Heparin

All mice were terminally anesthetized with 3–5% isoflurane followed by injection of pentobarbital (300 mg/kg body weight, i.p.; Streuli Pharma AG). Animals were transcardially perfused with ice-cold Ringer’s solution [containing 10⁵ IU/l heparin (Roche) and 0.25% NaNO₂]. Brains and spinal cords were quickly dissected and snap-frozen on dry ice to preserve RNA quality. The brains were immersed in 4% PFA overnight before being transferred to 30% sucrose in phosphate buffer (PB) for cryoprotection. Samples were blinded from the point of tissue harvesting.

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).

All mice were terminally anesthetized with 3–5% isoflurane followed by injection of pentobarbital (300 mg/kg body weight, i.p.; Streuli Pharma AG). For the anatomical studies, animals were transcardially perfused with Ringer's solution [containing 10⁵ IU/L heparin, (Roche) and 0.25% NaNO₂] followed by 4% phosphate-buffered formaline containing 5% sucrose. The brain and spinal cord were dissected, postfixed in the same solution for 16 h and transferred to 30% sucrose in PBS for cryoprotection. For the transcriptomic study, animals were transcardially perfused with ice-cold Ringer's solution only. To preserve RNA quality, all following procedures were done rapidly to minimize time between animal kill and tissue collection. Brains and spinal cords were dissected and the intermediate layer of spinal levels C5/C6 was dissected by crude trimming with blades und a dissection microscope. The spinal samples were snap frozen in liquid nitrogen and stored at −80°C until further processing. Adjacent spinal tissue was analyzed for spinal anatomy to ensure correctness of the spinal level. The brains were immersed in 4% PFA overnight before being transferred to 30% sucrose in phosphate buffer (PB) for cryoprotection. Samples were blinded from the point of tissue harvesting.