Fibroblast growth factor basic

The sphere-forming ability of colorectal tumor cells was evaluated by sphere formation assay as previously reported [22 (link)]. Briefly, one single tumor cell was sorted into one well of the Corning® Costar® ultra-low attachment 96-well plate (Sigma-Aldrich), which was filled with 200 μL of Dulbecco’s modified Eagle medium/F12 medium supplemented with 20 μg/mL fibroblast growth factor-basic, 1× B27, and 20 μg/mL epidermal growth factor (Life Technologies, Waltham, USA). Cells were cultivated in a 5% CO₂/37 °C for 7 days. The number of spheres with a diameter of >50 μm was counted and normalized to the Control group.

The human CRC cell line HCT-116 was obtained from American Type Cell Collection (ATCC, Manassas, VA, USA), and maintained in RPMI medium (ATCC) supplemented with 5% heat inactivated fetal bovine serum (FBS; Mediatech, Herndon, VA, USA), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were grown in a humidified incubator containing 5% CO₂ and 95% air at 37 °C, kept sub-confluent, and medium was changed every other day. All cells were assayed within 5–25 passages. Enrichment culture of tumor-derived colon-spheres was performed by incubating parental HCT-116 cells in serum-free medium (SFM) composed of DMEM/F-12 medium supplemented with 2% B-27 supplement, 20 ng/mL recombinant human epidermal growth factor, 10 ng/mL fibroblast growth factor-basic (Life Technologies, Grand Island, NY, USA), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 10 μg/mL insulin (Sigma-Aldrich) in ultra low-attachment plates (Corning, Lowell, MA, USA) at 37 °C. Plated under these anchorage-independent conditions in supplemented-SFM, tumor cells form floating spheres reported to represent the growth of CSC [27 (link),31 (link),54 (link)].

Fresh human bone marrow aspirate was purchased from Lonza (donor 18‐year‐old black female) and the hMSCs were isolated based on preferential adhesion to TCPS plates, using previous published protocols.20, 28 Freshly isolated hMSCs (P0) were detached with 0.05% trypsin–EDTA (Sigma) and subsequently centrifuged, counted, and frozen down in 80% fetal bovine serum (FBS; Invitrogen) and 20% dimethylsulphoxide and stored in liquid nitrogen. For passaging, hMSCs were cultured for 3 days on TCPS at an initial density of 4,000 cells/cm² in expansion media, detached with 0.05% trypsin‐EDTA, centrifuged, and replated at the same density. Expansion media consisted of low glucose (1 ng/mL glucose) Dulbecco's Modified Eagle Medium (ThermoFisher) supplemented with 10% FBS (ThermoFisher), 1 ng/ml fibroblast growth factor basic (Life Technologies), 50 U/ml penicillin (ThermoFisher), 50 μg/ml streptomycin (ThermoFisher), 0.5μg/ml of Amphotericin B (ThermoFisher). This method was repeated to generate desired passage numbers. For subsequent analyses, cells at desired passage numbers (P2 for early, P5–P7 for middle, and P11–P12 for late) were frozen in cell freezing medium (Sigma) and stored in liquid nitrogen.