A stress-reduced passaging technique improves the viability of human pluripotent cells

Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, reproducibility issues, often arising from variability during passaging steps, remain. Here, we describe an improved method for the subculture of human PSCs. The revised method significantly enhances the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing and directed differentiation. Furthermore, the method does not alter PSC characteristics after long-term culture and attenuates the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application

Microarray Analysis of Paramylon, Isolated from Euglena Gracilis EOD-1, and Its Effects on Lipid Metabolism in the Ileum and Liver in Diet-Induced Obese Mice

We previously showed that supplementation of a high fat diet with paramylon (PM) reduces the postprandial glucose rise, serum total and LDL cholesterol levels, and abdominal fat accumulation in mice. The purpose of this study was to explore the underlying mechanism of PM using microarray analysis. Male mice (C57BL/BL strain) were fed an experimental diet (50% fat energy) containing 5% PM isolated from Euglena gracilis EOD-1 for 12 weeks. After confirming that PM had an improving effect on lipid metabolism, we assessed ileal and hepatic mRNA expression using DNA microarray and subsequent analysis by gene ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The results suggested that dietary supplementation with PM resulted in decreased abdominal fat accumulation and serum LDL cholesterol concentrations via suppression of the digestion and absorption pathway in the ileum and activation of the hepatic PPAR signaling pathway. Postprandial glucose rise was reduced in mice fed PM, whereas changes in the glucose metabolism pathway were not detected in GO classification and KEGG pathway analysis. PM intake might enhance serum secretory immunoglobulin A concentrations via promotion of the immunoglobulin production pathway in the ileum

Effects of β-glucan Rich Barley Flour on Glucose and Lipid Metabolism in the Ileum, Liver, and Adipose Tissues of High-Fat Diet Induced-Obesity Model Male Mice Analyzed by DNA Microarray

We evaluated whether intake of β-glucan-rich barley flour affects expression levels of genes related to glucose and lipid metabolism in the ileum, liver, and adipose tissues of mice fed a high-fat diet. C57BL/6J male mice were fed a high-fat diet supplemented with high β-glucan barley, for 92 days. We measured the expression levels of genes involved in glucose and lipid metabolism in the ileum, liver, and adipose tissues using DNA microarray and q-PCR. The concentration of short-chain fatty acids (SCFAs) in the cecum was analyzed by GC/MS. The metabolic syndrome indices were improved by barley flour intake. Microarray analysis showed that the expression of genes related to steroid synthesis was consistently decreased in the liver and adipose tissues. The expression of genes involved in glucose metabolism did not change in these organs. In liver, a negative correlation was showed between some SCFAs and the expression levels of mRNA related to lipid synthesis and degradation. Barley flour affects lipid metabolism at the gene expression levels in both liver and adipose tissues. We suggest that SCFAs are associated with changes in the expression levels of genes related to lipid metabolism in the liver and adipose tissues, which affect lipid accumulation

Multi-omics approach reveals post-transcriptionally regulated genes are essential for human pluripotent stem cells

ヒトのiPS細胞やES細胞の生存に必要な遺伝子の発見 --これまで見過ごされていた、タンパク質レベルで量が制御されている遺伝子群の制御機構解明に向けて--. 京都大学プレスリリース. 2022-05-13.Discovery of genes required for survival of human iPS and ES cells. 京都大学プレスリリース. 2022-05-16.The effects of transcription factors on the maintenance and differentiation of human-induced or embryonic pluripotent stem cells (iPSCs/ESCs) have been well studied. However, the importance of posttranscriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 posttranscriptionally regulated genes with strict upregulation of the protein level in iPSCs/ESCs. Among them, we found 84 genes were vital for the survival of iPSCs and HDFs, including 20 genes that were specifically necessary for iPSC survival. These 20 proteins were upregulated only in iPSCs/ESCs and not in differentiated cells derived from the three germ layers. Although there are still unknown mechanisms that downregulate protein levels in HDFs, these results reveal that posttranscriptionally regulated genes have a crucial role in iPSC survival

A stress-reduced passaging technique improves the viability of human pluripotent cells.

Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, reproducibility issues, often arising from variability during passaging steps, remain. Here, we describe an improved method for the subculture of human PSCs. The revised method significantly enhances the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing and directed differentiation. Furthermore, the method does not alter PSC characteristics after long-term culture and attenuates the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application

Critical Roles of Translation Initiation and RNA Uridylation in Endogenous Retroviral Expression and Neural Differentiation in Pluripotent Stem Cells

Previous studies have suggested that the loss of the translation initiation factor eIF4G1 homolog NAT1 induces excessive self-renewability of naive pluripotent stem cells (PSCs); yet the role of NAT1 in the self-renewal and differentiation of primed PSCs is still unclear. Here, we generate a conditional knockout of NAT1 in primed PSCs and use the cells for the functional analyses of NAT1. Our results show that NAT1 is required for the self-renewal and neural differentiation of primed PSCs. In contrast, NAT1 deficiency in naive pluripotency attenuates the differentiation to all cell types. We also find that NAT1 is involved in efficient protein expression of an RNA uridyltransferase, TUT7. TUT7 is involved in the neural differentiation of primed PSCs via the regulation of human endogenous retrovirus accumulation. These data demonstrate the essential roles of NAT1 and TUT7 in the precise transition of stem cell fate

Rapid and Deep Profiling of Human Induced Pluripotent Stem Cell Proteome by One-shot NanoLC–MS/MS Analysis with Meter-scale Monolithic Silica Columns

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography–tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98 977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more “transcription regulation”-related proteins, while the fibroblast group contained more “transport”-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis

Rapid and Deep Profiling of Human Induced Pluripotent Stem Cell Proteome by One-shot NanoLC–MS/MS Analysis with Meter-scale Monolithic Silica Columns

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography–tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98 977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more “transcription regulation”-related proteins, while the fibroblast group contained more “transport”-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis

Mentha x rubra

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography–tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98 977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more “transcription regulation”-related proteins, while the fibroblast group contained more “transport”-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis