Identification of potential transcription factors that enhance human iPSC generation

Although many factors have been identified and used to enhance the iPSC reprogramming process, its efficiency remains quite low. In addition, reprogramming efficacy has been evidenced to be affected by disease mutations that are present in patient samples. In this study, using RNA-seq platform we have identified and validated the differential gene expression of five transcription factors (TFs) (GBX2, NANOGP8, SP8, PEG3, and ZIC1) that were associated with a remarkable increase in the number of iPSC colonies generated from a patient with Parkinson's disease. We have applied different bioinformatics tools (Gene ontology, protein–protein interaction, and signaling pathways analyses) to investigate the possible roles of these TFs in pluripotency and developmental process. Interestingly, GBX2, NANOGP8, SP8, PEG3, and ZIC1 were found to play a role in maintaining pluripotency, regulating self-renewal stages, and interacting with other factors that are involved in pluripotency regulation including OCT4, SOX2, NANOG, and KLF4. Therefore, the TFs identified in this study could be used as additional transcription factors that enhance reprogramming efficiency to boost iPSC generation technology.This study was supported by QBRI internal grant (QB16) and the Qatar University Student grant (QUST-2-CMED-2019-1)

Hyperosmotic Stress Induces a Specific Pattern for Stress Granule Formation in Human-Induced Pluripotent Stem Cells

Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells

The Anti-Tumor Agent Sodium Selenate Decreases Methylated PP2A, Increases GSK3βY216 Phosphorylation, Including Tau Disease Epitopes and Reduces Neuronal Excitability in SHSY-5Y Neurons

Selenium application as sodium selenate was repeatedly shown to have anti-carcinogenic properties by increasing levels of the serine/ threonine protein phosphatase 2A (PP2A) in cancer cells. PP2A has a prominent role in cell development, homeostasis, and in neurons regulates excitability. PP2A, GSK3β and Tau reside together in a complex, which facilitates their interaction and (dys)-function as has been reported for several neurological disorders. In this study we recorded maximum increase in total PP2A at 3 µM sodium selenate in a neuron cell line. In conjunction with these data, whole-cell electrophysiological studies revealed that this concentration had maximum effect on membrane potentials, conductance and currents. Somewhat surprisingly, the catalytically active form, methylated PP2A (mePP2A) was significantly decreased. In close correlation to these data, the phosphorylation state of two substrate proteins, sensitive to PP2A activity, GSK3β and Tau were found to be increased. In summary, our data reveal that sodium selenate enhances PP2A levels, but reduces catalytic activity of PP2A in a dose dependent manner, which fails to reduce Tau and GSK3β phosphorylation under physiological conditions, indicating an alternative route in the rescue of cell pathology in neurological disorders

SG assembly in human induced pluripotent stem cells is stress specific.

<p><b>(A)</b> Representative fluorescence microscopy images showing non-treated hiPSCs (No treat and 37^°C), cells treated with 125μM sodium arsenite and H₂O₂ (250μM and 2mM) or subjected to heat shock (42^°C) stained with the robust SG marker (G3BP (green)). Nucleus is stained in blue (Hoechst). Insets show magnified views of SGs. White arrows indicate SGs. Scale bar indicates 50μm. <b>(B)</b> Percentage of hiPSCs with G3BP positive SGs after 1h treatment with the indicated concentrations of sodium arsenite, or concentration range of H₂O₂ (25, 50, 100, 250, 500μM, 1mM, and 2 mM) or subjected to heat shock at the indicated temperatures. The average percentage of cells with SGs is shown. Error bars indicate the ± standard deviation from 3 independent experiments.</p

Down-regulation of some pluripotent protein expression is stress specific.

