Journal: Frontiers in Pharmacology

Article Title: Assessment and Comparison of the Efficacy of Methotrexate, Prednisolone, Adalimumab, and Tocilizumab on Multipotency of Mesenchymal Stem Cells

doi: 10.3389/fphar.2020.01004

Figure Lengend Snippet: Effect of MTX, PSL, adalimumab, and tocilizumab on MSC differentiation into adipocytes. (A) Typical imaging screening panel for quantification of mFABP4 expression. MSCs were seeded on 96-well plates, and 32 fields were captured in each well using a high-throughput image quantitation system. One of 32 fields is shown. (B, C) The titration curve of mFABP4 expression in MSCs treated with (B) (MTX or PSL, and (C) adalimumab or tocilizumab. The average change of fluorescent intensity was obtained from 96 images for each concentration. Results are presented as mean ± SEM. (D) Typical images of lipid droplets analysis in antirheumatic drug-treated MSCs. Following Oil Red O staining, lipid droplets present as red-stained areas. (E, F) Changes in positive area of lipid droplet in MSCs treated with (E) MTX or PSL, and (F) adalimumab or tocilizumab. The red-stained area was calibrated by the number of nuclei. Results are presented as mean ± SEM. mFABP4, mouse fatty acid binding protein; MSC, mesenchymal stem cell; MTX, methotrexate; PSL, prednisolone; SEM, standard error of the mean.

Article Snippet: For observation of MSC multipotency, a Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) was used according to manufacturer’s instructions ( ).

Techniques: Imaging, Expressing, High Throughput Screening Assay, Quantitation Assay, Titration, Concentration Assay, Staining, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Assessment and Comparison of the Efficacy of Methotrexate, Prednisolone, Adalimumab, and Tocilizumab on Multipotency of Mesenchymal Stem Cells

doi: 10.3389/fphar.2020.01004

Figure Lengend Snippet: Osteogenic differentiation potencies of antirheumatic drug-treated MSCs. (A) Typical imaging screening panel for quantification of osteocalcin expression. One of 32 screening panels is shown. (B , C) The titration curve of osteocalcin expression in MSCs treated with (B) MTX or PSL, and (C) adalimumab or tocilizumab. Average change of fluorescent intensity was obtained from 96 images for each concentration. Results are presented as mean ± SEM. (D) Typical images of ALP-activity analysis in antirheumatic drug-treated MSCs. Positive areas present as purple-stained areas (200×; scale bar: 40 μm). (E , F) Changes of relative positive area of ALP-activity assay in MSCs treated with (E) MTX or PSL, and (F) adalimumab or tocilizumab. The purple-stained area was segmented from the background and the change of relative area was quantified. More than four fields per section and an average of five sections for each concentration were used for semiquantitative analysis. Results are presented as mean ± SEM. ALP, alkaline phosphatase; MSC, mesenchymal stem cell; MTX, methotrexate; PSL, prednisolone; SEM, standard error of the mean.

Article Snippet: For observation of MSC multipotency, a Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) was used according to manufacturer’s instructions ( ).

Techniques: Imaging, Expressing, Titration, Concentration Assay, Activity Assay, Staining, ALP Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Assessment and Comparison of the Efficacy of Methotrexate, Prednisolone, Adalimumab, and Tocilizumab on Multipotency of Mesenchymal Stem Cells

doi: 10.3389/fphar.2020.01004

Figure Lengend Snippet: In vitro chondrogenic assessment of antirheumatic agent-treated MSC-spheroids. (A) Micromass formation by antirheumatic agent-treated spheroids after 28 days of induction culture. (B) Typical images of aggrecan and CD44 expression in MSC spheroids. Fixed micromasses were embedded and sectioned on a cryotome. Expression of aggrecan (green) and CD44 (red) were observed using immunohistochemical staining (200×; scale bar: 40 μm). (C–F) Quantification of volume of micromass formed by chondrospheroids treated with (C) MTX, (D) PSL, (E) adalimumab, and (F) tocilizumab. MRI imaging of micromass was acquired under a three-dimensional T2-weighted flash sequence protocol; coronal and sagittal images were collected and reconstructed to obtain the volume of chondrospheroids. Results are expressed as the mean ± standard deviation (SD) (n=5). MSC, mesenchymal stem cell; MRI, magnetic resonance imaging; MTX, methotrexate; PSL, prednisolone.

