Ethics statement
All methods were performed in accordance with relevant guidelines and regulations. This study complies with the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the International Peace Maternity and Child Health Hospital (No. 201411, Approval date: 27/10/2014). Written consent forms were signed by the participating subjects.
Isolation and culture of hAESCs
After written informed consent was provided by the donors, samples of human amnion membranes were obtained from healthy mothers who were confirmed to be negative for hepatitis A, B, C and D and HIV-I and Treponema pallidum(TPAB) antibodies, as described in our previous study42. Briefly, amniotic membranes were mechanically isolated from the placenta and immersed in PBS buffer to remove the remaining blood clots. Then, the amniotic membranes were digested with 0.25% trypsin at 37 °C for 20 min. After the membranes were removed, hAESCs were collected by centrifugation for 10 min. For cell culture, complete culture medium was prepared with DMEM/F12 containing 10% knockout serum replacement (KSR), 1% antibiotic–antimycotic, 1 mM sodium pyruvate, 1% nonessential amino acids, 2 mM L–glutamine (Thermo Fisher Scientific, Grand Island, NY, USA) and 10 ng/mL human EGF (Peprotech, Rocky Hill, NJ, USA). hAESCs were incubated in an atmosphere of 5.5% CO2 at 37 °C for 3 to 5 days until the cells attached to and filled the dish.
Isolation and culture of hWJMSCs
Human umbilical cords were obtained after full-term birth, and written informed consent was provided by the donors. The procedure was approved by the Institutional Patient and Ethics Committee of the International Peace Maternal and Child Health Hospital, Shanghai Jiao Tong University. The umbilical cords were washed to remove blood and clots and cut into 1.5 cm long pieces to isolate cells from Wharton’s jelly (WJ). Each piece was then cut longitudinally to remove the umbilical artery, vein and umbilical cord epithelium to obtain WJ. The remaining gelatinous tissue around the vessels was collected and crushed into small pieces (1 mm2). After the tissue was allowed to adhere to the culture plate, standard proliferation medium was gently added. After the first cellular outgrowth was detected, the tissue blocks were removed, and the cells were harvested for subsequent passaging. The hWJMSCs were isolated and expanded in an incubator at 37 °C with 5% CO2in MEMα supplemented with 10% FBS, and a 1% antibiotic–antimycotic mixture (Thermo Fisher Scientific, Grand Island, NY, USA), as described in a previous study35.
Flow cytometry
Multiple cell markers (CD31, Invitrogen, Cat# 11-0319-42; CD34, Invitrogen, Cat# 12-0349-41; CD45, Invitrogen, Cat# 11-0459-41; CD324, BioLegend, Cat# 324105; CD326, BioLegend, Cat# 324206; CD90, Invitrogen, Cat# 11-0909-42; CD73, Invitrogen, Cat# 11-0739-41; and CD105, BioLegend, Cat# 800503) were detected via flow cytometry to determine the basic characteristics of hWJMSCs and hAESCs. hWJMSCs and hAESCs were collected after an incubation with 10 ng/mL IFN-γ (Peprotech, Cat# 300-02-100) for 72 h to determine immunogenicity. Then, they were stained with anti-HLA-ABC (Invitrogen, Cat# 11-9983-41), anti-HLA-DR (BioLegend, Cat# 307604), anti-HLA-DQ (BioLegend, Cat# 318104), anti-HLA-G (BioLegend, Cat# 335909), anti-HLA-E (BioLegend, Cat# 342603) and relevant isotype control antibodies according to the manufacturer’s instructions and analysed by flow cytometry (FACS Calibur; BD Biosciences, Franklin Lakes, NJ). Analyses were performed using three biological replicates.
Immunostaining
After fixation with 4% paraformaldehyde in PBS for 15 min, the cells were permeabilized with 0.25% Triton X-100 in PBS for 5–10 min and blocked for 60 min with 5% goat serum. The cells were subsequently incubated for 60 min at room temperature with the following primary antibodies: anti-pancytokeratin antibody (Abcam, ab7753) and anti-E-cadherin antibody (Abcam, ab40772). The cells were then incubated for 2 h at room temperature with the corresponding secondary antibodies: Alexa Fluor 594-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat# 715-586-150) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cat# 711-546-152). Fluorescence images were acquired with a confocal microscope (Zeiss LSM 800, Carl Zeiss).
