Journal: Molecular Therapy. Nucleic Acids

Article Title: Ratio of miRNA-29 to miRNA-199 expression coordinates mesenchymal stem cell repair of bleomycin-induced pulmonary injury

doi: 10.1016/j.omtn.2025.102461

Figure Lengend Snippet: Bleomycin-induced lung injury, which is reduced after adipose-derived mesenchymal stem cell instillation (A) Representative μCT transverse and coronal lung sections acquired from aged (22-month-old) male C57BL/6 mice at baseline (left) and 7 days following intratracheal bleomycin (BLM, 2.0 U/kg) administration (right) demonstrating increased lung density and loss of airspaces. (B) Saline treatment did not result in evidence of lung injury on μCT scan at baseline (left) or 7 days post-instillation (right). Histological sections of lung tissue collected at day 21 post-BLM were stained with Masson’s trichrome as described in . (C and D) Representative photomicrographs (20× and 40× magnifications) of lung sections from saline-treated control mice (C) and BLM-treated mice (D). (E) Infusion of adipose-derived mesenchymal stem cells (ASCs) 12 days post-BLM instillation resulted in reduced severity of pulmonary fibrosis (PF). (F) Degree of PF on histological sections was measured by semi-quantitative Ashcroft score as described in . BLM-induced lung injury resulted in increased Ashcroft score compared to saline controls. Infusion with ASCs 12 days post-BLM injury resulted in decreased Ashcroft score. (G) Intratracheal BLM instillation increased lung collagen content as measured by hydroxyproline assays as described in . Mice treated with ASCs on day 12 post-BLM had decreased lung collagen content compared to BLM-only controls. Each data point represents an individual biological replicate (mouse); n = 6–10 mice/group. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (H) Ratio of pAKT to AKT protein expression in lung tissue of mice was quantified by western blot analysis at day 21 post-BLM sacrifice. Aged C57BL/6 mice treated with intratracheal BLM demonstrated increased pAKT/AKT protein expression compared to saline-treated controls. Lungs from mice treated with intravenous infusion of ASCs 12 days post-BLM-induced injury demonstrated decreased expression of pAKT/AKT compared to BLM-only group. Inset shows a representative western blot and β-actin loading control. Data are graphed as individual biological replicates ( n = 6–8 mice/group); ∗ p < 0.05.

Article Snippet: For mesenchymal differentiation potential, the Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s instructions.

Techniques: Derivative Assay, Saline, Staining, Control, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

doi: 10.3389/fimmu.2017.00147

Figure Lengend Snippet: Granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates c-kit and stem cell factor (SCF) expression by BM-derived DCs (BMdDCs) . (A–C) Effect of GM-CSF and cell density on c-kit expression by BMdDCs. BMdDCs were plated in 24-well plates and cultured for 2 days in complete Opti-MEM medium in four different conditions, that is at either 2 × 10 5 or 1.2 × 10 5 cell/well, and either with or without (w/o) GM-CSF at 20 ng/ml, as indicated. Cells were stained with fluorochrome-conjugated monoclonal antibodies (mAbs) and analyzed by flow cytometry. (A) Typical flow cytometric profiles, showing CD40 and MHCII expression by BMdDCs. Numbers represent percentages of cells in the indicated regions. (B) Typical histograms showing c-kit expression by MHCII int CD40 int and MHCII hi CD40 hi BMdDCs, gated as in (A) . Solid lines represent c-kit staining profiles, dashed lines represent isotype control mAb. Numbers indicate c-kit median fluorescence intensity (MFI) values. (C) Summary of c-kit expression results obtained from MHCII hi CD40 hi BMdDCs, gated as in (A) . c-kit MFI from individual samples and average values (bar). (D,E) Effect of GM-CSF on SCF expression by BMdDCs. (D) Cell lysates were prepared from day 0 BMdDCs and BMdDCs cultured for 2 days in 24-well plates at 1.2 × 10 5 cell/well in complete Opti-MEM medium with or w/o GM-CSF at 20 ng/ml, as indicated. SCF protein expression was analyzed by ELISA, testing 25 µg of cell lysate in 100 µl/well. Data are expressed as picograms per milliliter. Individual results from three experiments and average values (bar) are shown. (E) Day 0 and day 2 BMdDCs cultured in 24-well plates at 1.2 × 10 5 cell/well with or w/o GM-CSF at 20 ng/ml were analyzed by Real-Time PCR in triplicates. SCF mRNA expression was calculated relative to hprt1 in arbitrary units. For each experiment, day 2 c-kit/hprt1 levels were normalized with day 0. In (A,B) representative data of N = 4 experiments, in (C) N = 4 experiments, in (D) N = 3 experiments, in (E) mean ± SD of four experiments (* P ≤ 0.05; ** P ≤ 0.01).

Article Snippet: BMdDC culture supernatants (100 μl/well) and BMdDC lysates (25 μg of cell lysate/well) were tested by mouse SCF ELISA kit (Boster Immunoleader, Pleasanton, CA, USA).

Techniques: Expressing, Derivative Assay, Cell Culture, Staining, Bioprocessing, Flow Cytometry, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction