Cd105

BMSCs were flushed from the femurs and tibiae of SD rats with α-minimum essential medium (α-MEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 20% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). Cells were collected and seeded in flasks with α-MEM supplemented with 10% FBS and incubated at 37 °C in a humidified atmosphere of 5% CO₂. The medium was changed every 3 days. To identify the cell phenotype, flow cytometry analysis was performed to examine cell surface markers CD73, CD105, CD90, and CD45 (CD73, eBioscience, 11-0739-41; CD105, eBioscience, 12-1051-81; CD90, eBioscience, 11-0900-81; CD45, eBioscience, 11-0460-82, respectively) according to the recommendation of the International Society for Cellular Therapy [18 (link)]. After 2–3 passages, BMSCs negative for CD45 and positive for CD73, CD105, and CD90 were used.
H9C2 rat cardiomyoblasts were purchased from the Cell Bank of Chinese Scientific Academy (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% FBS.

The standard immunohistochemical test was considered positive when the
fluorescence was high (≥ 50%), medium (≥ 15 < 50%) or low (≥ 5 < 15%) and
negative when the mean fluorescence intensity was very low (< 5%). The
positive pattern antibodies were CD71 (FITC mouse anti-rat/BD Pharmigen, San
Diego, USA), CD73 (Purified mouse anti-rat/BD Pharmigen, San Diego, USA), CD90
(FITC mouse anti-rat/BD Pharmigen, San Diego, USA), CD105 (PE mouse
anti-rat/Life Technology, Carlsbad, CA) and CD106 (Purified mouse anti-rat/BD
Pharmigen, San Diego, USA). The negative pattern antibodies were CD31 (mouse PE
anti-rat/BD Pharmigen, San Diego, USA), CD34 (rabbit FITC anti-rat/Biorbyt,
Saint Louis, USA), CD40 (FITC hamster anti-rat/BD Pharmigen, Saint Louis, USA),
CD44 (RPE mouse anti-rat/AbD Serotec, Kidlington, UK), CD45 (FITC mouse
anti-rat/BD Pharmigen, Saint Louis, USA) and CD11b (Biotin mouse anti-rat/BD
Pharmigen, Saint Luis, USA).
A random sample of mesenchymal stem cells was cultured for differentiation in the
adipogenic, osteogenic and chondrogenic lines. This culture was performed only
to confirm the ability of the cells to differentiate but not to be used in the
experiment. The culture was performed according to a pre-established protocol in
the Molecular Biology and Cell Engineering Laboratory for mesenchymal stem cells
obtained from the adipose tissue. Evaluations were performed in a blind way.

Purity, identity and viability of stromal cells were characterized by flow cytometry as previously described^{39 (link)}. In brief, 5 × 10⁵ cells at passage 1 were resuspended in 50 µL phosphate buffered saline (PBS, Sigma Aldrich). Cells were mixed with 0.5 µL viability dye (Fixable Viability Dye eFluor™ 520, eBioscience, Thermo Fisher Scientifics, Waltham, MA, USA) and antibody mastermix for CD73, CD19, CD14, CD34, CD45, HLA-DR (all Becton Dickinson BD Biosciences, Franklin Lakes, NJ, USA), CD90 (Beckman Coulter, Brea, CA, USA) and CD105 (Life Technologies Corporation, Frederick, MD, USA). For determination of F-heparin internalization, 1 × 10³ cells per cm² were seeded. After 48 hours, cells were incubated for three hours with medium supplemented with 10% pHPL either without heparin, with 2 IU/ml unlabeled heparin (Biochrom) or with 2 IU/ml fluoresceinamine-labeled sodium heparin (F-heparin, PG Research, Tokyo, Japan). After washing cells twice with PBS, 2 × 10⁵ cells were resuspended in 100 µL 7AAD viability dye mastermix (BD). All cells were measured immediately (BD LSRFortessa™A) and results were analyzed with Kaluza Analysis Software (Beckman Coulter).