Rmtpo

Primary MKs, OBs, and ECs were isolated according to previously published methods21 (link), 31 (link)-34 (link). For MKs preparation, c-kit⁺ cells from mouse bone marrow (BM) were first sorted with flow cytometry. Then, the cells were grown in StemSpan SFEM medium (Stem Cell Technologies, Vancouver, BC, Canada) in the presence of 20 ng/mL rmTPO (Peprotech; Rocky Hill, NJ, USA) and 10 ng/mL IL-3 (Peprotech). After 9-10 days of culture, the primary MKs were purified with flow cytometry according to the expression of CD41 (MWREG30; Biolegend, San Diego, CA, USA) and CD42b (M040-1; Emfret Analytics, Eibelstadt, Germany). For OBs preparation, calvaria obtained from neonatal mice was sequentially digested with 200 U/mL collagenase (Roche, Indianapolis, USA). The samples were incubated at 37°C for five different time periods (0-10, 10-20, 20-35, 35-50 and 50-65 min), and fractions 3-5 were collected and used for the OBs culture. ECs isolated from the BM of 7- to 8-week-old C57BL/6 mice were purchased from BioChain (Z7030031; Newark, CA, USA). These cells were positive for CD31 (MEC13.3; Santa Cruz, Dallas, Texas, USA), CD105 (MJ7/18; Biolegend) and vascular endothelial growth factor receptor 1 (VEGF-R1; ab9540, Abcam), and negative for CD133 (315-2C11; Biolegend). All experiments were performed on fresh, low passage (p2-3) cells.

Bone marrow of C57BL/6 (Janvier Laboratories, Saint Berthevin Cedex, France and Central animal facility, Hannover Medical School, Hannover, Germany) mice was harvested from femora and tibiae and lineage negative (lin⁻) cells were purified using MACS separation (Lineage Cell depletion kit, Miltenyi, Bergisch Gladbach, Germany). Cells were cultured and prestimulated for 24 h in StemSpan medium (StemCell Technologies, Cologne, Germany) supplemented with 10 ng/ml rmSCF, 20 ng/ml rmTPO, 20 ng/ml rmIGF and 10 ng/ml rhFGF (all PeproTech) prior to transduction.

Single cells were deposited into 96 well U-bottom plate (TPP) using a BD FACS ARIA II cell sorter. Cells were cultivated in Iscove’s Modified Dulbecco’s Medium (Gibco) supplemented with 10% FCS (FBS superior, Biochrom), 1% Pen/Strep, stem cell factor (rmSCF 20 ng/ml, PeproTech), Thrombopoietin (rmTPO 20 ng/ml, PeproTech), Interleukin-3 (rmIL-3 20 ng/ml, PeproTech), Erythropoietin (rhEPO, 5U/ml, NeoRecormon, Roche). Culture wells were visually inspected and counted by light microscopy (PrimoVert, Zeiss) and assigned to categories on day 5 of culture (see Fig. 4d). Representative wells were stained with Hoechst 33,342 (5 µg/ml) and imaged by fluorescence microscopy (BZ-X710, Keyence) (Supplementary Fig. 5g).