Ethics declarations
This study was approved by the institutional regulations of the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval No. K202110-18). This study was conducted in accordance with the ARRIVE guidelines.
Isolation, culture, and identification of hADSCs
Human subcutaneous adipose tissue was obtained from the Department of Cosmetic Plastic Surgery at the First Affiliated Hospital of Xinjiang Medical University.Written informed consent was obtained from all volunteers prior to participation and all experiments were performed in accordance with relevant guidelines and regulations. A previously described ADSC isolation protocol was used20. Briefly, sterile adipose tissue was washed with phosphate-buffered saline (PBS) solution, then digested with 1 mg/mL type I collagenase (Worthington, USA) at 37 ℃ for 30 min. Blood vessels and connective tissue were removed from the adipose tissue with sterile tweezers and then the mixture was centrifuged at 1500 rpm for 10 min. The supernatant was discarded and cells were seeded in cell culture dishes and grown in low-glucose Dulbecco’s Modified Eagles Medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin in an incubator with a 5% CO2 atmosphere. The medium was replaced every other day. Cells were digested and passaged with 0.25% trypsin (Gibco, USA) until a confluence of 80–90% was reached. First-passage cells were labeled as P1.
The cultured hADSCs were identified by flow cytometry analysis. Third passage (P3) hADSCs were digested with 0.25% trypsin and resuspended in pre-cooled PBS buffer at a concentration of 3 × 106 cells/mL. Aliquots of 1 × 106 cells were incubated in the dark for 30 min at 4 ℃ with fluorochrome antibody, including mesenchymal stem cell markers (CD29-PE, CD44-FITC, and CD90-PE), hematopoietic markers (CD31-FITC, CD34-PE, and CD45-FITC), and a major histocompatibility antigen (HLA-DR). The antibodies used in the experiments are listed in Table S1. The cell suspension washed with 400 µL PBS and detected by flow cytometry (FACS AriaII, BD, USA).
When cultured ADSCs reached 50% confluence, the growth medium was replaced with osteogenic or adipogenic differentiation medium (Weitong, Shenzhen, China) to induce either osteogenesis or adipogenesis and to determine the ability of ADSCs to differentiate into mesodermal cell types (adipogenic and osteogenic cells). The differentiation medium was changed once every 2–3 d according to the manufacturer’s instructions. Oil red O staining was performed 10 d after adipogenic differentiation and Alizarin Red staining was performed 21 d after osteogenic differentiation. Lipid deposition and calcium nodules were recorded using a phase-contrast microscope (Leica DM6000B; Germany).
Differentiation of hADSCs into iHEPs in vitro
ADSCs were differentiated into iHEPs using a previously established protocol21. Briefly, second (P2) and third passage (P3) ADSCs were digested with 0.25% trypsin and seeded on the cell culture plates at density of 2 × 104 cells/cm2 and incubated in growth medium at 37 ℃ in a 5% CO2 atmosphere. Hepatogenic induction was adopted as a three-stage differentiation protocol for in vitro differentiation of ADSCs into iHEPs. Briefly, when ADSCs reached 50% confluence, the medium was changed to hepatocyte differentiation medium (HDM) in which serum was removed and supplemented with cytokines according to induction stage. Activin AA and FGF-4 (20 ng/mL) were supplemented in the pre-induced stage (days 1–3); EGF (20 ng/mL), HGF (20 ng/mL), and ITS were supplemented in the differentiation stage (days 4–7); and OSM (40 ng/mL), dexamethasone (1 µmol/L), and DMSO (1‰) were supplemented during the maturation stage (days 7–21). The medium was changed every 3 d during the induction period. A schematic of the induction protocol is shown in Supplementary Figure S1.
Identification of iHEPs
Morphological changes in the iHEPs at specific differentiation stages were recorded using a phase-contrast microscope (DM6000B; Leica, Germany). The percentage of iHEPs was determined by counting the number of epithelial-like cells and the total number of cells within a specific field of view at 200× magnification.
To assess the hepatic function of iHEPs, media from undifferentiated (day 0) and differentiated cells (days 7, 17, and 21) were discarded. The cells were then washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. Hepatic glycogen storage in ADSCs and iHEPs was visualized using a Periodic acid-Schiff (PAS) staining kit (Solarbio, Beijing, China) according to the manufacturer’s protocol. The glycogen-stained area was analyzed using Image Pro Plus software (version 6.0).
