Stem cell factor (scf)

Bones of Pf4-Cre⁺Gucy1b1^+/LoxP and Pf4-Cre⁺Gucy1b1^LoxP/LoxP mice were centrifuged at 2,500g for 1 min after removing proximal epiphyses^{47 (link)}. The obtained bone marrow was subjected to 1× RBC lysis buffer (catalog no. 420301; BioLegend), filtered through 70-μm mini-cell strainers and resuspended in IMDM medium (catalog no. 31980030; Thermo Fisher Scientific) supplemented with 10% FCS (catalog no. S0615; Sigma-Aldrich), penicillin-streptomycin (1:100, catalog no. 15070063; Thermo Fisher Scientific), thrombopoietin (200 ng ml⁻¹, catalog no. 130–096-301; Miltenyi Biotec) and stem cell factor (20 ng ml⁻¹, catalog no. 130–101-693; Miltenyi Biotec) to a concentration of 1 × 10⁷ cells per ml. Cells were cultivated in a humidified incubator with 5% CO₂ at 37 °C for 9 d and supplemented with fresh medium every third day.
Megakaryocytes were collected by performing two rounds of bovine serum albumin (BSA) density gradient filtration^{48 (link)}. Briefly, cells were resuspended in PBS and placed on top of two layers of a 1.5 and 3% BSA solution and incubated for 40 min. Sedimented cells were subjected to a second round of density gradient filtration, obtained purified megakaryocytes resuspended in 500 μl of TRIzol (catalog no. 15596026; Thermo Fisher Scientific) and stored at −80 °C for further processing.

Spin-EBs were formed using a 96-well ultra-low adherence plate (Costar 7007). iPSCs were washed with PBS and harvested by incubating the cells for 5 min at 37°C with 1 mL of warm TrypLE Express (Gibco by Life Technologies). Cells were counted, washed with PBS, and resuspended at a final concentration of 1.25 × 10⁵ cells/mL in EB media: mTeSR1 (STEMCELL Technologies), 50 ng/mL BMP-4 (GIBCO- PHC9534), 20 ng/mL stem cell factor (Miltenyi Biotec), 50 ng/mL vascular endothelial growth factor (GIBCO- PHC9394); 100 μL of cell suspension in EB media supplemented with 10 μmol/L Y-27632 was added per well, and the 96-well plate was centrifuged at 100 × g for 3 min and incubated for 4 days. EBs were fed at days 1 and 2 by aspirating 50 μL of medium and adding 50 μL of fresh EB medium.

In this experiment 1x10⁵ CD34⁺ cells were co-cultured in a volume of 500μl RPMI per well with or without MVs from 6 HD and 6 patients. After 24hours, 5x10³ cells were seeded into methylcellulose MACS Media with Stem cell Factor, GM-CSF, G-CSF, IL-3 and IL-6 (Miltenyi Biotec GmbH, Germany) to quantify the progenitor cell CFU-GM, as previously described[15 (link)].
These cultures were incubated in a humidified atmosphere at 37°C with 5% CO₂. After 14 days, CFU-GM colonies were scored with an inverted microscope. Results were expressed as the ratio between CFU-GM obtained with CD34⁺ cells that had been co-cultured with MVs from MDS or HD and the same CD34⁺ cells without MVs.

For MKs differentiation and maturation, cell line cultures were treated with 20 nM phorbol 12-myristate 13-acetate (PMA, Sigma) for 10 days. BMCs were incubated with 50 ng/ml recombinant mouse thrombopoietin (TPO, Miltenyi) in combination with 10 ng/ml Stem Cell Factor (SCF, Miltenyi), 10 ng/ml Interleukin-3 (IL-3, Invitrogen), 10 ng/ml IL-11 (Miltenyi) and 10 ng/ml IL-6 (Miltenyi) for 6 days.