Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors

Additional file 6: Figure S1. Derivation of iPS cells in defined culture conditions

Novel strong tissue specific promoter for gene expression in human germ cells

<p>Abstract</p> <p>Background</p> <p>Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene <it>NDUFV1 </it>containing an endogenous retroviral sequence.</p> <p>Results</p> <p>Among seven established human cell lines and five primary cultures, this modified <it>NDUFV1 </it>upstream sequence (mNUS) was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102), where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter). To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD) suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1), whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293). In further experiments, we tested mNUS-driven expression of a hyperactive <it>Sleeping Beauty </it>transposase (SB100X). The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter.</p> <p>Conclusions</p> <p>We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream <it>NDUFV1 </it>transcriptional start site plays a crucial role in the activity of this gene promoter <it>in vitro </it>in the majority of tested cell types (10/12), and an important role - in the rest two cell lines.</p

EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line

<div><p>The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant–EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis.</p></div

Primer sequences.

<p>Primer sequences.</p

Effect of different inhibitors on serum starved, EGF treated, EGFRwt expressing AD293 cells.

<p>Effect of different inhibitors on serum starved, EGF treated, EGFRwt expressing AD293 cells.</p

Wild-type EGFR increases proliferation rate in complete medium.

<p>(A,B) AD293 cells overexpress EGFRvIII, EGFRwt or both receptor variants. Cells were cultured in complete medium, collected and lysed. Western blots show levels of protein expression of total EGFR (sc-03 antibody), phospho-EGFR (tyr1173 antibody), phospho-AKT, AKT, phospho-STAT5 and Actin in four different cell lines (A). Real-time PCR was used to evaluate relative expression of total EGFR (wt and vIII) against <i>HPRT1</i> housekeeping gene in AD293 cell lines (B). <i>Statistical significance calculated against a value of specific gene in AD293par</i>, <i>ns</i>: <i>P>0</i>.<i>05; ***</i>: <i>P<0</i>.<i>001</i>. (C) EGFRwt expressing cells have the highest proliferation rate. Cells were seeded in complete medium and after 6 hours were moved to Nikon BioStation CT for live observation. Student’s t-test: ns: P>0.05; ***: P<0.001. (D) EGFRvIII expressing cells have constitutive expression of cyclin D1 independently on EGF treatment. Cells were serum starved for 24 hours, then supplemented with DMSO (control), EGF (50 ng/ml) or EGF/erlotinib (15 μM). After 24 hours cells were lysed and real-time RT-PCR was conducted. <i>Statistical significance calculated against a value for AD293par from the same group</i>, <i>ns</i>: <i>P>0</i>.<i>05; *</i>: <i>P<0</i>.<i>05; ***</i>: <i>P<0</i>.<i>001</i>. (E, F) EGFRwt has a peak activity after 1 hour of EGF treatment. Cells were serum starved for 24 hour and then serum free medium with EGF (20 ng/ml) was added. After specified time cells were lysed and blotted for total EGFR, phospho-EGFR and Actin (E). Bands intensity was calculated with ImageJ software (F). (G) AD293 cells overexpressing EGFRvIII have constitutively activated AKT and STAT5, while EGFRwt induces such activation after EGF treatment. Cells were serum starved for 24 hour and then serum free medium with EGF (20 ng/ml) or EGF/erlotinib (15 μM) was added. After 1 hour cells were lysed and blotted for phospho-EGFR, phospho-AKT, AKT, phospho-STAT5 and Actin.</p

Serum starved EGF-treated cells expressing wild-type EGFR undergo apoptosis, what is prevented by erlotinib.

<p>(A, B) AD293par cells survive EGF treatment in contrast to massively detaching cells overexpressing EGFRwt. Medium was changed to serum free, DMSO/EGF/EGF+erlotinib was added and cells were photographed every 6 hours. Photos present cells at time 0 and at 48 hour. (C) Most of AD293 cells with wild-type EGFR treated with EGF become apoptotic. Medium was changed to serum free, DMSO/EGF/EGF+erlotinib/erlotinib was added, cells were harvested after 48 hours, stained with Annexin V FITC/propidium iodide and analyzed by flow cytometry. (D) Analysis of compacted state of chromatin in apoptotic cells confirmed the link between EGF activated EGFRwt and apoptosis. Medium was changed to serum free, DMSO/EGF/EGF+erlotinib/erlotinib was added, cells were stained after 24 hours with propidium iodide and Hoechst 33342 and analyzed under fluorescence microscopy. <i>Blue arrow</i>: <i>viable cell; green arrow</i>: <i>early apoptotic cell; yellow arrow</i>: <i>late apoptotic cell; red arrow</i>: <i>necrotic cell</i>.</p

Inhibitors of PI3K, AKT and rapamycin do not prevent apoptosis in wild-type EGFR-expressing cells treated with EGF.

<p>(A) Rapamycin does not influence STAT5 and AKT activity, contrary to GDC-0941. Cells were serum starved for 24 hour and then serum free medium was supplemented with DMSO, EGF (20 ng/ml), EGF (20 ng/ml) + inhibitors (NU-7441 1 μM; GDC-0941 1 μM; rapamycin 1 μM). After 1 hour, cells were lysed and blotted for phospho-EGFR, phospho-STAT5, phospho-AKT, AKT and Actin. (B) PI3K inhibition slightly decreased and mTOR inhibition increased expression of cyclin D1. Cells were serum starved for 24 hours and DMSO, EGF (20 ng/ml), EGF (20 ng/ml) + inhibitors (erlotinib 15 μM; NU-7441 1 μM; GDC-0941 1 μM; rapamycin 1 μM) were added. After 24 hours cells were lysed and real-time RT-PCR was conducted. (C) PI3K and mTOR inhibitors do not reverse an effect of EGF. Medium was changed to serum free with DMSO/GDC-0941/rapamycin, and every 24 hours cells were photographed and counted. (D) AKT inhibitor MK2206 did not rescue cells after EGF treatment. Medium was changed to serum free and supplemented with EGF (20 ng/ml) and DMSO or inhibitors (erlotinib 15 μM or MK2206 2 μM). Cells, run in triplicates, were photographed every 6 hours and counted (time points shown: 0 h, 24 h, 48 h and 72 h). <i>Significance calculated against time 0 within same cell line and same treatment</i>, <i>ns</i>: <i>P>0</i>.<i>05; ***</i>: <i>P<0</i>.<i>001</i>.</p