Hepatic-specific gene analysis for ALB, HNF-1B, Transferrin, ASGPR1, AAT, and MRP2 expression on different days (7, 17, and 21) was performed using quantitative real-time (qRT) PCR. Total RNA from undifferentiated (day 0) cells or iHEPs was isolated using TRIzol reagent, and RNA concentration and purity were determined using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Total RNA (1 µg) was reversed transcribed to single-stranded cDNA using the Prime Script RT reagent Kit (RR047A; Takara, Japan) to a final volume of 20 µL. Quantitative real-time PCR was performed using TB Green Premix Ex Taq (RR820A, Takara, Japan) on an ABI StepOnePlus Real-Time PCR System (Q6; Thermo Fisher Scientific, USA). Relative mRNA expression of targeted genes was calculated using the 2−ΔΔCt method by normalization with GAPDH. Primer sequences used for this assay are listed in Table S2.
Immunofluorescence staining
P3 ADSCs or iHEPs at different stages were washed with pre-cooled PBS buffer and then fixed with 4% paraformaldehyde for 15 min at room temperature. Next, cells were permeabilized and incubated in PBS with 0.01% TritonX-100 for 5 min. Non-specific binding sites were blocked with 3% BSA in PBS for 60 min. Cells were incubated at 4 ℃ overnight with primary antibodies specific for mesenchymal stem cells (CD29, CD44, CD90, and CD105) and hepatocyte markers (ALB). The cells were than washed with PBS for three times and visualized with goat anti-rabbit or chicken secondary fluorescence conjugated antibodies (either Alexa Fluor 488 or 596). The antibodies used for immunofluorescence staining are shown in Table S1. Nuclei were visualized by DAPI (Sigma, USA) counterstaining. The cells were observed using a laser-scanning confocal microscope (SP8; Leica, Germany) and quantitatively analyzed using ImageJ software (version 1.52a; NIH, USA).
Parasites and animal experiments
Eight-week-old female C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained on a 12 h/12 h light/dark cycle with ad libitum access to food and water at the Laboratory Animal Center of Xinjiang Medical University.
Grouping and treatment
Mice were randomly divided into three groups (n = 6 per group) as follows: (1) Sham operation + vehicle injection (sham group); mice were injected with 200 µL of normal saline via the hepatic portal vein. (2) E. multilocularis infection + vehicle injection (Em group); mice were inoculated with protoscoleces (PSCs) in saline via the hepatic portal vein. (3) E. multilocularis infection + iHEPs treatment (Em + iHEPs group); mice were infected with E. multilocularis PSCs for 8 weeks, and 5 × 105 DiI-labelled iHEPs in 200 µL PBS was transplanted into the infected mice via the tail vein. Mice in the sham and Em groups were injected with 200 µL PBS via the tail vein. Four weeks after transplantation, mice in each group were sacrificed to collect liver and blood samples for further analysis.
Establishment of E. Multilocularis-infected mouse model
We isolated E. multilocularis PSCs from Mongolian gerbils naturally infected for 24 weeks and used them as sources of E. multilocularis, as previously described22. Briefly, PSCs were counted and adjusted to a concentration of 10,000 PSCs/mL after washing several times with PBS. Parasite viability was determined using eosin staining. Only PSC batches with 95% viability were used. Mice were gas-anesthetized with 2% isoflurane and kept under anesthesia during the procedure. PSCs in saline (200 µL; 2000 PSCs per mouse) were injected via the hepatic portal vein. The sham group was injected with an equivalent volume of saline.
Transplantation of DiI-labelled iHEPs into E. Multilocularis-infected mice
Cell labeling and tracking in the livers of E. multilocularis-infected mice have been described in our previous study23. Briefly, iHEPs were harvested and resuspended in DMEM supplemented with DiI (5 µg/mL) and labeled for 30 min in the dark at room temperature on a rotatory shaker at 110 rpm. The labeling efficiency and cell viability were assessed by fluorescence microscopy and trypan blue staining. DiI-labelled iHEPs (5 × 105 cells) suspended in 200 µL PBS buffer were injected into the tail vein of mice that were previously infected with EmPSCs for 8 weeks. Mice in the Em and sham groups were injected with 200 µL PBS. All mice were sacrificed by inhalation of pure CO2 at 4 weeks post-transplantation. Liver and blood samples were collected for further examination.
