Measurement of AhR Ligands in the Tissues of Colon Cancer Patients with XRE Luciferase Reporter

The Aryl hydrocarbon receptor (AhR) ligands exhibiting modulating activity represents a new class of anticancer agents that can be directed towards several tumors. We have examined AhR expression in human colon cancer and adjacent non-tumor tissue. AhR expression level was about 2-7 times higher in tumor tissue samples than in the adjacent non-tumor samples (in 82% of all the samples). We were unable to find any increase of ABCG2 expression on the level of the transcription, while the expression of MDR2 was increased in half of the tumors compared to the levels of expression in normal adjacent tissue. We have used FICZ as a potent high affinity ligand of the AhR to calibrate the reporter cell line HEK293T-AhR-luc as a potent high affinity ligand of the AhR. The concentration of xenobiotic response element (XRE) ligands is higher, than in the blood of healthy people in 86% of the patients. The proposed test system will allow the use of the AhR ligand level as an additional diagnostic marker in the treatment of colon cancer

Differentiation of Human Pluripotent Stem Cells Into Definitive Endoderm Cells in Various Flexible Three-Dimensional Cell Culture Systems : Possibilities and Limitations

Publisher Copyright: © Copyright © 2021 Bogacheva, Harjumäki, Flander, Taalas, Bystriakova, Yliperttula, Xiang, Leung and Lou.The generation of human stem cell-derived spheroids and organoids represents a major step in solving numerous medical, pharmacological, and biological challenges. Due to the advantages of three-dimensional (3D) cell culture systems and the diverse applications of human pluripotent stem cell (iPSC)-derived definitive endoderm (DE), we studied the influence of spheroid size and 3D cell culture systems on spheroid morphology and the effectiveness of DE differentiation as assessed by quantitative PCR (qPCR), flow cytometry, immunofluorescence, and computational modeling. Among the tested hydrogel-based 3D systems, we found that basement membrane extract (BME) hydrogel could not retain spheroid morphology due to dominant cell–matrix interactions. On the other hand, we found that nanofibrillar cellulose (NFC) hydrogel could maintain spheroid morphology but impeded growth factor diffusion, thereby negatively affecting cell differentiation. In contrast, suspension culture provided sufficient mass transfer and was demonstrated by protein expression assays, morphological analyses, and mathematical modeling to be superior to the hydrogel-based systems. In addition, we found that spheroid size was reversely correlated with the effectiveness of DE formation. However, spheroids of insufficient sizes failed to retain 3D morphology during differentiation in all the studied culture conditions. We hereby demonstrate how the properties of a chosen biomaterial influence the differentiation process and the importance of spheroid size control for successful human iPSC differentiation. Our study provides critical parametric information for the generation of human DE-derived, tissue-specific organoids in future studies.Peer reviewe

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

BACKGROUND: Cancer stem cells\u27 (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs\u27 subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. METHODS: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). RESULTS: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. CONCLUSION: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment

Thyroid Hormone Effect on the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

Human induced pluripotent stem cells (hiPSCs) hold great potential as an unlimited source for obtaining hepatocyte-like cells (HLCs) for drug research. However, current applications of HLCs have been severely limited by the inability to produce mature hepatocytes from hiPSCs in vitro. Thyroid hormones are one of the hormones that surge during the perinatal period when liver maturation takes place. Here we assessed the influence of thyroid hormone on hepatic progenitor differentiation to HLCs. We analyzed gene and protein expression of early and late hepatic markers and demonstrated the selective activity of thyroid hormone on different genes. Particularly, we demonstrated thyroid hormone-dependent inhibition of the fetal hepatic marker AFP. Our study sheds light on the role of thyroid hormone during liver differentiation and maturation