Assessment of Genetic Stability in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Using Droplet Digital PCR.

Unintended genetic modifications that occur during the differentiation and proliferation of human induced pluripotent stem cells (hiPSCs) can lead to tumorigenicity. This is a crucial concern in the development of stem cell-based therapies to ensure the safety and efficacy of the final product. Moreover, conventional genetic stability testing methods are limited by low sensitivity, which is an issue that remains unsolved. In this study, we assessed the genetic stability of hiPSCs and hiPSC-derived cardiomyocytes using various testing methods, including karyotyping, CytoScanHD chip analysis, whole-exome sequencing, and targeted sequencing. Two specific genetic mutations in KMT2C and BCOR were selected from the 17 gene variants identified by whole-exome and targeted sequencing methods, which were validated using droplet digital PCR. The applicability of this approach to stem cell-based therapeutic products was further demonstrated with associated validation according to the International Council for Harmonisation (ICH) guidelines, including specificity, precision, robustness, and limit of detection. Our droplet digital PCR results showed high sensitivity and accuracy for quantitatively detecting gene mutations, whereas conventional qPCR could not avoid false positives. In conclusion, droplet digital PCR is a highly sensitive and precise method for assessing the expression of mutations with tumorigenic potential for the development of stem cell-based therapeutics.

Genetic variants shape rheumatoid arthritis-specific transcriptomic features in CD4+ T cells through differential DNA methylation, explaining a substantial proportion of heritability.

OBJECTIVE: CD4+ T cells have been suggested as the most disease-relevant cell type in rheumatoid arthritis (RA) in which RA-risk non-coding variants exhibit allele-specific effects on regulation of RA-driving genes. This study aimed to understand RA-specific signatures in CD4+ T cells using multi-omics data, interpreting inter-omics relationships in shaping the RA transcriptomic landscape. METHODS: We profiled genome-wide variants, gene expression and DNA methylation in CD4+ T cells from 82 patients with RA and 40 healthy controls using high-throughput technologies. We investigated differentially expressed genes (DEGs) and differential methylated regions (DMRs) in RA and localised quantitative trait loci (QTLs) for expression and methylation. We then integrated these based on individual-level correlations to inspect DEG-regulating sources and investigated the potential regulatory roles of RA-risk variants by a partitioned-heritability enrichment analysis with RA genome-wide association summary statistics. RESULTS: A large number of RA-specific DEGs were identified (n=2575), highlighting T cell differentiation and activation pathways. RA-specific DMRs, preferentially located in T cell regulatory regions, were correlated with the expression levels of 548 DEGs mostly in the same topologically associating domains. In addition, expressional variances in 771 and 83 DEGs were partially explained by expression QTLs for DEGs and methylation QTLs (meQTLs) for DEG-correlated DMRs, respectively. A large number of RA variants were moderately to strongly correlated with meQTLs. DEG-correlated DMRs, enriched with meQTLs, had strongly enriched heritability of RA. CONCLUSION: Our findings revealed that the methylomic changes, driven by RA heritability-explaining variants, shape the differential expression of a substantial fraction of DEGs in CD4+ T cells in patients with RA, reinforcing the importance of a multidimensional approach in disease-relevant tissues.

Genome-wide copy number variation study reveals KCNIP1 as a modulator of insulin secretion.

Copy number variations (CNVs) have emerged as another important genetic marker in addition to SNP for understanding etiology of complex diseases. In light of this, we performed a genome-wide CNV study to identify type 2 diabetes (T2D)-associated CNV using an array comparative genomic hybridization from 3180 subjects for T2D cases (n=863) and controls (n=2,317). Thus, five CNV regions having a p-value threshold ≤0.05 were identified and evaluated by validation with quantitative PCR and comparison with previously reported CNV regions in the Database of Genomic Variants. Furthermore, we performed a functional experiment to assess the biological significance of a gene encompassing a CNV region. The inhibition of KCNIP1 led to increased insulin secretion in a glucose-dependent manner, but had no effect on insulin gene transcription as well as cell apoptosis. Taken together, these data indicate that KCNIP1 from CNV study might function as a T2D-susceptibility gene whose dysregulation alters insulin production.

Effect of Xenogeneic Substances on the Glycan Profiles and Electrophysiological Properties of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Background and Objectives: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) hold great promise as a cellular source of CM for cardiac function restoration in ischemic heart disease. However, the use of animal-derived xenogeneic substances during the biomanufacturing of hiPSC-CM can induce inadvertent immune responses or chronic inflammation, followed by tumorigenicity. In this study, we aimed to reveal the effects of xenogeneic substances on the functional properties and potential immunogenicity of hiPSC-CM during differentiation, demonstrating the quality and safety of hiPSC-based cell therapy. Methods and Results: We successfully generated hiPSC-CM in the presence and absence of xenogeneic substances (xeno-containing (XC) and xeno-free (XF) conditions, respectively), and compared their characteristics, including the contractile functions and glycan profiles. Compared to XC-hiPSC-CM, XF-hiPSC-CM showed early onset of myocyte contractile beating and maturation, with a high expression of cardiac lineage-specific genes (ACTC1, TNNT2, and RYR2) by using MEA and RT-qPCR. We quantified N-glycolylneuraminic acid (Neu5Gc), a xenogeneic sialic acid, in hiPSC-CM using an indirect enzyme-linked immunosorbent assay and liquid chromatography-multiple reaction monitoring- mass spectrometry. Neu5Gc was incorporated into the glycans of hiPSC-CM during xeno-containing differentiation, whereas it was barely detected in XF-hiPSC-CM. Conclusions: To the best of our knowledge, this is the first study to show that the electrophysiological function and glycan profiles of hiPSC-CM can be affected by the presence of xenogeneic substances during their differentiation and maturation. To ensure quality control and safety in hiPSC-based cell therapy, xenogeneic substances should be excluded from the biomanufacturing process.

Design and synthesis of novel hydroxyalkylaminomethylchromones for their IL-5 inhibitory activity.

A series of hydroxyalkylaminomethylchromone analogs 3 were prepared and evaluated as inhibitors of interleukin-5. The most active analog 3d inhibited interleukin-5 activity with an IC50 of 17.5 µM. The structural requirements of chromone analogs possessing the inhibitory activity against IL-5 could be summarized as: (i) the cyclohexylmethoxy group at 5th position of the A ring, (ii) the planarity of chromone ring, (iii) hydrophobic unit around the B ring with hydroxyl functional group, (iv) the hydrophobic unit which does not have to be a planar and (v) the length of carbon units between amino and hydroxyl group is limited to two.

Identification of metabolic markers predictive of prediabetes in a Korean population.

Prediabetes (PD) is a high-risk state of developing type 2 diabetes, and cardiovascular and metabolic diseases. Metabolomics-based biomarker studies can provide advanced opportunities for prediction of PD over the conventional methods. Here, we aimed to identify metabolic markers and verify their abilities to predict PD, as compared to the performance of the traditional clinical risk factor (CRF) and previously reported metabolites in other population-based studies. Targeted metabolites quantification was performed in 1723 participants in the Korea Association REsource (KARE) cohort, from which 500 normal individuals were followed up for 6 years. We selected 12 significant metabolic markers, including five amino acids, four glycerophospholipids, two sphingolipids, and one acylcarnitine, at baseline, resulting in a predicted incidence of PD with an area under the curve (AUC) of 0.71 during follow-up. The performance of these metabolic markers compared to that of fasting glucose was significantly higher in obese patients (body mass index: BMI ≥ 25 kg/m2, 0.79 vs. 0.58, P < 0.001). The combination with metabolic markers, CRF, and fasting glucose yielded the best prediction performance (AUC = 0.86). Our results revealed that metabolic markers were not only associated with the risk of PD, but also improved the prediction performance in combination with conventional approaches.

Metabolomics profiles associated with diabetic retinopathy in type 2 diabetes patients.

