Rational Design and Synthesis of Post-Functionalizable Peptide Foldamers as Helical Templates

In this study, we developed post-functionalizable helical peptides composed of Leu, Aib, and Azl residues. We show that the synthesized peptides <b>1</b> and <b>2</b> form helical structures, and may be modified using specific side chain or several functional groups by the click reaction without influencing their secondary structures

Detection of undifferentiated hiPSCs by qRT-PCR assay.

<p>(A) The relative mRNA expressions in primary RPE cells of Lin28, Oct-3/4, Sox2, Nanog, Rex1, Klf4, and c-Myc were determined by qRT-PCR analysis. (B–D) qRT-PCR analysis of hiPSCs spiked into primary RPE cells and five lots of primary RPE cells. Single-cell hiPSCs (1%, 2.5×10³ cells; 0.1%, 2.5×10² cells; 0.01%, 25 cells) were spiked into 2.5×10⁵ primary RPE cells, and total RNA was isolated from the mixed cells. The mRNA levels of Nanog (B), Oct3/4 (C) and Lin28 (D) are shown as a relative expression. Limit of detection was calculated as the mean plus 3.3 fold the standard deviation of the measurement of the five lots of primary RPE cells. (E) Lin28 expression of hiPSCs differentiating into RPE and purified hiPSC-derived RPE cells (passage 3 and 4). All values are expressed as mRNA levels relative to those in undifferentiated hiPSCs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037342#s2" target="_blank">Results</a> are means ± standard deviation (n = 3).</p

Comparison of the tumorigenicity-associated assays.

*<p>Not based on the calculation found in Reference #21 because the background signal from the negative controls (primary RPE cells) was not detectable.</p

Detection of undifferentiated hiPSCs by flow cytometry assay.

<p>(A) Flow cytometry analysis of hiPSCs (blue) and primary RPE cells (red). Cells were fixed, permiabilized and stained with anti-TRA-1-60, anti-TRA-1-81, anti-Sox2, anti-Oct3/4 and anti-Nanog antibodies labeled with fluorophore. (B) Five lots of primary RPE cells were analyzed by flow cytometry with anti-TRA-1-60 antibody. (C) HiPSCs (0.1%, 2.5×10² cells; 0.01%, 25 cells) were spiked into primary RPE cells (2.5×10⁵ cells) and analyzed by flow cytometry with anti-TRA-1-60 antibody. (D) Flow cytometry analysis of hiPSC-derived RPE cells was performed with anti-TRA-1-60 antibody. Ten thousand cells (A) and 1×10⁵ cells (B–D) were used for one assay of flow cytometry analysis. The numbers indicate the quantity of cells contained in the gate.</p

Soft agar colony formation assay of hiPSCs and teratocarcinoma PA-1 cells.

<p>(A) Phase-contrast images of hiPSCs, primary RPE cells, hiPSC-derived RPE cells and PA-1cells spiked into primary RPE cells (1%) cultured in soft agar medium for 30 days. Arrows indicate the colonies of PA-1 cells. (B) PA-1 cells (1%, 100 cells; 0.5%, 50 cells; 0.25%, 25 cells; 0%, 0 cells) were spiked into 1.0×10⁴ primary RPE cells and grown in soft agar for 10, 20 and 30 days. Cell growth was quantified using a CytoSelect kit. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037342#s2" target="_blank">Results</a> were expressed as a relative fold change of the value of blank well. Statistical significance was determined using two-way ANOVA and Bonferroni's post-hoc test (*<i>P</i><0.05 compared with the 0% control). (C) HiPSC-derived RPE cells, three lots of primary RPE cells and PA-1 cells spiked into primary RPE cells were grown in soft agar for 30 days. Quantification of the results is described in (B). Limit of detection was calculated as the mean plus 3.3 fold the standard deviation of the measurement of the three lots of primary RPE cells. Error bars represent the standard deviation of the measurements (n = 3).</p

Electrophysiological Characteristics of Human iPSC-Derived Cardiomyocytes for the Assessment of Drug-Induced Proarrhythmic Potential

<div><p>The aims of this study were to (1) characterize basic electrophysiological elements of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that correspond to clinical properties such as QT-RR relationship, (2) determine the applicability of QT correction and analysis methods, and (3) determine if and how these in-vitro parameters could be used in risk assessment for adverse drug-induced effects such as Torsades de pointes (TdP). Field potential recordings were obtained from commercially available hiPSC-CMs using multi-electrode array (MEA) platform with and without ion channel antagonists in the recording solution. Under control conditions, MEA-measured interspike interval and field potential duration (FPD) ranged widely from 1049 to 1635 ms and from 334 to 527 ms, respectively and provided positive linear regression coefficients similar to native QT-RR plots obtained from human electrocardiogram (ECG) analyses in the ongoing cardiovascular-based Framingham Heart Study. Similar to minimizing the effect of heart rate on the QT interval, Fridericia’s and Bazett’s corrections reduced the influence of beat rate on hiPSC-CM FPD. In the presence of E-4031 and cisapride, inhibitors of the rapid delayed rectifier potassium current, hiPSC-CMs showed reverse use-dependent FPD prolongation. Categorical analysis, which is usually applied to clinical QT studies, was applicable to hiPSC-CMs for evaluating torsadogenic risks with FPD and/or corrected FPD. Together, this results of this study links hiPSC-CM electrophysiological endpoints to native ECG endpoints, demonstrates the appropriateness of clinical analytical practices as applied to hiPSC-CMs, and suggests that hiPSC-CMs are a reliable models for assessing the arrhythmogenic potential of drug candidates in human.</p></div

Relationship between field potential duration (FPD)/corrected FPD (FPDc) and interspike interval (ISI) of 96 samples of hiPSC-CM.

<p>The data in sham treatment from 4 facilities were plotted (n = 96) in Fig 2a (FPD-ISI), Fig 2b (FPDcF-ISI) and Fig 2c (FPDcB-ISI), respectively. The solid and dashed lines indicate the linear regression line and 95% confidence bands, respectively. The equation, R² value, and root mean squared prediction error (RMSE) are shown in the figures.</p

Categorical analyses of absolute FPD values after E-4031 application.

<p>Categorical analyses of absolute FPD values after E-4031 application.</p

Effects of <i>I</i>_Kr and <i>I</i>_Ks inhibitors on field potential duration (FPD)-interspike interval (ISI) plots.

<p>FPD-ISI plots are shown before and after compound application both in terms of absolute values (left) and % change (right), for DMSO (Fig 3a), E-4031 (Fig 3b), cisapride (Fig 3c) and chromanol 293B (Fig 3d). The solid lines and dashed lines indicate the linear regression lines and 95% confidence bands, respectively. Data for the middle concentrations are omitted for clarity. ‘Slope’ refers to the slope of the regression line.</p

Assay design and representative field potential (FP) waveforms before/after drug application.

<p>Schematic depicting the measurement schedule for the hiPSC-CM/MEA assay (Fig 1a). The upper panels show FP waveforms and parameters, and the lower panels indicate the arrhythmogenic waveform of EAD (left, dashed circle) and TA (right, dashed circle) (Fig 1b). Example FP waveforms after application of DMSO, E-4031, cisapride or chromanol 293B (Fig 1c).</p