cross the placenta, are detected in cord blood, and have first trimester effects that should be predicted by screening cultured ESCs that emulate first trimester embryogenesis. 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.
Interestingly, as the cell cycle is slowed down experimentally with hyperosmotic stress, it has been reported that G1 delay is associated with increased endoderm differentiation and less neuronal differentiation Coronado et al. Thus, it will be important to test whether experimental stressors also override stemness and proliferation signals from LIF and imbalance forced differentiation similarly to hyperosmotic stress.
Unlike previous growth measurements of stressed ESCs, time-lapse measurements given here were done by the analysis of confluence. The discrepancy is likely due to the live imager use of confluence of an ESC epithelium that is squamous near Tzero and columnar near Tfinal and that the higher density of the columnar is missed by the confluence-based cell number and doubling rate estimates.
These discrepancies can be remedied by use of a technique that counts the number of cells instead of measuring confluence. For this initial report, the conclusions reached should be valid but would be more accurate with object-based, rather than confluence-based, estimates of growth rates.
Interestingly, the use of a live imager to monitor cell cycle progression of FUCCI ESCs has not been reported, and cell cycle timing is usually evaluated by synchronizing the cells by serum starvation, Zielke and Edgar, and not partially synchronized by medium change as performed here.
This exit or abnormal delay in non-green G1 does occur with serum starvation prior to cell cycle synchronization, which occurs when G1 phases as short as one h in FUCCI-ESCs have been reported, but serum starvation is delayed for 24 h Sakaue-Sawano et al. The delay in the non-green G1 phase could be due to increased lactate acidosis, due to aerobic glycolytic anabolic metabolism enabling rapid ESC cycles, or to depleted nutrition.
Lactate acidosis is a problem as a report suggests that pluripotent stem cells which grow at high densities are hindered by increasing acidosis, and growth is enabled by buffering acid with sodium bicarbonate Liu et al. Many countries have VAM committees. Taken together, the data here suggest that several measurements of normal and toxicant-modulated growth of ESCs are enabled by live imager time-lapse data for 1 confluence-based accumulated growth assay, 2 predictive cell cycle progression assay of the suppression of cell cycle progression which diminishes future accumulation, and 3 direness index Yang et al.
The use of these assays should be applied to more toxicants to inform which toxicants endanger ESCs at lowest levels and fastest endpoints. Additional endpoints of transcriptomic, epigenomic, and metabolomic nature should be used to complement growth analysis and inform teratogenic and transgenerational effects.
We thank Chris Kassotis for comments on the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the study and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers.
Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Front Toxicol. Published online Nov Harris , 4 Douglas M. How can we assist you? What are you mainly interested in? What other areas are you interested in?
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