Detection of ALT and AST in serum
Blood samples were harvested and centrifuged at 3000 rpm for 15 min, and serum was collected to measure alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels using commercial detection kits (Mindray, Shenzhen, China) according to the manufacturer’s instructions.
Histopathological examination and immunohistochemical staining
The liver samples from the sacrificed mice were carefully checked for hepatic lesions as a preliminary assessment of the severity of E. multilocularis infection. The liver samples were weighed, and the area of hepatic parasitic lesions was measured and recorded in square millimeters2. Each liver sample was subsequently divided into two parts: liver samples allocated for histopathological and immunohistochemical analyses, and liver samples frozen in liquid nitrogen for RNA and protein extraction. The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4-µm thick sections. The sections were then subjected to hematoxylin and eosin (H&E) staining to assess inflammatory cell infiltration and Masson or Sirius red staining to evaluate the liver fibrosis stage.
For frozen samples, the liver samples were cryoprotected with 30% sucrose solution and fixed in 4% paraformaldehyde for 4 h, then embedded in Optimal Cutting Temperature compound (OCT, Leica, Germany) to prepare 10-µm thick cryosections using the CM1850 cryostatmodel (Leica, Wetzlar, Germany).
Paraffin sections were deparaffinized with water and Sirius red or Masson staining was performed to evaluate collagen deposition using a commercially available staining kit (G1340, Solarbio, China) according to the manufacturer’s instructions. The extent of fibrosis was analyzed at 200× magnification in three fields of view per section per sample. The area of positive staining was quantitatively analyzed using Image-Pro Plus software (version 6.0.0.260; Media Cybernetics, USA).
Immunohistochemical staining was performed according to a previously reported protocol24. Briefly, the sections were deparaffinized and processed with citric acid buffer (ZSGB-BIO, Beijing, China) for heat-mediated antigen-retrieval. Sections were blocked with PBS containing 10% goat serum for 1 h at room temperature and incubated at 4°C overnight with primary antibodies (rabbit anti-mouse PCNA, 1:2000; rabbit anti-mouse CD31, 1:50; and rabbit anti-mouse α-SMA, 1:1000) in PBS buffer. Subsequently, the sections were incubated with goat anti-rabbit-HRP antibody for 30 min at 37°C. The 3, 3’ diaminobenzidine (DAB) substrate kit (ab64238, Abcam) was then used to stain the sections according to the manufacturer’s instructions. Images were captured using a light microscope (DM3000; Leica, Germany), and positively stained cells and microvessel density was analyzed at a magnification of 200× in three to five fields of view per section per sample by manual counting. The average optical density (AOD = IOD/Area) of α-SMA were quantified to determine the differences in each group. The antibodies used for immunohistochemical staining are listed in Table S1.
qRT-PCR analysis
Total RNA was extracted from the mouse liver tissue of each group using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA was subjected to reverse transcription to form cDNA, and qRT-PCR was performed as previously described for the in vitro experiment (identification of induced hepatocytes). Primer sequences for target genes (β-catenin) are listed in Table S2.
Western blot analysis
Total proteins from the liver tissues were extracted using RIPA lysis buffer supplemented with PMSF (Invitrogen). Proteins were quantified using a BCA Protein Assay Kit (23225; Thermo Fisher Scientific, USA). Equal amounts of protein lysate (20 µg) were separated by 10% sodium dodecyl sulfate and transferred to 0.22-µm polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking membranes with 5% skim milk in TBST for 1 h. The proteins were incubated with primary antibodies (anti-β-catenin, 8480 S, CST, anti-GAPDH, ab181602, and Abcam) overnight at 4 °C, followed by incubation with secondary antibodies (Goat anti-rabbit IgG H&L/HRP, ZB2301, and ZSGB-BIO; Beijing, China) for 1 h at room temperature. Protein detection was performed using an enhanced chemiluminescence western blotting reagent (BL520A; Biosharp, Anhui, China). We normalized the protein density data to GAPDH levels.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0 (version 8.0.1; CA, USA). The normal distribution of the data was assessed using the Shapiro–Wilk (SW) normality test, with all samples yielding a P-value > 0.05. Results are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to compare the means across three groups or more, with statistical significance set at *P < 0.05; **P < 0.01; ***P < 0.001.
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- Source: https://www.nature.com/articles/s41598-024-77555-8