The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture.

The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications

Evaluation of the impacts of the COVID-19 pandemic on the development of the unemployment rate in Slovakia: counterfactual before-after comparison

Research background: The COVID-19 pandemic, which hit the world in the first quarter of 2020, has impacted almost every area of people's lives. Many states have introduced varying degrees of measures to prevent its spread. Most of these measures were, or still are, aimed at reducing or completely stopping the operation of shops and services, or in some cases, also the large manufacturing companies. However, as many companies have failed to cope with these restrictions, unemployment has risen in almost all EU countries. A similar situation was also observed in Slovakia, where the mentioned measures also had a significant impact on unemployment. Purpose of the article: In this study, we deal with the quantification of the impact of a pandemic, or more precisely, anti-pandemic measures, on the development of the registered unemployment rate in Slovakia. Methods: This quantification is based on the counterfactual method of before-after comparison, which is one of the most widely used methods in the field of impact assessments and brings very accurate results, based on real data. In the analysis, we use officially published data on the unemployment rate in Slovakia during the years 2013?2020 on a monthly basis. Such a long time series, using statistical methods of its decomposition and modelling of its trend, will allow predicting the development of the unemployment rate in Slovakia, assuming a counterfactual situation of no pandemic, and compare this development with the actual situation that occurred during 2020. Findings & Value added: The study results indicate an increase in the unemployment rate in Slovakia during 2020 by 2?3% compared to the trend of its development, which would have occurred without a pandemic. Given the counterfactual method used, this difference can be described as the impact of the COVID-19 pandemic. The results of the study can be used in practice in the design and implementation of measures introduced to mitigate the impacts of the pandemic on unemployment and, in the long-term perspective, also to eliminate these effects as much as possible. It can also be used as a theoretical tool in conducting impact assessments, which have so far been carried out very rarely in Slovakia

MethylScreen technology principles and data interpretation.

<p>(A) Representative qPCR standard curve for PAX6 obtained from 100, 50, 10, 1 and 0.1 ng of control DNA per reaction. (B₁) <i>UTF1</i> MethylScreen qPCR results for CCTL-12, IPSCF, CBIA-11, A549 and KG-1 genomes. Changes in c_t between enzyme treated and non-treated templates are depicted: Rs-R0 is represented by white columns (HhaI reaction), Rd-R0 is represented by grey columns (McrBC reaction), and Rsd-R0 is represented by black columns (both HhaI and McrBC reaction). (B₂) <i>UTF1</i> DNA methylation profile for five cell lines. c_t values from four restriction reactions (B₁) were converted to DNA methylation occupancy, expressed as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. (C) MethylScreen results obtained from sample mixtures with an increasing ratio of hypermethylated DNA (0–100%, x-axis). The colour legend is identical for (B₂), and the values are averages for <i>PAX6</i> and <i>TSPYL5</i> genes after serial mixing of CCTL-14 (unmethylated) and A549 (hypermethylated) samples and NDFs (unmethylated) and CCTL-14 (hypermethylated) samples, respectively.</p

Gene expression levels in hPSCs and dermal fibroblasts, as determined by quantitative real-time PCR.

<p>Expression levels of eleven genes were compared i) among the hiPSC lines, hESC lines and dermal fibroblasts (NDFs and ADFs together) (A-C) and ii) within hiPSC lines between low and high passage numbers (D-F). Analysed hiPSC lines and the passage numbers are reported in the graph legend. The values are relative to the values from hESCs (set at 1). Values are the mean + SD, and asterisks indicate the significance between indicated samples: *p < 0.05; **p < 0.005; ***p < 0.0005.</p

hPSC lines used in this study.

<p>hPSC lines used in this study.</p

PCR primers used for MethylScreen reactions.

<p>PCR primers used for MethylScreen reactions.</p

DNA methylation profile of selected genes in hPSC lines.

<p>DNA methylation occupancy is reported as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. The profile is shown for hESC lines (A), hiPSC lines from NDFs (B), hiPSC lines from ADFs (C, D) and hiPSC lines from PBMC CD34⁺ cells (E, F) generated by STEMCCA lentivirus (STE), Sendai virus (SEN) and the episomal vector (EPI) reprogramming. Values are the averages from hPSC lines within the line group where appropriate.</p

Hierarchical clustering of hPSC lines and dermal fibroblasts based on quantitative real-time PCR data.

<p>The colour-coded scale on the left of the picture indicates the expression value for each gene; low expression is represented by green, and high expression is represented by red. The values are relative to the values from CCTL-14 (set at 1).</p

DNA methylation profile of selected genes in hiPSCs source cells.

<p>DNA methylation occupancy is reported as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. The profile is shown for NDFs (A), ADFs (B), and PBMC CD34⁺ cells (C).</p

The Manufacture of Xeno- and Feeder-Free Clinical-Grade Human Embryonic Stem Cell Lines: First Step for Cell Therapy

Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP