Clinical-Grade Human Pluripotent Stem Cells for Cell Therapy: Characterization Strategy

Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans’ lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and ′for information only′ characterization methods with release criteria for the establishment of clinical-grade hPSC lines

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

Truncated vitronectin with E-cadherin enables the xeno-free derivation of human embryonic stem cells

Abstract Human embryonic stem cells (hESCs) have unique abilities that enable their use in cell therapy, disease modeling, and drug development. Their derivation is usually performed using a feeder layer, which is undefined and can potentially cause a contamination by xeno components, therefore there is a tendency to replace feeders with xeno-free defined substrates in recent years. Three hESC lines were successfully derived on the vitronectin with a truncated N-terminus (VTN-N) in combination with E-cadherin in xeno-free conditions for the first time, and their undifferentiated state, hESC morphology, and standard karyotypes together with their potential to differentiate into three germ layers were confirmed. These results support the conclusion that the VTN-N/E-cadherin is a suitable substrate for the xeno-free derivation of hESCs and can be used for the derivation of hESCs according to good manufacturing practices

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

The Role of RNA Polymerase II Contiguity and Long-Range Interactions in the Regulation of Gene Expression in Human Pluripotent Stem Cells

The eukaryotic nucleus is a highly complex structure that carries out multiple functions primarily needed for gene expression, and among them, transcription seems to be the most fundamental. Diverse approaches have demonstrated that transcription takes place at discrete sites known as transcription factories, wherein RNA polymerase II (RNAP II) is attached to the factory and immobilized while transcribing DNA. It has been proposed that transcription factories promote chromatin loop formation, creating long-range interactions in which relatively distant genes can be transcribed simultaneously. In this study, we examined long-range interactions between the POU5F1 gene and genes previously identified as being POU5F1 enhancer-interacting, namely, CDYL, TLE2, RARG, and MSX1 (all involved in transcriptional regulation), in human pluripotent stem cells (hPSCs) and their early differentiated counterparts. As a control gene, RUNX1 was used, which is expressed during hematopoietic differentiation and not associated with pluripotency. To reveal how these long-range interactions between POU5F1 and the selected genes change with the onset of differentiation and upon RNAP II inhibition, we performed three-dimensional fluorescence in situ hybridization (3D-FISH) followed by computational simulation analysis. Our analysis showed that the numbers of long-range interactions between specific genes decrease during differentiation, suggesting that the transcription of monitored genes is associated with pluripotency. In addition, we showed that upon inhibition of RNAP II, long-range associations do not disintegrate and remain constant. We also analyzed the distance distributions of these genes in the context of their positions in the nucleus and revealed that they tend to have similar patterns resembling normal distribution. Furthermore, we compared data created in vitro and in silico to assess the biological relevance of our results

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

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

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