Cell culture models for SARS-CoV-2 infectivity and systemic complications

COVID-19 was declared by WHO as a pandemic since March 2020. The vaccination program has been implemented worldwide. Specific antiviral drugs such as remdesivir, molnupiravir and ritonavir-based nirmatrelvir were effective against SARS-CoV-2 infection. However, the new SARS-CoV-2 variants have been elevated due to viral mutation causing vaccine resistance and rapid spreading. Long-term COVID-19 complications have been life-threatening in some recovery cases. To overcome viral adaptation, cell culture model is essential to comprehend SARS-CoV-2 infection, pathophysiology, complications, and drug target alterations. The classical 2D culture cell was frequency used for viral propagation and high-throughput screening. Modern 3D culture has recapitulated key cellular and molecular events of tissue physiology. Here, we reviewed the cell lines, 3D culture, organoid and relevant models for the aforementioned applications

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

<p>Abstract</p> <p>Background</p> <p>The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.</p> <p>Results</p> <p>The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.</p> <p>Conclusion</p> <p>The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.</p

Proteomic Profiles of Mesenchymal Stem Cells Induced by a Liver Differentiation Protocol

The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs

Coagulant activity of recombinant human factor VII produced by lentiviral human F7 gene transfer in immortalized hepatocyte-like cell line.

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into hepatocyte-like cells, indicating that these cells may be the new target cell of interest to produce biopharmaceuticals. Our group recently established a hMSC-derived immortalized hepatocyte-like cell line (imHC) that demonstrates several liver-specific phenotypes. However, the ability of imHC to produce coagulation factors has not been characterized. Here, we examined the potential for imHC as a source of coagulation protein production by investigating the ability of imHC to produce human factor VII (FVII) using a lentiviral transduction system. Our results showed that imHC secreted a low amount of FVII (~22 ng/mL) into culture supernatant. Moreover, FVII from the transduced imHC (0.11 ± 0.005 IU/mL) demonstrated a similar coagulant activity compared with FVII from transduced HEK293T cells (0.12 ± 0.004 IU/mL) as determined by chromogenic assay. We demonstrate for the first time, to the best of our knowledge, that imHC produced FVII, albeit at a low level, indicating the unique characteristic of hepatocytes. Our study suggests the possibility of using imHC for the production of coagulation proteins

Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3⁺CD56⁺ Subset through the Co-Culturing Dendritic Cells

<div><p>Cytokine-induced killer (CIK) cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs). We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6) in DCs at the expense of Th2 inducing phenotype (IL-13) and regulatory phenotype (PD-L1, IDO). Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet) and the downregulation of the Th2 signature (GATA-3) and the Th17 marker (RORC) on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.</p></div

The FACS analysis for the maturity of macrophages, iDCs, and mDCs after sunitinib exposure.

<p>The markers for the maturity of DCs (A) are CD80, CD83 and CD86. The macrophage markers (B) are CD14 and CD40. The selected immunosuppressive molecules (C) are IDO and IL-10.</p

Additional file 1: of A robust model of natural hepatitis C infection using hepatocyte-like cells derived from human induced pluripotent stem cells as a long-term host

Primer sets and conditions used in quantitative real-time PCR (qPCR). (DOCX 15 kb

Suite de la defense de l'Esprit des loix, ou Examen de la replique du gazetier ecclesiastique a la Defense de l'Esprit des loix

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