<p>(<b>A</b>) RT-PCR on mRNA extracted from non-treated hiPSCs (No treat and 37^°C) or cells subjected to sodium arsenite, H₂O₂, and heat shock, to detect pluripotent genes, <i>OCT4</i>, <i>SOX2</i>, <i>NANOG</i>, <i>KLF4</i>, <i>L1TD1</i>, and <i>LIN28A</i>. GAPDH is used as a loading control. (<b>B</b>) Representative Immuno-blots illustrating the protein levels of OCT4, SOX2, NANOG, KLF4, L1TD1, and LIN28A. (<b>C</b>) Bar graphs represent the levels (normalized with β-actin loading controls) of the tested proteins. Asterisks indicate statistical significance p values; *p<0.05, **p<0.001 and ***p<0.0001. The values are expressed in mean ± SEM from 3 experiments.</p

Analysis of pluripotent stem cell proteins for the presence of RNA binding domains.

<p>Analysis of pluripotent stem cell proteins for the presence of RNA binding domains.</p

Effects of oxidative and thermal stresses on stress granule formation in human induced pluripotent stem cells

<div><p>Stress Granules (SGs) are dynamic ribonucleoprotein aggregates, which have been observed in cells subjected to environmental stresses, such as oxidative stress and heat shock (HS). Although pluripotent stem cells (PSCs) are highly sensitive to oxidative stress, the role of SGs in regulating PSC self-renewal and differentiation has not been fully elucidated. Here we found that sodium arsenite (SA) and HS, but not hydrogen peroxide (H₂O₂), induce SG formation in human induced (hi) PSCs. Particularly, we found that these granules contain the well-known SG proteins (G3BP, TIAR, eIF4E, eIF4A, eIF3B, eIF4G, and PABP), were found in juxtaposition to processing bodies (PBs), and were disassembled after the removal of the stress. Moreover, we showed that SA and HS, but not H₂O₂, promote eIF2α phosphorylation in hiPSCs forming SGs. Analysis of pluripotent protein expression showed that HS significantly reduced all tested markers (OCT4, SOX2, NANOG, KLF4, L1TD1, and LIN28A), while SA selectively reduced the expression levels of NANOG and L1TD1. Finally, in addition to LIN28A and L1TD1, we identified DPPA5 (pluripotent protein marker) as a novel component of SGs. Collectively, these results provide new insights into the molecular cues of hiPSCs responses to environmental insults.</p></div

Emetine inhibits stress granule formation in stressed human induced pluripotent stem cells.

<p><b>(A</b>) Cells treated with sodium arsenite or <b>(B)</b> heat shock (42^°C) followed by emetine treatment (SA+Em; 42 ^oC+Em) or recovered from HS (42 ^oC+Rec) and stained with SG marker eIF3b (red). Nucleus is stained in blue (Hoechst). Insets show magnified views of SGs. White arrows indicate SGs and yellow arrowhead indicate PBs. Scale bar indicates 20μm.</p

Sodium arsenite and heat shock treatments induce eIF2α phosphorylation and localize stress granule proteins in human induced pluripotent stem cells.

<p><b>(A and B)</b> SA and HS treatments induce eIF2α phosphorylation and SG formation in hiPSCs. <b>(A)</b> Representative Immuno-blots that show the levels of peIF2α (Ser51) and eIF2α in non-treated cells (No treat and 37^°C) or cells treated with 125μM sodium arsenite and 250μM H₂O₂, or subjected to heat shock <b>(</b>42^°C). <b>(B)</b> Bar graph representing the ratio of (normalized against β-actin loading controls) peIF2α (Ser51)/eIF2α for each treatment. Values are expressed as mean ± SEM from 4–5 experiments. Asterisks indicate a statistically significant change, *p<0.05, **p<0.001, and ***p<0.0001. <b>(C-E)</b> Representative fluorescence microscopy images showing hiPSCs treated with sodium arsenite (125μM), subjected to heat shock (42^°C), or left untreated (No treat; 37^°C), and stained with SG markers <b>(C)</b> TIAR (red)/eIF4E (green)/ eIF4A (white), <b>(D)</b> eIF3b (red)/eIF4G (green)/PABP (white), and <b>(E)</b> eIF3b (red)/YB1 (white) or PB marker (SK1, green). Insets are reproduced at the right as replicate views of SGs showing each marker separately and the merged view (yellow). Scale bar indicates 5μm.</p