Experimental animals

The C57BL/6 male mice (6–8 weeks old) used in this study were purchased from Shanghai Laboratory Animal Center (SLAC), Shanghai, China. Mice were housed in the Laboratory Animal Research Center, Tongji University, and maintained under controlled temperature (22 °C ± 1 °C) and humidity conditions with a 12:12 h light:darkness cycle. Mice were euthanized by cervical dislocation and the death was confirmed by cardiorespiratory arrest. All animal experiments were conducted following the institutional animal ethics guidelines and were approved by the Animal Use Committee of Tongji University (TJAB04523102).

Cell culture

UC-MSCs were cultured in MSC medium (ScienCell #7501) supplemented with 5% FBS, 1 × mesenchymal stem cell growth supplement (MSCGS), and 1 × penicillin/streptomycin (PS) solution.

Construction of MI model and UC-MSC transplantation

Animals were randomly assigned to different cage locations to prevent positional effects. To establish a mouse model of MI, adult male C57BL/6 mice were anesthetized with 2% isoflurane inhalation, followed by ligation of the left main descending coronary artery (LCA), as previously described [24]. Immediately after MI, 2 × 105 UC-MSCs suspended in 30 μL saline were injected intramyocardially into the infarct border zone at three distinct sites. Control MI animals received saline injection only, while sham-operated animals underwent all surgical procedures except for LCA ligation and intramyocardial injection.

In the experiment evaluating the therapeutic effects of MSCs from different donors on MI, 38 adult male C57BL/6 mice were randomly assigned to the following groups: the Sham group (n = 7), the MI + Saline group (n = 7, with 2 mice dying during surgery, 7/9), the MI + MSC-CX group (n = 7, injected with 2 × 105 MSC-CX, with 1 mouse dying during surgery, 7/8), the MI + MSC-CJ group (n = 7, injected with 2 × 105 MSC-CJ), and the MI + MSC-GY group (n = 7, injected with 2 × 105 MSC-GY). A total of 35 mice survived, resulting in a survival rate of 92.1%. After evaluation on day 3, all 28 surviving infarcted mice had an LVEF below 40%.

In the experiment evaluating the therapeutic impact of N-CADHERIN+/CD168− subpopulation within MSCs on MI, 30 adult male C57BL/6 mice were randomly assigned to the following groups: the MI + Saline group (n = 6, with 2 mice dying during surgery, 6/8), the MI + N-CADHERIN+/CD168− MSC-GY group (n = 6, injected with sorted N-CADHERIN+/CD168− cells from MSC-GY, with 2 mice dying during surgery, 6/8), the MI + others group (n = 6, injected with remaining MSC-GY cells excluding the N-CADHERIN+/CD168− subpopulation, with 1 mice dying during surgery, 6/7), and the MI + N-CADHERIN+/CD168− MSC-CX group (n = 6, injected with sorted N-CADHERIN+/CD168− cells from MSC-CX, with 1 mice dying during surgery, 6/7). A total of 24 mice survived, resulting in a survival rate of 80%. After evaluation on day 3, all 24 surviving infarcted mice had a LVEF below 40%.

In both experiments, all surgeries and injections were performed by a single experienced operator in a blinded manner. Additionally, The LVEF measurements and data analyses were conducted with the investigator blinded to the group assignments, ensuring unbiased assessment of cardiac function across all groups.

Assessment of cardiac function

Echocardiography was performed to assess the cardiac function of MI mice 28 days after cell transplantation (Visual Sonics Vevo 2100 system equipped with a 40-MHz 550 s probe). LVEF, fraction shortening, end-diastolic diameter, end-systolic diameter were measured and calculated using cardiac echocardiography software [25].

Masson trichrome staining

Masson trichrome staining was performed as previously described [24, 26]. Briefly, the mouse hearts (4 weeks after MI) were fixed in 4% paraformaldehyde for 24 h at 4 °C and then dehydrated using a sucrose gradient. Hearts were sectioned from below the ligation site to the apex at 8 μm thickness, with samples taken at 500 μm intervals for staining (Yeasen). Infarct size was quantified as the average of five sections using the formula: infarct size = [infarct perimeter (infarct epicardium + infarct endocardium) × 100]/left ventricle perimeter (left ventricle epicardium + left ventricle endocardium).

Immunostaining

Immunostaining was performed as previously described [27]. Fixed slides were blocked for 1 h at room temperature in PBS containing 10% donkey serum and 0.1% Triton X-100. Slides were incubated overnight at 4 °C with primary antibodies, followed by incubation with fluorescent secondary antibodies and Hoechst33342 for nuclear staining at room temperature for 1 h. CD31 and α-SMA positive cells were quantified from three randomly selected sections per slide.

Flow cytometry (FACS)

UC-MSCs were dissociated with TrypLE (Gibco), washed with PBS, and 3 × 105 cells were incubated with 100 μL PBS containing 1 μL CD168 (RHAMM)-FITC antibody (bs-4736R-FITC, Bioss) and 10 μL CDH2 (N-CADHERIN)-PE antibody (11039-R001-PE, Sino Biological) for 30 min at 4 °C. Cells were analyzed using a BD FACSVerse, and data were processed with FlowJo software. For sorting of the N-CADHERIN+/CD168− MSC subpopulation. MSCs were stained with CD168 (RHAMM)-FITC antibody (4 μL/106 cells) and CDH2 (N-CADHERIN)-PE antibody (30 μL/106 cells) for 30 min at 4 °C. After washing with PBS, the N-CADHERIN+/CD168− subpopulation was sorted by BD FACSAria II.

Tracing of MSCs

MSCs were transfected with lipophilic tracker-DiR (DiIC18(7)) (APEx BIO, B8806; 10 μM in PBS) labeling for 20 min before implantation into the hearts. At day 1 and 7 after transplantation, mice were anesthetized with isoflurane and photographed with an IVIS Lumina XRMS Series III instrument. The region of interest (ROI) of DiR-labeled MSCs was measured using living image4.4 software (Radiant Efficiency).

Production of UC-MSCs conditioned medium

UC-MSCs (2 × 106 cells) from different donors were cultured in 10 cm dishes until 80–90% confluent. Cells were washed with PBS and switched to low glucose DMEM. After 5 days, supernatants were collected and stored at 4 °C for further use.

Tube formation assay

Tube formation assay was performed as previously described [22]. Basement membrane matrix (Matrigel®, BD Biosciences) was solidified in 24-well plates at 37 °C for 30 min. HUVECs (8 × 104 cells/well) were plated on Matrigel in 1640 medium with 10% FBS and 30% conditioned medium from UC-MSCs. Tube formation was imaged after 8 h, and structures were quantified using Image J.

Cell migration assay

HUVECs (4 × 104 cells/well) were cultured in 24-well plates for 24 h. After creating a scratch, cells were cultured in RPMI 1640 medium with 30% UC-MSC conditioned medium for 16 h. Images of migrated cells were taken at 0, 8, and 16 h, and the migration rate was calculated using Image J.

Cell proliferation assay

HUVECs (8 × 104 cells/well) were cultured in 12-well plates in RPMI 1640 medium with 30% UC-MSC conditioned medium for 48 h. Cell numbers were counted at 0, 24, and 48 h using CounterStar software.

scRNA-seq analysis

Single-cell RNA sequencing was conducted using 10 × Genomics technology. Data were processed into a Seurat object, filtering out cells with fewer than 200 features or more than 5% mitochondrial gene expression. Data normalization and feature selection identified 2,000 highly variable features. Principal Component Analysis (PCA) reduced dimensionality to 30 components, which were used for clustering at a resolution of 0.25. UMAP and t-SNE techniques visualized cell populations.

Statistical analysis

All data were presented as mean ± S.E.M. of three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test for more than two groups (GraphPad Prism software). The significance levels were set at *, #P < 0.05, **, ##P < 0.01, and ***, ###P < 0.001. The work had been reported in line with the ARRIVE guidelines 2.0.