Interleukin 6 (il 6)

Normal, human CD34⁺ HSPCs were purified by positive selection using the Midi MACS (magnetic-activated cell sorting) LS⁺ separation columns and isolation Kit (Miltenyi Biotec # 130–042-401) starting with mononuclear cells that were isolated from CB by Ficoll-Hypaque Plus (GE Healthcare #17–1440-03) density centrifugation. CD34⁺ cells were cultured in X-vivo 15 medium (Lonza #04–744Q) and 20% BIT 9500 medium (STEMCELL Technologies #09500), supplemented with SCF (100 ng/ml), FLT-3 ligand (10 ng/ml), IL-6 (20 ng/ml), and TPO (100 ng/ml) as the basic culture. All cytokines were purchased from PeproTech.

Colony-forming cells (CFCs) were assayed in semisolid media as previously described.^{1 (link)-4 (link)} Briefly, 5 × 10² cells were plated per dish in duplicate cultures containing 1mL IMDM with 1.1% methylcellulose supplemented with 30% FBS, 5 × 10⁻⁵ M 2-ME (StemCell Technologies, Vancouver, BC, Canada), 100 ng/mL SCF, 100 ng/mL FL, 50 ng/mL IL-3, 50 ng/mL IL-6, 50 ng/mL granulocyte-macrophage colony-stimulating factor, and 5 U/mL erythropoietin. All cytokines were purchased from Cell Genix. The colonies were enumerated after 14 days using standard criteria. Fold expansion was calculated by dividing the number of total CFU-mix at Day 9 by the total number of CFU-mix at Day 0.

Blood or bone marrow was subjected to Ficoll separation followed by red blood cell lysis. An AutoMACS Pro Separator (Miltenyi Biotech, Bergisch Gladbach, Germany) was employed to purify CD34⁺ cells from MF patient samples and cord blood (CB) (St. Louis Cord Blood Bank, SSM Health Cardinal Glennon Children’s Hospital). Primary cells were cultured in RPMI medium supplemented with 10% FBS and cytokines (CC100: SCF, FLT3L, IL-3, IL-6; StemCell Technologies, Vancouver, Canada). We typically culture cells for 24 hours prior to experiments to minimize the influence of drugs present in the plasma. “Ruxolitinib exposed” refers to the status of the patient from which the sample was collected, and is defined by the patient’s physician, with adherence to published guidelines for diagnosis and disease monitoring (26 (link)–29 (link)). Except where noted as “responsive”, this term includes primary and secondary occurrences of relapse, refractory, and resistant disease, collectively known as failure. Written informed consent was obtained from all donors (The University of Utah IRB #45880). Patient information is summarized in Supplemental Table 1.

Human CD34+CD38+ cells were maintained in SFEM (Stem Cell Technologies) enriched with CC100 cocktail of cytokines (SCF, 100 ng/ml; FLT3L, 100 ng/ml; IL-3, 20 ng/ml; IL-6, 20 ng/ml; Stem Cell Technologies) for 12 hr after thawing. Cells were then washed to eliminate the CC100 cytokines and were plated at a concentration of 10⁶ cells/ml in SFEM with the addition of either 100 ng/ml of G-CSF (Peprotech) or 10,000 U/ml of M-CSF (R&D Systems, USA) to induce myeloid differentiation, or 10 ng/ml of EPO (Peprotech) to induce erythroid differentiation. The cytokines were re-added at every medium-change.