Human placenta mesenchymal stem cell-derived exosomes delay H2O2-induced aging in mouse cholangioids

Abstract

Background: Cholangiocyte senescence is an important pathological process in diseases such as primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Stem cell/induced pluripotent stem cell-derived exosomes have shown anti-senescence effects in various diseases. We applied novel organoid culture technology to establish and characterize cholangiocyte organoids (cholangioids) with oxidative stress-induced senescence and then investigated whether human placenta mesenchymal stem cell (hPMSC)-derived exosomes exerted a protective effect in senescent cholangioids.

Methods: We identified the growth characteristics of cholangioids by light microscopy and confocal microscopy. Exosomes were introduced concurrently with H₂O₂ into the cholangioids. Using immunohistochemistry and immunofluorescence staining analyses, we assessed the expression patterns of the senescence markers p16^INK4a, p21^WAF1/Cip1, and senescence-associated β-galactosidase (SA-β-gal) and then characterized the mRNA and protein expression levels of chemokines and senescence-associated secretory phenotype (SASP) components.

Results: Well-established cholangioids expressed cholangiocyte-specific markers. Oxidative stress-induced senescence enhanced the expression of the senescence-associated proteins p16^INK4a, p21^WAF1/Cip1, and SA-β-gal in senescent cholangioids compared with the control group. Treatment with hPMSC-derived exosomes delayed the cholangioid aging progress and reduced the levels of SASP components (i.e., interleukin-6 and chemokine CC ligand 2).

Conclusions: Senescent organoids are a potential novel model for better understanding senescence progression in cholangiocytes. hPMSC-derived exosomes exert protective effects against senescent cholangioids under oxidative stress-induced injury by delaying aging and reducing SASP components, which might have therapeutic potential for PSC or PBC.

Keywords: Cholangiocytes; Exosomes; Organoids; Primary sclerosing cholangitis; Senescence.

Fig. 1

Murine cholangiocyte organoids originating from mature cholangiocytes. a Schematic of cholangioid development. Primary cholangiocytes collected from wild-type C57BL/6 mice by liver duct cell digestion were embedded in Matrigel and covered with expansion medium, allowing them to grow into organoids. b Representative image of long-term cultured cholangioids (P1, passage 1; P2, passage 2; P4, passage 4) (scale bar, 50 μm). c Confocal images of cholangioids expressing specific cholangiocyte markers CK19 (green) and CK7 (red), with blue DAPI staining (scale bar, 20 μm). Left: organoids in two-dimensional form; right: organoids in 3D form. d CK19-positive (green) cholangioids expressing proliferating protein Ki67 (red) (scale bar, 50 μm). e Representative immunohistochemistry images of epithelial cell adhesion molecule (EpCAM) in cholangioids (scale bar, 50 μm)

Fig. 2

Features of long-term cultured cholangioids. a Organoids (P1 day 12) observed by light microscopy. Cells scattered inside and outside the organoid lumen (top, right bottom); shrunken organoids (left bottom) (scale bar, 100 μm). b CK7-positive cholangiocytes within organoids (P0 day 11). Cells remained close together and developed into more than one layer, as observed by confocal laser scanning microscopy (scale bar: left, 50 μm; right, 100 μm). c SA-β-gal staining of cholangioids (P1 day 12), some senescent cholangiocytes (green, top) and completely senescent cholangioids (bottom) (scale bar, 100 μm). d Viability/cytotoxicity assay of organoids (P1 day 4). Dead cells (red) aggregated inside the organoid lumen (green) (scale bar, 50 μm). e Organoids (P1 day 2) observed by light microscopy. White arrow indicates single cholangiocyte, and black arrow indicates organoids grown from single cholangiocytes (scale bar, 200 μm)