Diabetic retinopathy (DR) is a common complication of diabetes, and it is the consequence of microvascular retinal changes due to high glucose levels over a long time. Metabolomics profiling is a rapidly evolving method used to identify the metabolites in biological fluids and investigate disease progression. In this study, we used a targeted metabolomics approach to quantify the serum metabolites in type 2 diabetes (T2D) patients. Diabetes patients were divided into three groups based on the status of their complications: non-DR (NDR, n = 143), non-proliferative DR (NPDR, n = 123), and proliferative DR (PDR, n = 51) groups. Multiple logistic regression analysis and multiple testing corrections were performed to identify the significant differences in the metabolomics profiles of the different analysis groups. The concentrations of 62 metabolites of the NDR versus DR group, 53 metabolites of the NDR versus NPDR group, and 30 metabolites of the NDR versus PDR group were found to be significantly different. Finally, sixteen metabolites were selected as specific metabolites common to NPDR and PDR. Among them, three metabolites including total DMA, tryptophan, and kynurenine were potential makers of DR progression in T2D patients. Additionally, several metabolites such as carnitines, several amino acids, and phosphatidylcholines also showed a marker potential. The metabolite signatures identified in this study will provide insight into the mechanisms underlying DR development and progression in T2D patients in future studies.

Obesity susceptible novel DNA methylation marker on regulatory region of inflammation gene: results from the Korea Epigenome Study (KES).

INTRODUCTION: Obesity is growing global health concern and highly associated with increased risk of metabolic diseases including type 2 diabetes. We aimed to discover new differential DNA methylation patterns predisposing obesity and prioritize surrogate epigenetic markers in Koreans. RESEARCH DESIGN AND METHODS: We performed multistage epigenome-wide analyses to identify differentially expressed CpGs in obesity using the Illumina HumanMethylationEPIC array (EPIC). Forty-eight CpGs showed significant differences across three phases: 902 whole blood DNAs from two cohorts (phase 1: n=450, phase 2: n=377) and a hospital-based sample (phase 3: n=75). Samples from phase III participants were used to examine whether the 48 CpGs are significant in the fat tissue and influenced gene expression. Furthermore, we investigated the epigenetic effect of CpG loci in childhood obesity (n=94). RESULTS: Seven of the 48 CpGs exhibited similar changes in the fat tissue along with gene expression changes. In particular, hypomethylated CpG (cg13424229) on the GATA1 transcription factor cluster of CPA3 promoter was related to its increased gene expression and showed consistent effect in childhood obesity. Interestingly, subsequent analysis using RNA sequencing data from 21 preadipocytes and 26 adipocytes suggested CPA3 as a potential obesity-related gene. Moreover, expression patterns from RNA sequencing and public Gene Expression Omnibus showed the correlation between CPA3 and type 2 diabetes (T2D) and asthma. CONCLUSIONS: Our finding prioritizes influential genes in obesity and provides new evidence for the role of CPA3 linking obesity, T2D, and asthma.

Metabolomics profiles associated with HbA1c levels in patients with type 2 diabetes.

Glycated hemoglobin (HbA1c) is an indicator of the average blood glucose concentration. Failing to control HbA1c levels can accelerate the development of complications in patients with diabetes. Although metabolite profiles associated with HbA1c level in diabetes patients have been characterized using different platforms, more studies using high-throughput technology will be helpful to identify additional metabolites related to diabetes. Type 2 diabetes (T2D) patients were divided into two groups based on the HbA1c level: normal (HbA1c ≤6%) and high (HbA1c ≥9%) in both discovery and replication sets. A targeted metabolomics approach was used to quantify serum metabolites and multivariate logistic regression was used to identify significant differences between groups. The concentrations of 22 metabolites differed significantly between the two groups in the discovery set. In the replication set, the levels of 21 metabolites, including 16 metabolites identified in the discovery set, differed between groups. Among these, concentrations of eleven amino acids and one phosphatidylcholine (PC), lysoPC a C16:1, were higher and four metabolites, including three PCs (PC ae C36:1, PC aa C26:0, PC aa C34:2) and hexose, were lower in the group with normal HbA1c group than in the group with high HbA1c. Metabolites with high concentrations in the normal HbA1c group, such as glycine, valine, and PCs, may contribute to reducing HbA1c levels in patients with T2D. The metabolite signatures identified in this study provide insight into the mechanisms underlying changes in HbA1c levels in T2D.

Association of Metabolites with Obesity and Type 2 Diabetes Based on FTO Genotype.

