Stem cell factor (scf)

Cells (bone marrow and PBMCs) were thawed in a 37 °C water bath for 1.5 min and then washed with Iscove’s Modified Dulbecco’s Medium (IMDM). The ACK (ammonium-chloride-potassium) lysing buffer was used to remove red blood cells from unfractionated bone marrow samples. Cells were then washed and placed in an expansion medium containing serum-free expansion medium (SFEM; Stem Cell Technologies, Vancouver, BC, Canada) supplemented with granulocyte macrophage colony-stimulating factor (20 ng/mL; eBioscience, San Diego, CA), stem cell factor (SCF; 45 ng/mL; Stem Cell Technologies) and interleukin-3 (20 ng/mL). In the last step, plasma from a healthy control study donor was added at 5% concentration per well, followed by incubation at 37 °C with 5% CO₂.
On day 7, non-adherent cells were washed in IMDM and 0.05 × 10⁶ cells/well of a 12 well plate were seeded in SFEM supplemented with human recombinant Epo (1 U/mL; Stem Cell Technologies), holotransferrin (0.3 mg/mL; R&D Systems, Minneapolis, MN) and stem cell factor (10 ng/mL; Stem Cell Technologies) and 5% plasma from a control subject was added followed by incubation at 37 °C with 5% CO_2.

CD34-positive cells were immunomagnetically separated from aspirated bone marrow of CML patients at diagnosis as previously published [21 (link)] and grown in presence of low cytokines concentration (FLT3 ligand 5 ng/mL, stem cell factor 5 ng /mL, IL-3 and IL-6 1 ng/mL, all from Stem Cell technologies) to avoid impairing BCR-ABL1-dependent proliferation. To isolate Ph+ B-ALL cells we used peripheral blood of patient at diagnosis showing 80% of lymphoblastic cells obtained by gradient separation using Ficoll Paque Premium, cultivated in RPMI supplemented with 10% of non-inactivated fetal bovine serum (FBS) (EuroClone), 2 mM of glutamine, 100 µg/mL and 50 µg/mL of streptomycin (all from Sigma-Aldrich). Nilotinib was provided by Novartis while Venetoclax was purchased from Santa Cruz.

BM-MSC derived from JAK2V617F patients and HD were seeded at a density of 1X10⁵cells/well in 24-well plates and cultured overnight. Purified CD34⁺cells (1x10⁵-2x10⁵) from BM of MPN patients and HD were co-cultured using a transwell membrane of 0.04μm (Costar, Corning NY, USA). After 48 hours of co-culture, HSPC were recovered and 5x10³ cells were seeded into methylcellulose MACS Media with Stem Cell Factor, GM-CSF, G-CSF, IL-3 and IL-6 (MethoCult H4534-Stem Cell Technologies, Vancouver, Canada) to quantify only the progenitor cell colony-forming unit–granulocyte/macrophage (CFU-GM), according to the manufacturer’s instructions. After 14 days, CFU were enumerated and classified by morphology as previously described[27 (link)].

Single cell culture was performed according to a previous study [17 (link)]. Briefly, individual cells isolated from different sources were placed into each well of 96-well microplates, ranging from 192 to 960 wells, as per the number of cells obtained from each patient (Additional file 2). Individual CD34 cells were cultured in serum-free medium containing 100 ng/mL stem cell factor, 100 ng/mL Flt-3, 100 ng/mL thrombopoietin, and 50 ng/mL granulocyte colony-stimulating factor (G-CSF) (all from Stem Cell Technologies, Vancouver, British Columbia, Canada). After culture for 5 days, each well of the microtiter plate was examined with an inverted microscope (Olympus IX50, Melville, NY) to determine growth and plating efficiency of the single CD34 cells. The growth and proliferative capacities of normal hematopoietic stem cells and ASCs were determined as a function of plating efficiency (the number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Growth was quantified and graded with the following scoring system according to cell number in each CD34 clone: grade 1, 5 or less cells/well; grade 2, 6 to 10 cells/well; grade 3, 11 to 20 cells/well; grade 4, 21 or more cells/well.

A transgenic NKX2-5-eGFP/w hESC line that faithfully reports endogenous NKX2.5 expression by GFP was described previously (Elliott et al, 2011 (link)). Undifferentiated hESCs were maintained on irradiated mouse embryonic fibroblasts, and cardiac differentiation was induced using a spin EB protocol. Briefly, hESCs were harvested and resuspended on day 0 in BPEL medium (Ng et al, 2008 (link)) containing 20–30 ng/ml hActivin-A (R&D Systems), 20–30 ng/ml bone morphogenetic protein 4 (R&D Systems), 40 ng/ml stem cell factor (Stem Cell Technologies), 30 ng/ml vascular endothelial growth factor (R&D Systems) and 1.5 μmol/l CHIR 99021 (Axon Medchem). EBs were refreshed on day 3 with BPEL and then transferred to gelatin-coated dishes on day 7.
To induce atrial specification in hESCs, cardiac differentiation was initiated as described above and 1 μmol/l all-trans retinoic acid (RA) (Sigma) was added on day 4 of differentiation. Cells were refreshed with BPEL on day 7 of differentiation.

Purified CD34⁺ HPCs were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO). Recombinant cytokines, including stem cell factor, Flt-3 ligand, and thyroperoxidase were purchased from Stem Cell Technologies (Vancouver, BC, Canada) and were used at concentrations of 100 ng/mL. The cells were incubated in a fully humidified incubator at 37° C in an atmosphere containing 5% CO₂.

Sorted cells (1 × 10³ cells) were seeded in a 24-well ultra-low adherent plate (Corning) in 0.5 mL of mixed medium to perform sphere formation detection. The medium contained 32% MethoCult medium, 20% MammoCult basal human medium with a final concentration of 2% MammoCult proliferation supplements and 48% DMEM supplemented with final concentrations of 100 pg/mL EGF, 50 ng/mL bFGF, 5 ng/mL stem cell factor, 1 μM hydrocortisone, and 5 mg/mL insulin, all obtained from STEMCELL Technologies. The cells were cultured at 37 °C in a 1% O₂ and 5% CO₂ humidified atmosphere for 14 d. The number of tumor spheres was counted under microscope.