Fig. 3

Establishment of senescent cholangioids by H₂O₂ treatment. a Schematic of the treatment of expansion medium (EM) containing 50 nM H₂O₂ was classified into the Sen group. The Ctrl group was treated with EM only. Medium was changed at intervals of 24 or 48 h. b Representative light microscopy image of H₂O₂-stimulated organoids, showing cytological features characteristic of senescence (reduced size, darkened spheroids) (scale bar, 200 μm). c Typical organoids in the Sen and Ctrl groups expressing cell cycle arrest protein p21^WAF1/Cip1 (red) after exposure to oxidative stress for 120 h; CK19 protein was stained cyan and cell nuclei were stained blue (scale bar, 50 μm). d SA-β-gal-positive organoids (green) in the Sen and Ctrl groups following oxidative stress induction for 120 h, observed by light microscopy (scale bar, 100 μm). e mRNA expression levels of SASP components and chemokines (IL-6, CCL2, CXCL2, CXCL16, and CX3CL1) increased significantly at 120 h, compared with 24, 48, and 96 h. Data are presented as mean ± standard deviation (SD) (***P < 0.001, **P < 0.01, n = 4; ordinary one-way ANOVA)

Fig. 4

Exosomes were derived from hPMSCs and co-cultured with organoids. a Schematic representation of exosome collection. b Western blotting: hPMSC-derived exosomes expressing exosome-specific markers CD9 and CD81. c hPMSC-derived exosomes observed by transmission electron microscopy (scale bar, 200 nm). d Schematic of exosome treatment. Organoids treated with expansion medium (EM) containing 50 nM H₂O₂ and 0.1 μg/ml exosomes were classified into the Exo group. Cholangioids were collected and analyzed at 48, 72, and 120 h. e Presence of the exosome-specific marker CD9 (red) in cholangioids (CK19, green) of the Exo group after culture for 72 and 120 h, demonstrated by immunofluorescence analysis (scale bar, 100 μm)

Fig. 5

hPMSC-derived exosomes have a protective effect in senescent cholangioids. a Comparison of organoids among the Ctrl, Sen, and Exo groups at 0 h (top), 48 h (middle), and 120 h (bottom). Typical senescent organoids at 120 h are indicated by white circles (scale bar, 200 μm). b Comparison of the immunohistochemical staining results for proliferation marker Ki67, as well as the senescence-associated markers p16^INK4a and p21^WAF1/Cip1 among the Ctrl, Sen, and Exo groups at 120 h (× 40 objective, scale bar, 200 μm; rectangle indicates × 80 objective). c–e Semi-quantitative analysis of p16^INK4a, p21^WAF1/Cip1, and Ki67 immunohistochemical staining results at 120 h. These data were acquired with a × 20 objective, and Image-Pro Plus software was used to analyze 10 randomly selected areas (two-tailed t test, mean ± standard error of the mean, n = 10, ***P < 0.001, **P < 0.01, *P < 0.05). f Percentages of SA-β-gal-positive organoids in 30 randomly selected areas, counted under a × 20 objective with bright field illumination (two-tailed t test, mean ± SD, n = 30, ***P < 0.001, **P < 0.01). g Representative CK19-positive cholangioids (cyan) in 3D form. The area of p21^WAF1/Cip1-positive staining was greater in the Sen group than in the other two groups, while the Exo group showed the smallest area (scale bar, 100 μm). h SA-β-gal-positive cholangioids. Organoids were collected and analyzed at 120 h (green, scale bar, 50 μm)

Fig. 6

Exosomes reduced the expression of SASP components and chemokines in senescent cholangioids. a mRNA expression levels of SASP components (IL-6, CCL2, CXCL2, CXCL9, CXCL16, CX3CL1, and CXCL1), which decreased significantly at 120 h in the Exo group compared with the Sen group. Data are presented as mean ± SD (n = 4). b Fold changes of CXCL10 mRNA expression in organoids at different time points. Data are presented as mean ± SD (n = 4). c Tumor necrosis factor-α protein concentrations in culture supernatant at 24, 48, 96, and 120 h. Data are presented as mean ± SD (n = 3). d–g IL-6, CCL2, CXCL1, and CXCL10 protein concentrations in culture supernatant at 120 h, which were significantly decreased in the Exo group compared with the Ctrl and Sen groups. Data are presented as mean ± SD (n = 3). One-way ANOVA, ***P < 0.001, **P < 0.01, *P < 0.05