Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse Embryonic Stem Cells

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways

Tau tubulin kinase 1 and 2 regulate ciliogenesis and human pluripotent stem cells–derived neural rosettes

Abstract Primary cilia are key regulators of embryo development and tissue homeostasis. However, their mechanisms and functions, particularly in the context of human cells, are still unclear. Here, we analyzed the consequences of primary cilia modulation for human pluripotent stem cells (hPSCs) proliferation and differentiation. We report that neither activation of the cilia-associated Hedgehog signaling pathway nor ablation of primary cilia by CRISPR gene editing to knockout Tau Tubulin Kinase 2 (TTBK2), a crucial ciliogenesis regulator, affects the self-renewal of hPSCs. Further, we show that TTBK1, a related kinase without previous links to ciliogenesis, is upregulated during hPSCs-derived neural rosette differentiation. Importantly, we demonstrate that while TTBK1 fails to localize to the mother centriole, it regulates primary cilia formation in the differentiated, but not the undifferentiated hPSCs. Finally, we show that TTBK1/2 and primary cilia are implicated in the regulation of the size of hPSCs-derived neural rosettes

Generation of human iPSCs from fetal prostate fibroblasts HPrF

Human induced pluripotent stem cell line was generated from commercially available primary human prostate fibroblasts HPrF derived from a fetus, aged 18–24 weeks of gestation. The fibroblast cell line was reprogrammed with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting the expression of factors of pluripotency on a single-cell level, and in vivo using teratoma formation assay. This iPS cell line will be a useful tool for studying both normal prostate development and prostate cancer disease

Generation of human iPSCs from human prostate cancer-associated fibroblasts IBPi002-A

A human induced pluripotent stem cell line was generated from cancer-associated fibroblasts of a 68-years old patient with diagnosed prostate adenocarcinoma (PCa). The fibroblast cell line was reprogrammed with Epi5™ Episomal iPSC Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting expression of factors of pluripotency on a single-cell level, and also in vivo using teratoma formation assay. This new iPS cell line may be used for differentiation into different prostate-specific cell types in differentiation studies

The acceleration of cardiomyogenesis in embryonic stem cells <i>in vitro</i> by serum depletion does not increase the number of developed cardiomyocytes - Fig 4

<p>A number of viable cardiomyocytes determined as the level of ATP in the HG8 clone of the R1 ES cell (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173140#sec002" target="_blank">methods</a>) ratio in the culture derived from 5- and 8-day old EBs (A and B). Selection of cardiomyocytes by G418 antibiotics started on days 14 (Days of selection: 14–20) and 20 (Days of selection: 20–26) of differentiation, and continued for 6 days in both cases. Data are expressed as mean ± SEM (n = 4). Differences between samples were analyzed by paired T-test and considered statistically significant for p ≤ 0.05; they are marked with asterisks. The purity of the selected cardiomyocytes is also shown. Representative picture of non-selected (C, D) and selected (E, F) cardiomyocytes stained using the anti-MHC antibody MF20 (C, E) on day 20 of differentiation. Cell nuclei are counterstained by DAPI (D, F). Scale bar = 100 μm.</p

Probes and sequences of primers used in quantitative RT-PCR.

<p>Probes and sequences of primers used in quantitative RT-PCR.</p

The acceleration of cardiomyogenesis in embryonic stem cells <i>in vitro</i> by serum depletion does not increase the number of developed cardiomyocytes - Fig 2

<p>Gene expressions of transcriptional factor Nkx2.5, Actn2, Myh6, Myh7, Myl2 and Myl7, recognized as markers of cardiomyocyte differentiation in mouse ES R1 cells that were differentiated for 20 days <i>in vitro</i> and primarily cultivated in the form of EBs under non-adherent conditions for 5 days or 8 days (A). The ratios of Myls expression to Nkx2.5 expression are also shown (B). Data are expressed as mean ± SEM (minimum n = 4). Differences between samples were analyzed by paired T-test and considered statistically significant for p ≤ 0.05; they are marked with asterisks.</p

Electrophysiological characteristics of differentiated cardiomyocytes.

<p>Beating frequency (A) and duration of peak width (B) analyses by Ca²⁺ flux in formed 5EB and 8EB cardiomyocytes on day 20 of differentiation. Data are expressed as mean ± SEM (n = a minimum of 4). Differences between samples were analyzed by paired T-test and considered statistically significant for p ≤ 0.05; they are marked with asterisks. Representative action potential recordings from cells stimulated with suprathreshold current pulses (0.5 ms, 4–10 nA) at 1 Hz; a cells with atrial (C) and ventricular (D) action potential morphology. In the majority of cells, we did not observe any spontaneous electrical activity during 20-s recordings (E; upper panel); occasionally, a single spontaneous AP appeared during this period (E; lower panel); <i>V</i>_m−membrane voltage. Whole cell membrane current during a 5-s ramp pulse at voltages between -110 and +40 mV (F); <i>I</i>_m−membrane current.</p

Flow-cytometric analysis of the Nkx2.5-GFP positive cell ratio in cultures derived from 5- and 8-day-old EBs.

<p>Samples were analyzed on days 8, 15 and 20 of differentiation (A). Number of cardiomyocytes formed per one EB (400 cells per EB were seeded) on days 15 and 20 of differentiation (B, C). Data are expressed as mean ± SEM (n = 4). Differences between samples were analyzed by ANOVA with Bonferroni's Multiple Comparison Test (A) and paired T-test (B, C) and considered statistically significant for p ≤ 0.05; they are marked with asterisks.</p

The experimental setup employed for comparison of the effects of adhesion, FBS supplementation, and NAC supplementation analyzed by determination of the expression of cardiomyogenic markers at the end of the differentiation experiment (8 days).

<p>Supplementation was altered for the last 3 days of cultivation under adherent or non-adherent conditions for EBs cultivation, after 5 days of cultivation in the form of EBs in the presence of FBS (A). Gene expressions of transcriptional factor Nkx2.5, Actn2, Myh6, and Myh7, recognized as markers of cardiomyocyte differentiation–data are presented as the effect of individual variables: the adhesion of EBs on days 5 or 8 of differentiation (B), the presence (S) or absence (SF) of FBS (C), and the presence ((+)NAC) or absence ((-)NAC) of N-Acetyl Cysteine (D). Data are expressed as mean ± SEM (n = 4). Differences between samples were analyzed by Kruskal-Wallis test and post-hoc Dunns test and considered statistically significant for p ≤ 0.05; they are marked with asterisks.</p