Phase contrast microscopy

FACS-sorted CD44^high/CD44^low CAF cells (5000 cells/ml) were cultured in ultra-low attachment plates (Corning) in serum-free DMEM-F12 (Life Technologies) containing stem cell culture supplements. After 10 days in culture, spheres >50 μm were counted under phase-contrast microscopy (Olympus), and the size and number of spheroids were calculated from 5 random fields using Image J (images.nih.gov). The number of CD44^high spheres >50 μm formed under the sphere culture condition was 8–12 per field, while the number of CD44^low spheres formed >50 μm was 0–2 per field. The spheres less than 50 μm were not counted. Three independent experiments were conducted.

Passage 3 (P3) MSCs, P10 MSCs, and P10 MSCs (knockdown by c-Cbl LNA Gapmers) were cultured in the EC basal medium containing 0.75% fetal bovine serum (FBS) without growth factors for 24 h. The conditioned culture medium was collected and stored at 4°C for subsequent experiments.
Ninety-six-well plates were coated with 50 µl growth factor reduced Matrigel (Biocoat Incorporation, NY, USA) per well. HUVECs were randomly divided into five groups, which were suspended in different culture medium at the density of 1 × 10⁵/ml, including the positive control group (suspended in EC complete medium), negative control (NC) group (suspended in EC basal medium supplemented with 0.75% FBS alone), P3 group (suspended in P3 conditioned culture medium), P10 group (suspended in P10 conditioned culture medium), P10 + SCR group (suspended in P10 + scramble LNA Gapmers conditioned culture medium), and P10 + c-Cbl KD group (suspended in P10 + c-Cbl LNA Gapmers conditioned culture medium). Each group was added with 100 µl cells per well and incubated at 37°C with 5% CO₂. After incubation for 8 h, tube formation was captured with phase-contrast microscopy (Olympus, Tokyo, Japan) and three fields of each picture were randomly selected for further analysis with ImageJ software.

The senescence of BM-MSCs was determined by using an senescence associated β-galactosidase (SA-β-Gal) staining kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, BM-MSCs that were cultured in a six-well plate were washed with PBS and then fixed in fixative solution for 15 min at room temperature. After that, each well was added with 1 ml staining working solution and maintained at 37°C without CO₂ for 12 h. The SA-β-gal-positive senescent MSCs were photographed with phase-contrast microscopy (Olympus) and the percentage was calculated from five randomly chosen fields of each well.

Cell motility was determined by measuring the movement of cells to close an artificial wound. Cells were seeded in 24‐well plates at 80% confluence. Cells were wounded with a 200‐μL pipette tip, washed with PBS, and incubated with medium containing 1% FBS. The distance traveled by cells was monitored by phase‐contrast microscopy (Olympus, Tokyo, Japan) at indicated time points.

SC morphology was assessed by phase contrast microscopy (Olympus, Tokyo, Japan) at 100× magnification. Cells obtained by enzymatic digestion were resuspended in SC culture medium. Most cells adhered to the laminin-coated flasks within 48 hours and had one of two distinct shapes corresponding to two cell types: SCs were small, bipolar or tripolar, and refractile, while fibroblasts had a flat, polygonal shape with an oval nucleus and blunt cytoplasmic processes.