Fig 1.
Generation of hPFs and acquisition of lymphangiogenic potential.
(A) Representative phase-contrast images of hPFs without stimulation (left) and hPFs stimulated with TNF-α and IL-4 (hPF + CKs, right). (B) Flow cytometry of CD90 and VCAM-1 expression on the cell surface of hPFs and hPF + CKs, with CD90 and VCAM-1 plotted on the x- and y-axes, respectively.The percentages of cells in each quadrant are as follows:・hPFs: CD90 ⁺ VCAM-1⁺ = 15.56%, CD90 ⁺ VCAM-1⁻ = 84.13%, CD90 ⁻ VCAM-1⁺ = 0.06%, CD90 ⁻ VCAM-1⁻ = 0.25%・hPF + CKs: CD90 ⁺ VCAM-1⁺ = 28.45%, CD90 ⁺ VCAM-1⁻ = 70.20%, CD90 ⁻ VCAM-1⁺ = 0.24%, CD90 ⁻ VCAM-1⁻ = 1.11% (C) mRNA expression levels of ADM and VEGFC in hPFs and hPF + CKs determined by RT-qPCR. Data are presented as mean ± SEM. ***P < 0.001, ****P < 0.0001 by unpaired t-test. (D) Tube formation assay using co-culture with HMVECs. Immunofluorescence images: Vimentin (red), VE-Cadherin (green), and Hoechst 33258 (blue).
Fig 2.
Immunogenicity of adult human pulmonary fibroblasts.
Flow cytometry of immune-related markers HLA class I, HLA class II, CD80, and CD86 in hPFs and hPF + CKs.
Fig 3.
Efficacy of allogeneic rPF + CKs in a rat model of pulmonary fibrosis.
(A) Experimental timeline of the allogeneic transplantation study. (B) Representative phase-contrast images of rPFs without stimulation (left) and rPFs stimulated with TNF-α and IL-4 (rPF + CKs, right), captured at ×5 (upper) and ×10 (lower) magnifications. Cytokine stimulation induced morphological elongation in rPFs, particularly evident at higher magnification. (C) Flow cytometry of VCAM-1 expression on the cell surface of rPFs and rPF + CKs.
Fig 4.
Engraftment and therapeutic effects of pulmonary fibroblasts.
(A) IVIS imaging performed one day after cell administration showing pulmonary localization of transplanted fibroblasts. (B) Representative Masson trichrome-stained lung sections from each group (Day 0, saline (Ctrl), rPF, and rPF + CKs). For each group, whole lung images (top row) and corresponding magnified views (bottom row) are shown to highlight differences in fibrosis and tissue architecture. (C) Quantification of fibrotic area. Data are presented as mean ± SEM. Statistical analysis was performed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. *p < 0.05, **p < 0.01 vs. indicated groups.
Fig 5.
Gene expression analysis and Lyve-1 localization in lung tissue.
(A) RT-qPCR analysis of fibrosis- and lymphangiogenesis-related genes in lung tissues. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparison test. (B) Representative double-staining images combining WGA immunofluorescence (green) and Lyve1 ISH (red), with DAPI counterstain (blue). (C) Quantification of ISH signal (Total Intensity) in lung sections following rPF and rPF + CKs treatment.Total fluorescence intensity per image was measured in each group (Day 0, rPF, rPF + CKs, and Ctrl). Sample sizes were as follows: Day 0 = 9 images, Ctrl = 7 images, rPF = 7 images, rPF + CKs = 6 images. Welch’s t-test was performed to compare each group to the Ctrl. *p < 0.05 vs. Ctrl (Welch’s t-test).
Fig 6.
Plasma biomarkers of pulmonary fibrosis.
(A) Effect of allogeneic rPF or rPF + CKs cell treatment on plasma levels of surfactant protein D (SP-D) levels. (B) Effect of allogeneic rPF or rPF + CKs cell treatment on plasma fibrinogen levels.Pulmonary fibrosis was induced in mice (Day 0), followed by treatment with saline (Ctrl), allogeneic pulmonary fibroblasts (rPF), or cytokine-enriched fibroblasts (rPF + CKs).Plasma samples were collected on day 14 and analyzed for fibrinogen concentration using ELISA.Sample sizes (animal numbers) were as follows: SP-D: Day 0 = 8, Ctrl = 8, rPF = 8, rPF + CKs = 8 Fibrinogen: Day 0 = 7, Ctrl = 7, rPF = 7, rPF + CKs = 7.