Article Snippet: For observation of MSC multipotency, a Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) was used according to manufacturer’s instructions ( ).

Techniques: In Vitro, Expressing, Immunohistochemical staining, Staining, Imaging, Sequencing, Standard Deviation, Magnetic Resonance Imaging

Journal: Frontiers in Pharmacology

Article Title: Assessment and Comparison of the Efficacy of Methotrexate, Prednisolone, Adalimumab, and Tocilizumab on Multipotency of Mesenchymal Stem Cells

doi: 10.3389/fphar.2020.01004

Figure Lengend Snippet: Establishment an in vivo chondrogenic drug-screening system. (A) Cartilaginous mass formation in chondrospheroid-engrafted mice. Chondrospheroids with and without scaffold were implanted to NOD/SCID mice preengrafted with healthy-donor derived PBMCs. Four weeks later, cartilaginous particles had formed. (B) Typical images of aggrecan and CD44 expression in MSC spheroids before engrafting and 4 weeks after chondrospheroid transplantation. Expression of aggrecan (green) and CD44 (red) were observed using immunohistochemical staining (200×; scale bar: 40 μm). (C) Cartilage regenerative capability of chondrospheroids. Predifferentiated chondrospheroids were transplanted into a human xenografted RA model. Implanted RA-patient derived synovium, cartilage, and bone were explanted 8 weeks after chondrospheroid transplantation. Hematoxylin and eosin staining was performed on sections; typical images are shown (200×; scale bar: 20 μm; arrow: synovial invasion to cartilage). (D–G) Quantification of volume of cartilaginous particles formed in mice treated with (D) MTX, (E) PSL, (F) adalimumab, or (G) tocilizumab. Antirheumatic agents were continuously subcutaneously infused for 30 days in chondrospheroid-engrafted mice. MRI imaging of explanted micromass was acquired to evaluate the volume of chondrospheroids. Results are described as the median and interquartile range (difference between 25 th and 75 th percentiles) (n=5). MSC, mesenchymal stem cell; MTX, methotrexate; PBMCs, peripheral blood mononuclear cells; PSL, prednisolone.

Article Snippet: For observation of MSC multipotency, a Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) was used according to manufacturer’s instructions ( ).

Techniques: In Vivo, Drug discovery, Derivative Assay, Expressing, Transplantation Assay, Immunohistochemical staining, Staining, Imaging

Journal: Scientific Reports

Article Title: Derivation of Ethnically Diverse Human Induced Pluripotent Stem Cell Lines

doi: 10.1038/srep15234

Figure Lengend Snippet: Ethnically diverse parental fibroblasts and induced Pluripotent Stem Cell Lines.

Article Snippet: Analysis of lineage commitment to differentiation was done using antibodies to OTX2 (ectoderm), SOX17 (endoderm), and Brachyury (mesoderm; 1:100 each) provided in the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN).

Techniques:

Journal: Scientific Reports

Article Title: Derivation of Ethnically Diverse Human Induced Pluripotent Stem Cell Lines

doi: 10.1038/srep15234

Figure Lengend Snippet: ( a ) Schematic of the process flow to generate LTA-PDMS grids used for high throughput embryoid bodies (EBs) templating (top image), a PDMS mold of arrays prepared on a 4 inch wafer (middle), and 5 day time course of EBs formation in 200 μm LTA-PDMS grid wells (bottom image; (N > 400; scale bar is 200 μm). ( b ) EBs characterization by Hoechst, immunocytology for pluripotency markers Sox2 or SSEA-4, and Actin (phalloidin) staining. The F3.5.2 ED-iPSCs EBs shown was templated in a 200 μm well to day 5. Scale bar is 200 μm. ( c ) Representative images of differentiated 200 μm ED-iPSCs EBs using a Human pluripotent stem cell identification kit. Left column image shows bright field of pluripotent ED-iPSCs 2D colonies. Differentiated ED-iPSCs were fixed for ICC analysis at day 4 for the ectoderm and endoderm lineages and at day 3 for the mesoderm lineage. Antibodies used for immunocytology were indicated at the top of each column. Scale bars are 200 μm. ( d ) Colocalization of Hoescht nuclear stain and germ layer specific markers, listed in each image, to the nucleii of differentiated iPSC cells. Negative control is secondary antibody only. Representative images for germ layer commitment are shown for the iPSC line A2.2.2 at day 4 for ectoderm and endoderm and at day 3 for mesoderm. Scale bars are 200 μm.

