Thrombopoietin

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.

FA-like CD34⁺ cells were generated by transducing HD CB CD34⁺ cells with a shFANCA/EGFP LV (47 (link)). This LV carries the sh11 and sh7 hairpin RNAs (shRNAs) that target human FANCA and expresses the EGFP marker gene. The shFANCA/EGFP LV was generated in the puc2CL11Egw.sh7FA-A7 with 2 sites of interference over the FANCA messenger. The shSCR/EGFP LV was used as an interference control and contained a scrambled sequence and the EGFP marker gene. The functionality of the shFANCA/EGFP LV has been previously demonstrated (47 (link)). HD CB CD34⁺ cells were stimulated for 2 days in IMDM plus GlutaMAX supplemented with 20% FBS and the hematopoietic growth factors stem cell factor (SCF) (30 ng/mL), thrombopoietin (TPO) (10 ng/mL), and FMS-like tyrosine kinase 3 ligand (FLT3-L) (10 ng/mL) (Miltenyi Biotec) in a humidified atmosphere at 37°C and 5% CO₂. Cells were transduced with the shSCR/EGFP or shFANCA/EGFP LVs using 2 infection cycles of 24 hours in plates precoated with Retronectin (Takara). Transduced cells were then incubated for 4–12 days in culture under conditions used for prestimulation. Levels of NKG2D-Ls in cells transduced with the shFANCA/EGFP LV were compared with shSCR/EGFP-transduced cells showing similar levels of EGFP expression, and also with EGFP^– cells.