Fgf 1

Hematopoietic progenitor cells were expanded for up to seven days as described previously [34] (link) in StemSpan culture medium supplemented with 10 ng/mL stem cell factor (SCF; PeproTech GmbH, Hamburg, Germany), 20 ng/mL thrombopoietin (TPO; PeproTech), 10 ng/mL fibroblast growth factor 1 (FGF-1; PeproTech) and 10 µg/mL heparin (Roche GmbH, Mannheim, Germany) [32] (link). For co-culture experiments, addition of cytokines was not performed as MSCs alone activate proliferation. Culture medium was always supplemented with 10% serum of individual MDS patients or control samples as described in our previous work [31] (link).

MSCs were isolated from 20 mL BM aspirates collected from the iliac crest of healthy, consenting adult donors. The Mater Health Services Human Research Ethics Committee and the Queensland University of Technology Human Ethics Committee approved aspirate collection (Ethics No. 1541A). MSCs were isolated as described previously.^{18 (link)} MSCs were expanded in medium containing low-glucose Dulbecco's modified Eagle's medium (DMEM; Life Technologies), 10% fetal bovine serum (FBS; Life Technologies), 10 ng/mL fibroblast growth factor-1 (FGF-1; PeproTech), and 100 U/mL penicillin/streptomycin (PenStrep; Life Technologies) in a 2% O₂ and 5% CO₂ atmosphere at 37°C. MSCs were used up to passage 4 for experiments.

Human adult BM MSC isolation, culture and characterization were performed as previously described [14]. Ethics approval for aspirate collection was granted by the Mater Health Services Human Research Ethics Committee and the Queensland University of Technology Human Ethics Committee (Ethics number: 1000000938). Briefly, mononuclear cells were isolated from 20 ml of BM aspirates using density gradient centrifugation. MSC were enriched using plastic adherence overnight in a 20% O₂ and 5% CO₂ atmosphere at 37 °C in MSC expansion medium containing: low glucose DMEM (Life Technologies), 10% fetal bovine serum (FBS; Life Technologies), 10 ng/mL fibroblast growth factor-1 (FGF-1; Peprotech), and 100 U/ml penicillin/streptomycin (PenStrep; Life Technologies). After discarding the loose cells and replenishing the medium, MSC were further expanded in a 2% O₂ and 5% CO₂ atmosphere at 37 °C. Cells were passaged at 80% confluence using 0.25% trypsin/EDTA (Life Technologies) and new flasks were re-seeded at ~1,500 cells per cm². MSC were used at passage 3. Using flow cytometry, cells were characterized for their expression of CD44, CD90, CD73, CD105, CD146, CD45, CD34, HLA-DR, and tri-lineage differentiation capacity, as described previously43 (link).

The drugs listed below were acquired from Selleck chem: FGFR inhibitors PD166866 (specific to FGFR1), Alofanib (targeting FGFR2), H3B-6527 (inhibiting FGFR4), and AZD4547 (effective against FGFR1-3), pan-FGFR inhibitor TAS-120, JAK inhibitors Solcitinib (inhibiting JAK1) and AZD1480 (inhibiting JAK2), UC2288 (p21 inhibitor), STAT inhibitors Fludarabine (inhibiting STAT1),
Stattic (inhibiting STAT3) and BAY2353 (Niclosamide, inhibiting STAT3), SGC-CBP30 (potent CREBBP/EP300 inhibitor), and ulixertinib (ERK1/ERK2 inhibitor). Human EGF protein and FGF ligands including FGF1, FGF2, FGF4, FGF7, FGF9, FGF8a, FG19 and FGF21 were purchased from PeproTech. The breast cancer cell lines CAMA1, MDA-MB-134, MCF7, and T47D were obtained from the American Type Culture Collection (ATCC). The CAMA1 and MCF7 cell lines were grown in DMEM with a 10% FBS and 1% antibiotic-antimycotic solution, while T47D and MDA-MB-134 were cultured in RPMI with 10% FBS and 1% antibiotic-antimycotic solution. Regular testing for mycoplasma contamination was conducted using the commercially available Myco Alert kit from Lonza. All cell lines utilized in this research have undergone authentication by ATCC, and only cells with a low number of passages were employed in experiments to ensure work confidence.

Primary keratinocytes were incubated overnight in keratinocyte serum‐free medium without EGF. EGF (Sigma) or FGF1 (Peprotech) was added to a final concentration of 10 ng/mL and incubated for 24 hours. After 20 hours, BrdU (Sigma) was added to the cell culture medium at a final concentration of 100 μmol/L followed by incubation for 4 hours at 37°C and 5% CO₂. Then, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes at RT. Afterwards, they were permeabilized and DNA was denatured using 0.1% Triton X‐100 in 2 mol/L HCl for 30 minutes. Cells were then incubated in boric buffer (100 mmol/L boric acid, 75 mmol/L NaCl, 25 mmol/L sodium tetraborate, pH 8.5) for neutralization for 5 minutes and blocked using 1% BSA for 30 minutes, followed by incubation with a FITC‐coupled anti‐BrdU antibody (11202693001, Sigma) at 4°C overnight. Cells were then washed with PBS, and a Cy2‐conjugated secondary antibody (1:500, Jackson ImmunoResearch Laboratories, Inc) was added to amplify the signal. Nuclei were counterstained with Hoechst 33342 (1:3000, Sigma).

Cells were treated according to certain conditions before sphere culture with or without TGF-β1. Treated cells (1×10²) were seeded in ultra-low attachment 96-well plates (Corning) in serum-free DMEM/F12 (Gibco) supplement with insulin (5 μg/ml, HY-P0035, MCE), epidermal growth factor (EGF, 20 ng/ml, PeproTech), 2% B27 supplement (Invitrogen, CA, USA), and fibroblast growth factor-1 (FGF-1, 20 ng/ml, PeproTech). The cells were cultured for 7 days and then photographed using a microscope. The number of spheres (diameter > 50 μm) in each well was finally counted.