Stro 1

Magnetic bead selection was performed according to the manufacturer’s instructions (Life Technologies, Grand Island, NY). Freshly isolated myometrial and fibroid cell suspensions were incubated with biotinylated and conjugated antibodies to CD-44 (BD Biosciences, San Jose, CA) and Stro-1 (R&D systems, Minneapolis, MN), diluted in isolation buffer containing Phosphate Buffered Saline (PBS, Sigma- Aldrich, St. Louis, MO), and supplemented with 0.1% Bovine Serum Albumin (BSA, Sigma- Aldrich, St. Louis, MO) and 2 mM of Ethylene diamine tetraacetic acid, EDTA. Dynabeads FlowComp (Life Technologies, Grand Island, NY) were then added and tubes containing myometrial and fibroid cells were placed in a magnet to separate the candidate Stro-1/CD44 positive (Stro-1⁺/CD44⁺) cells from the supernatant containing non target cells, repeating this step in triplicate to remove any residual beads. Finally, Stro-1⁺/CD44⁺ myometrial (Stro-1⁺/CD44⁺MyoF), fibroid (Stro-1⁺/CD44⁺F) cells, (considered as putative myometrial/fibroid stem cells) and primary myometrial/ fibroid (PrMyoF/ PrF) cells, (categorized by the absence of Stro-1 and CD44), were cultured separately in Dulbecco’s modified Eagle’s medium (DMEM-F12, Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (FBS, Stem Cell Technologies, Canada) and 1% antibiotic-antimycotic (Life Technologies, NY), and plated in collagen coated dishes.

A total of 5 × 10⁵ cells per tube were incubated for 15 min at 4 °C with 5 μL of CD44, CD45, CD73 or CD90 (BD Biosciences, Mountain View, CA, USA), STRO-1 (Invitrogen) or IgG (BD Biosciences), conjugated with the fluorochromes FITC or APC (BD Biosciences). For detection of ALDH1 activity, the Aldefluor kit (Stem Cell Technologies, Durham, NC, USA) was employed according to the manufacture’s protocol. Briefly, a total of 1 × 10⁶ cells were suspended with activated Aldefluor substrate (BODIPY-amino acetaldehyde - BAAA) or the negative control (diethylaminobenzaldehyde - DEAB) for 45 min at 37 °C. Data acquisition was performed on cells in the fifth passage, using the BD FACSAria II flow cytometer. The Aldefluor kit was also used to select the population with high ALDH1 enzymatic activity.

Immunofluorescence (STRO-1 and CD34) staining was used to identify the cells. hPDLSCs (passage 2) cultured for 7 days were fixed in 4 % paraformaldehyde (Millipore Sigma) for 30 min, permeabilized with 0.1 % Triton-X100 (Thermo Scientific), and blocked with 10 % bovine serum albuminin (BSA, Jackson Immunoresearch, West Grove, PA, USA) in PBS for 1 h. Then the cells were incubated with the primary antibody-STRO-1 (1: 50, Invitrogen) and CD34 (1:100, Abcam, Cambridge, MA, USA) for 1 h. After washing with a wash buffer, the cells were incubated with FITC-labeled second antibody (1:500 in PBS, Invitrogen) and protected from light for 1 h. Then the cells were counter-stained with DAPI (1:1000, Invitrogen) for 5 min, and examined using a fluorescence microscope (Eclipse TE-2000S, Nikon).