Liquid chromatography and tandem mass spectrometry (LC–MS/MS)
The cell samples were divided into two groups, hWJMSCs and hAESCs, with three biological replicates (n = 3). All the cells were seeded in culture dishes overnight and cultured for another 48 h before harvest. The culture media were discarded, and the dishes were placed on absorbent paper to drain the medium. The cell surface was subsequently washed with precooled PBS solution 2–3 times to wash away the culture medium. PBS that had been precooled at 4 °C was added to the Petri dish. The dish was gently shaken, and the cells were scraped off with a clean cell scraper (quickly). A pipette was used to transfer the cells into a precooled centrifuge tube. After centrifugation at 300 × g for 5 min, cell pellet was obtained, and the supernatant was removed. Total protein was subsequently extracted in radioimmunoprecipitation assay (RIPA) buffer (Solarbio Life Science, China) supplemented with a protease inhibitor on ice-cold plates. Specifically, 300 L of RIPA working mixture was added, mixed thoroughly, steel balls were added, the samples were ground at a low temperature of 45 Hz for 4 min and the samples were centrifuged at 4 °C for 10 min at 12000 rpm; the samples were ultrasonicated in an ice water bath with a cell crusher for 20 min to fully crack the samples. The mixture was centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was transferred to a new EP tube. Then, part of each sample was used to determine the protein concentration (Enhanced BCA Protein Assay Kit, Beyotime, China). The products were subjected to liquid chromatography‒tandem mass spectrometry (LC‒MS/MS) analysis. TMT was performed and analysed by Luming Biotechnology (Shanghai, China).
Quantitative real-time RT‒PCR
The total RNA was extracted with a Super FastPure Cell RNA Isolation Kit (Vazyme, Nanjing, China) as described in the manufacturer’s instructions. One microgram of RNA was subjected to cDNA synthesis with HiScript III RT SuperMix for qPCR (Vazyme, Nanjing, China) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Quantitative real-time PCR was performed with the Bio-Rad iCycler real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and the primers listed in Supplementary Tables 3, and the analysis was performed in triplicate in at least three independent experiments. Fold changes in the expression level of each gene were calculated using CT values. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize expression levels.
Immunomodulation assay
RAW264.7 cells (ATCC) were seeded in a 6-well plate at a density of 1 × 106 cells/well and incubated overnight. Then, 1 µg/mL lipopolysaccharide (LPS) (Sigma) was used to induce macrophage polarization for 24 h with or without co-culture with hAESCs or hWJMSCs. Then, RAW264.7 cells were collected for a qPCR analysis of proinflammatory factors, including IL-β, IL-6 and TNF-α, according to the manufacturer’s protocol (Lianke Bio). All primer sequences are presented in Supplementary Table 3.
Fibroblast activation assay
Human fibroblast IMR90 (Meisen Cell, Zhejiang, China) were starved overnight at 70% confluency in serum-free medium. The differentiation of myofibroblasts was stimulated with 10 ng/ml TGF-β1 (Abclonal, Wuhan, China), and then the cells were co-cultured with hAESCs or hWJMSCs. Following culture for 24 h, the cells were collected for qPCR (for α-SMA, COL1, CTGF and FN1). All primer sequences are presented in Supplementary Table 3.
Human endothelial cell tube formation assay
The basement membrane matrix Matrigel (Corning, Cat# 354277) was added to a 24-well plate and solidified by incubation at 37 °C for 30 min. Human umbilical vein endothelial cells (HUVECs) (4 × 104 cells per well) were plated onto a 24-well plate containing Matrigel with EGM-2 medium (Lonza, Cat# CC-3156) and incubated at 37 °C for 6 h in a humidified incubator. The experimental groups were co-cultured with hAESCs or hWJMSCs, and the tube structures were evaluated via microscopy. Tube formation was quantified by measuring the number of branches produced.
Wound healing assay
Endothelial cells (ECs) were seeded onto 6-well plates (10 × 104 cells/well) with EGM-2 medium and grown to confluence. A straight line was then scratched with a 1-mL pipette tip to conduct a “wound healing” assay. Subsequently, the experimental groups were co-cultured with hAESCs or hWJMSCs, and the migration area was digitally photographed at 0 and 24 h; the area of the cell-free gap was calculated using ImageJ (version 6.0, NIH). The extent of wound healing (% closure) was determined and reported as a percentage of closure relative to the initial wound size at 0 h.
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- Source: https://www.nature.com/articles/s41598-024-79063-1