The single nucleotide polymorphism rs9939609 of the gene FTO, which encodes fat mass and obesity-associated protein, is strongly associated with obesity and type 2 diabetes (T2D) in multiple populations; however, the underlying mechanism of this association is unclear. The present study aimed to investigate FTO genotype-dependent metabolic changes in obesity and T2D. To elucidate metabolic dysregulation associated with disease risk genotype, genomic and metabolomic datasets were recruited from 2,577 participants of the Korean Association REsource (KARE) cohort, including 40 homozygous carriers of the FTO risk allele (AA), 570 heterozygous carriers (AT), and 1,967 participants carrying no risk allele (TT). A total of 134 serum metabolites were quantified using a targeted metabolomics approach. Through comparison of various statistical methods, seven metabolites were identified that are significantly altered in obesity and T2D based on the FTO risk allele (adjusted p < 0.05). These identified metabolites are relevant to phosphatidylcholine metabolic pathway, and previously reported to be metabolic markers of obesity and T2D. In conclusion, using metabolomics with the information from genome-wide association studies revealed significantly altered metabolites depending on the FTO genotype in complex disorders. This study may contribute to a better understanding of the biological mechanisms linking obesity and T2D.

MicroRNA-650 in a copy number-variable region regulates the production of interleukin 6 in human osteosarcoma cells.

Copy number variation is a well-known genetic variation. microRNAs (miRNAs/miRs) are non-coding RNAs that mediate gene expression by regulating target mRNAs. In the present study, copy number deletions encompassing miRNA coding regions were investigated to determine the association between the deletion of miRNA and its phenotypic effects. A total of 38 human miRNAs in copy number variants were identified and miR-650, which is functional in the human osteosarcoma MG-63 cell line, was selected. Overexpression of miR-650 decreased the expression of inhibitor of growth family member 4 (ING4) in the MG-63 cells and increased interleukin (IL)6 transcription, as well as IL6 secretion in IL1B-stimulated cells. Furthermore, miR-650 downregulated the amount of nuclear factor of κ light polypeptide gene enhancer in B cells inhibitor α and increased the transcriptional activity of nuclear factor (NF)κB. Downregulation of ING4 also increased the production of IL6, similar to miR-650 overexpression. Taken together, these data indicate that miR-650 plays a significant role in the production of IL6 by regulating ING4 expression and NFκB signaling in IL1B-stimulated MG-63 osteosarcoma cells.

Identification of differentially-expressed genes by DNA methylation in cervical cancer.

To identify novel cervical cancer-related genes that are regulated by DNA methylation, integrated analyses of genome-wide DNA methylation and RNA expression profiles were performed using the normal and tumor regions of tissues from four patients; two with cervical cancer and two with pre-invasive cancer. The present study identified 19 novel cervical cancer-related genes showing differential RNA expression by DNA methylation. A number of the identified genes were novel cervical cancer-related genes and their differential expression was confirmed in a publicly available database. Among the candidate genes, the epigenetic regulation and expression of three genes, CAMK2N1, ALDH1A3 and PPP1R3C, was validated in HeLa cells treated with a demethylating reagent using methylation-specific polymerase chain reaction (PCR) and quantitative PCR, respectively. From these results, the expression of the CAMK2N1, ALDH1A3 and PPP1R3C genes are were shown to be suppressed in cervical cancers by DNA methylation. These genes may be involved in the progression or initiation of cervical cancer.

Synthesis and evaluation of novel chromone analogs for their inhibitory activity against interleukin-5.

A novel series of chromone analogs were synthesized and evaluated for their inhibitory activity against interleukin-5. Among them compounds 5-Cyclohexylmethoxy-3-(4-hydroxybenzyl)-4H-chromen-4-one (6a, 98% inhibition at 30 microM, IC50<3.0 microM) and 5-Cyclohyxylmethoxy-3-(hydroxymethyl)-4H-chromen-4-one (8a, 84% inhibition at 30 microM, IC50=7.6 microM) showed most potent activity. The structural requirement of chromone analogs possessing the inhibitory activity against IL-5 could be summarized as: (i) importance of hydrophobic group such as cyclohexylmethoxy at 5th position of ring A, (ii) requirement of ring B with small size of hydrogen bonding group with electron donating property such as phenolic hydroxyl group at 4th position and (iii) planarity of the chromen-4-one ring.