Article Snippet: Analysis of lineage commitment to differentiation was done using antibodies to OTX2 (ectoderm), SOX17 (endoderm), and Brachyury (mesoderm; 1:100 each) provided in the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN).

Techniques: High Throughput Screening Assay, Staining, Negative Control

Journal: Scientific Reports

Article Title: Consumption of interesterified palm oil leads inflammation of white adipose tissue and triggers metabolic disturbances in mice on a high-fat diet

doi: 10.1038/s41598-024-63488-9

Figure Lengend Snippet: Biochemical analysis of plasma from animals submitted to the experimental protocol.

Article Snippet: The plasma concentrations of insulin (EZRMI-13-K, Millipore, MO, USA), leptin (EZML-82K, Millipore, MO, USA), adiponectin (EK0596, Boster Biological Engineering Co., Ltd., Wuhan, China), interleukin 6 (IL-6, BMS603-2, Invitrogen, CA, USA), tumor necrosis factor (TNFα, #88-7324; Thermo Fisher Scientific, MA, USA), interleukin 1 beta (IL-1β #88-7013A-76, Thermo Fisher Scientific, MA, USA), glucagon-like peptide-1 (GLP-1, #BMS2194, Thermo Fisher Scientific, MA, USA) and glucose-dependent insulinotropic popypeptide (GIP, # EZRMGIP-55K, Millipore, MO, USA) were measured using mouse ELISA kits following the manufacturer's instructions.

Techniques: Clinical Proteomics

Journal: Scientific Reports

Article Title: Consumption of interesterified palm oil leads inflammation of white adipose tissue and triggers metabolic disturbances in mice on a high-fat diet

doi: 10.1038/s41598-024-63488-9

Figure Lengend Snippet: Palm oil and interesterified palm oil in a high-fat diet model caused morphofunctional changes in white adipose tissue. Hypertrophy resulting from the accumulation of lipids increases the production of inflammatory cytokines (IL-6, TNFα, MCP-1) and immune cells (F4-80), in addition to altering the leptin/adiponectin ratio, which is associated with metabolic dysregulation and the increased risk of chronic diseases. When the influx of fatty acids surpasses the storage capacity of adipose tissue, it leads to lipotoxicity and ectopic lipid accumulation. This accumulation promotes functional changes in the liver and pancreas. In liver tissue, lipogenesis (Srepb-1, Fas, Pparγ) is increased, whereas beta-oxidation (Cpt1-α, Pparα, Pgc1-α) is decreased, which further promotes lipid accumulation in the liver and can cause hepatic steatosis. In the pancreas, in addition to lipid accumulation, hyperglycemia caused by the diet causes pancreatic hypertrophy, with an increase in pancreatic mass (Ki-67) and insulin production, exacerbating the worsening of glucose metabolism. Therefore, the consumption of palm oil and interesterified palm oil in a high-fat diet model causes metabolic changes in the metabolism of carbohydrates and lipids, due to morpho-functional changes in adipose tissue and ectopic accumulation in other metabolic tissues (liver and pancreas).

Article Snippet: The plasma concentrations of insulin (EZRMI-13-K, Millipore, MO, USA), leptin (EZML-82K, Millipore, MO, USA), adiponectin (EK0596, Boster Biological Engineering Co., Ltd., Wuhan, China), interleukin 6 (IL-6, BMS603-2, Invitrogen, CA, USA), tumor necrosis factor (TNFα, #88-7324; Thermo Fisher Scientific, MA, USA), interleukin 1 beta (IL-1β #88-7013A-76, Thermo Fisher Scientific, MA, USA), glucagon-like peptide-1 (GLP-1, #BMS2194, Thermo Fisher Scientific, MA, USA) and glucose-dependent insulinotropic popypeptide (GIP, # EZRMGIP-55K, Millipore, MO, USA) were measured using mouse ELISA kits following the manufacturer's instructions.

Techniques: Functional Assay