Hbfgf

Trypsinized resuspended cells were rinsed with N2-supplemented DMEM/F12 (Invitrogen, Carlsbad, CA, USA), containing 20 ng/mL human recombinant epidermal growth factor (hrEGF; Biovision, Atlanta, GA, USA) and 10 ng/mL human basic fibroblast growth factor (hbFGF; Invitrogen). Cells (5 × 10³/well) were seeded in ultra-low attachment 24-well plates (Corning Inc., Corning, NY, USA), and then treated with crude acetone extract of P. granulata, physciosporin, or DMSO (0.01%) 3 h after seeding. The cells were then incubated for 14 days at 37 °C under 5% CO₂. The images of sphere were taken by inverted phase-contrast microscopy (Nikon, Kawasaki, Japan), and the relative sphere formation ability was calculated through the IMT iSolution software (IMT iSolution Inc., Northampton, NJ, USA) measuring the pixel intensity of the sphere area randomly in each plate. Data are presented as the average of three independent experiments.

Human osteosarcoma U2OS cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) and penicillin (100 IU/ml)/streptomycin (100 μg/ml) at 37°C in 5% CO₂.
For sphere culture, U2OS cells (1 × 10³) were suspended in the cancer stem cell-conditioned medium (CSC-CM) composed of serum-free DMEM/F12 (Invitrogen, Carlsbad, CA, USA) with 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml hrEGF (Invitrogen), 20 ng/ml hbFGF (Invitrogen), 2% B27 (Invitrogen), 0.4% BSA (Invitrogen), and 4 μg/ml insulin (Sigma-Aldrich) to form spheres. The sphere-forming U2OS cells obtained from the sphere formation culture were called OSLCs as described by Zou et al. [11 (link)].

Cells were trypsinized at that point washed with N2-supplemented DMEM/F12 (Invitrogen, Carlsbad, CA, USA). Human basic fibroblast growth factor (hbFGF; 10 ng/mL; Invitrogen) and human recombinant epidermal growth factor (hrEGF; 20 ng/mL; Biovision, Atlanta, GA, USA) are added in N2-supplemented DMEM/F12^{96 (link)}. After CSC221 cells seeded at a density of 5 × 10³ cells/well in ultra-low attachment 24-well plates (Corning Inc., Corning, NY, USA). After 14 days of incubation, spheres were quantitated by inverted phase contrast microscopy. The relative sphere formation ability was calculated through the IMT iSolution software (IMT iSolution Inc., Northampton NJ, USA)⁹⁷ measuring the pixel intensity of the sphere area randomly in each plate. Data are presented as the average of three independent experiments.

A431 cancer cells were treated with hBM-MSC-CM or human fibroblast-CM as well as control media (DMEM) for 4 days, then were trypsinized, washed and passed through a 40 μM cell strainer to obtain single cell suspension. Cells were re-suspended into a six-well ultra-low attachment plate (Cat NO. 29443-030VWR) at 10,000 single cells/well and cultured in DMEM/F12 medium with 20 ng/ml hEGF, 20 ng/ml hbFGF, and 2% B-27 (Life Technologies Corp.) at 37°C in 5% CO₂ (serum free spheroid medium). Medium was changed once a week. Two weeks later, individual spheres were counted under an inverted microscope at 40x magnification. The percentage of cells capable of forming spheres was calculated as follows: [(number of spheres formed/number of cells plated) × 100].

Sphere cultures were performed as described previously^{31 (link)}. Cells were plated at 1000 cells per well in six-well ultra-low attachment plates (Corning Inc., Corning, NY) and cultured in a DMEM/F12 medium with 10 ng/mL hEGF, 10 ng/mL hbFGF, and 2% B-27 (Life Technologies Corp.) at 37 °C in 5% CO₂. On day 7, the number of colonies was counted under an inverted contrast microscope.

Undifferentiated RUES2 hESCs (Female line, Rockefeller University, NIH registry number 0013) were plated at 1.6x10⁵ cells/cm² on Matrigel (BD) coated plates and maintained in an undifferentiated state with mouse embryonic fibroblast (MEF) conditioned media containing 5 ng/mL hbFGF (Peprotech, 100-18B). Directed differentiations using a monolayer platform were performed based on previous reports [27 (link)] with a modified protocol. Undifferentiated hESCs were plated as single cells as described previously and upon reaching appropriate confluency, treated with the Wnt/β-catenin agonist CHIR-99021 (1 μM, Cayman chemical, 13122) for 24 hours. Cells were then exposed to Activin A (R&D SYSTEMS, 338-AC-050) (100 ng/mL) in RPMI/B27 medium (day 0). After 17 hours, media was changed to RPMI/B27 medium containing BMP4 (R&D SYSTEMS, 314-BP-050) (5 ng/mL) and CHIR-99021 (1 μM, Cayman chemical,13122). On day 3, media was changed to RPMI/B27 medium containing the Wnt/β-catenin antagonist XAV-939 (1 μM; Tocris, 3748). Media was then changed on day 5 to RPMI/B27 medium. From day 0 to day 5, the B27 supplement utilized did not contain insulin (Invitrogen, 0050129SA). From day 7–14 a B27 supplement with insulin was used (Invitrogen, 17504044). For assays assessing the onset and rate of beating, cultures were analyzed independently during differentiation, with each well counted as n = 1.